Curation Manual2.0

From curation_manual
Jump to: navigation, search

Contents

Use of this Manual

The following is a collection of notes from curation meetings with the Epitope Council and is a suggested guideline for capturing immunological data into the IEDB.

Purpose

The purpose of this manual is to ensure consistency and accuracy of literature based curation. All IEDB curators must:

1. Read the manual

2. Read the manual

3. Refer to the manual

4. Follow the manual

5. When experimental scenarios are encountered in the literature that do not conform to the guidelines found in the manual, these issues are to be discussed in the curation meeting to foster development of new guidelines or to adapt our current rules in order to accurately capture epitope related data.



How to Curate

General Considerations

The Curation Notes focus on detailed rules for handling data related to specific topics such as epitope structure, B and T cell responses, and negative data. When approaching a curation, one must take into consideration the main conclusions of the paper as revealed in the title and abstract. Rather than being buried in the comments, the main conclusions should be clear in the database and easily available to the user in a search. Be careful to avoid omission of critical data by blindly following curation rules. Be true to the integrity of the data and always consider the end user’s perspective.

Prevailing Rules

Certain database rules supersede all other criteria. Consider the full scope of the data prior to curation.

  • Due to the broad specificity of MHC molecules, all MHC binding data is captured. All MHC data receive separate entries and are not candidates for bulk curation. Note that if a mutated MHC molecule is mutated at a residue known, shown, or stated by the authors to contact the epitope directly, the mutated MHC molecule will be added to the MHC allele finder as a new MHC molecule labeled as a mutant. This data cannot be bulked under the wild type MHC molecule.
  • All naturally processed epitope data is captured. All naturally processed data receive a separate entry and are not a candidate for bulk curation.
  • Do not capture peptides tested as positive or negative controls. Exceptions to this rule occur when the control peptide is derived from the same source antigen or organism as the curated epitopes and its experimental data is considered to be interesting reference data.
  • Do not repeat data. If the database fields are used properly, each type of data should be entered only once, in its proper field.
  • Comments are to only be used when needed to clarify the information present in the other fields, not to repeat the information present in the other fields.

Minimum Criteria for Curation

Presence of Epitope

Polyclonal data

In general, polyclonal data generated without using the epitope as either the immunogen nor the antigen is not entered in the database for that specific epitope. Always consider if the epitope is responsible for the outcome of the assay. Look at the control experiments in order to determine if the epitope played a role in the immune response being measured.

Exmaple 1:

Assay 1 IVP1 = epitope, Ag = source organism, ELISA negative

Assay 2 IVP1 = epitope, IVP2 = source organism, Ag = source organism, ELISA positive

In this scenario, it is unclear if the epitope led to the positive outcome in assay 2. Only assay 1 can be curated for the epitope.


Exmaple 2:

Assay 1 IVP1 = epitope, Ag = source organism, ELISA negative

Assay 2 IVP1 = epitope, IVP2 = source organism, Ag = source organism, ELISA positive

Assay 3 IVP1 = source organism, Ag = source organism, ELISA negative

In this scenario, it is clear that the epitope led to the positive outcome in assay 2. Assays 1 and 2 should be curated under the epitope.


The table below serves as a guide to determine curatable contexts of polyclonal responses.


Table 5. Curatability of polyclonal T Cell/Antibody/B Cell Contexts
Immunogen 1 Antigen
Epitope Source Antigen Source Organism Other
Epitope Curate Curate Curate Curate
Source Antigen Curate Do not curate* Do not curate* Do not curate*
Source Organism Curate Do not curate* Do not curate* Do not curate*
Other Curate Do not curate* Do not curate* Do not curate*


Immunogen 1 = either in vivo process 1 immunogen or Primary induction in vitro immunogen


Important Exception: When curating a deduced epitope, and neither the immunogen nor the antigen is the epitope, the assays performed to deduce the epitope should be curated and the antigen epitope relation should be either <Fragment of Source Antigen> or <Other Structure from Source Organism>.


Important Exception: When curating contexts with a second in vivo process, some allergy assays are curatable when the only use of the epitope is the in vivo process 2 immunogen. Additionally, treatment/protection, tolerance, and exacerbation assays are curatable when the epitope is responsible for the treatment/protection, tolerance induction, or exacerbation of disease even when administered as in vivo process 2 immunogen.


Important Exception: If a disease (or other in vivo process) that is unrelated to the epitope is deemed important to capture, and this disease occurred in the host prior to administration of the epitope or epitope related immunogen, an exception to the above table should be made. In vivo 1 and in vivo 2 processes should always be captured in the order in which they occurred.

For example, if one donor group has HIV and this disease effects the outcome of the assay to an administered and unrelated immunogen, a polyclonal response with IV1 Imm = HIV, IV2 Imm = epitope, Ag = Source Ag is curatable.


Monoclonal data

Curatability of Monoclonal Contexts (monoclonal antibodies and T cell clones): If the specificity of the monoclonal receptor is shown, known, or implied (for example, the T cell clone is generated by in vitro stimulation with the epitope) and the clone or mAb is tested for reactivity to source protein or organism or to a different antigen, this data should be curated for the epitope even though neither the immunogen nor the antigen is the epitope. Essentially everything that a monoclonal T cell receptor or a monoclonal antibody binds is curatable under the epitope to which that receptor is specific.

Negative monoclonal data is not curated unless it was expected to be positive and the negative outcome is interesting.

Data Not Shown

Data not shown in the manuscript will not be entered into the database as a separate context, but may be commented on under similar contexts. When authors discuss results without actually showing the data in that particular reference, one may infer that the same protocols described in the reference were used to obtain those results.

  • When data is given as supplementary data, it will be curated.


Qualitative Measurement

This is a required field. Curators must always be able to complete this field in order to curate the assay. If the outcome is indeterminate, the curator must contact the authors before curation. If the outcome of an assay is said to be negative by the authors, but there are more than 0 subjects responding, leave the 'Number of Subject Responded' field blank. Enter any details in the assay comments field.

Before You Curate

Before you curate:

• Read the entire paper

• Determine all epitope sequences before you start and label the manuscript with the epitope #s –MANDATORY

• Analyze all figures (and text) for the important fields as below

• Have a plan for how many epitopes, how many assays, what type of assays, the qualitative outcome for each

• When bulking, enter all figures & tables that are bulked in the data location field (Required)

• If you need alleles, strains, nonseq objects, etc added, you should first determine if you can select a “dummy” value to be changed later or not.

After reading, develop a curation plan. For all figures, determine these important fields before curating:

  • Host Organism
  • Immunogen
  • Imm Process
  • Antigen
  • MHC (for T cell assays only)
  • Assay Type
  • Outcome

Determine how many epitopes, assays per epitope, assay types, and planned bulking prior to curation. This will allow you to optimize the copy and explode features of the curation interface.

Special Note:
Do not hit the enter key when entering data into any free text field!


Review Process

Curators

Use cover sheet to ask questions & bring up confusing issues. If you did something “special” explain why.

If you do not know how to do something, you should ask before curating. Try to ask the correct senior curator-we all have our “specialties”.

Always print a new PDF & return old PDF when asked to recurate & return to PR.

Prioritize recurations over initial curations

Label the manuscript with the epitope #s and label when you bulk figures. Label every monoclonal antibody and T cell line/clone with the epitope(s) it "belongs" to.

Always attach all cited refs -this is MANDATORY. The reviewer should not have to look up refs for you and the reviewer MUST check all cited refs as part of the review process.

Properly utilize the Data location field -it is visible to the public. If you bulk, enter all figures and tables that are depicted in that assay. Use proper structure (Figures 1, 2, and 3, tables I and IV).


Reviewers

Answer questions that are brought up & decide if it’s a minor misunderstanding or a real unresolved issue.

Ask other reviewers about any issue that you are not certain about before responding to the curator (communication is good!)

Do not sign the cover sheet if you need to see the curation again

Submit unresolved issues with all needed information to Nima by Monday for curation meeting discussion. Provide the current guideline(s), PMID, & describe what the problem is with some ideas about solutions.

Keep review time to a minimum, if you have time off or are otherwise unable to complete a review on time, have it reassigned. If there is a delay, communicate with the curator.


Check:

for accurate capture of all data

that data is curated under the correct epitope (optimal/minimal)

proper Imm/Ag epitope relations-compare sequence, protein, organism names & strains with the epitope

that every Epitope, Imm & Ag has an evidence code

that in vivo process type is correct-refer to manual

that ever APC or effector cell has all three cell fields filled out

that every possible antibody field is completed

for proper bulking (MHC in T cell assays, assay type in B cell assays, etc)

Ask other curators when you are not sure! 2nd opinions are good

Polyclonal data is not curated if neither Imm & Ag are the epitope

Neither Ag nor Imm are the epitope=needs comment.

If bulking=needs comment.

Check for typos, grammar, etc. It looks bad when it is missed!

The goal is more consistent review! We must work together and share our experiences/opinions.


Review Metrics

For consistent metrics regarding review, please use the following guidelines:

  • No recuration -no changes at all, corrections to typos, very minor changes that are due to problems with the system, waiting on additions to Finders, suggested subjective changes, etc.
  • Minor recuration -forgotten/wrong minor fields (cell type, adjuvant, carrier etc), needs/edit comments, misunderstanding of new field, complex/confusing issue, bulking issues etc.
  • Major recuration -forgotten/wrong major field (immunogen), did not curate figures/epitopes, misinterpretation of the paper, misunderstanding regarding well established rules.

Helpful Websites

  • FINDERS-If you ever want to look at the information present in any of the finders, once logged into the curation system, go to the link under the "support" tab:

http://curation.iedb.org/testfinders.do


  • NCBI-BLAST-If any peptide sequence does not match a source protein in the IEDB molecule finder, the next place to look is NCBI:

http://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Proteins&PROGRAM=blastp&BLAST_PROGRAMS=blastp&PAGE_TYPE=BlastSearch&SHOW_DEFAULTS=on


  • NCBI-Taxonomy-If you are having trouble finding an organism species or strain in the organism finder, try NCBI. If you have the tax ID, the search may be easier. If you need to request a new IEDB strain, please look up the parent tax ID of the strain.

http://www.ncbi.nlm.nih.gov/sites/entrez?db=taxonomy


  • CELL LINES-for information on the tissue type, cell type, and source organism of common cell lines/clones:

http://www.biotech.ist.unige.it/cldb/cname-1c.html

http://www.atcc.org/Default.aspx?base


  • MHC Alleles-MHC nomenclature can be confusing and authors often describe the same allele with different names. If you do not find an allele that you need in the finder, research the nomenclature to be certain that there are no common synonyms. With mice, you may want to describe the MHC types present and will need to look up the haplotype of the mouse strain. Also, for T cell assays, you will need to know if mice share the same alleles in order to bulk or not bulk assays.

http://www.anthonynolan.org.uk/research/hlainformaticsgroup/lists/the-naming-of-hla-antigens.htm

http://jaxmice.jax.org/query/f?p=205:1:5019279104898472738

http://imgt.cines.fr/textes/IMGTrepertoireMHC/Polymorphism/haplotypes/mouse/MHC/Mu_haplotypes.html


  • Helpful rat allele information -Some papers refer to rat alleles by the name of their mouse counterparts, instead of using the rat allele name. Here is the translation:

Mouse I-A = rat RT1B

Mouse I-E = rat RT1D


  • OTHER-If you have ideas about other helpful websites, please email rvita@liai.org to submit them.

Shortcuts

Shortcut keys have been created for use while curating

Ctrl Alt S = Submit

Ctrl Alt M = Modify

Ctrl Alt C = Copy

Ctrl Alt E = Explode

Comments

There are comment fields throughout the database in order to give the curator the ability to capture VITAL information that would otherwise not be captured by the available fields of the database. The following guidelines should be used when entering comments.

Comments are not required and should always be as brief as possible. Comments should only contain information specific to the pertinent field. In other words, the Comments on Assay field should provide only details pertinent to the assay, its results, or interpretation.

  • Comments should stand alone and be self-explanatory. The database user should not need to obtain the actual reference in order to understand the comments. Do not refer to figures in a manuscript in the comments fields unless the accompanying comment remains self-explanatory even if the data location were to be removed. The preferred format for alluding to other figures or tables is in parentheses or braces at the end of the sentence.
  • Be tactful and neutral. Refer to any unavailable or unclear information in an inoffensive manner. The fact that particular information is not available in the manuscript does not require mention unless it would be confusing for the end user otherwise. These fields are not meant to explain curation strategies to reviewers, but to convey critical information to end users.
  • Avoid the use of uncommon abbreviations. All users should be able to understand the comments. Use your judgment when using abbreviations and reviewers should also check for uncommon abbreviations in the comments.
  • Comments should be written in proper American English to ensure consistency and allow for standardized querying. Check spelling and grammar and use complete sentences (noun and verb). Remember to use articles such as of and the. Capitalize the first letter of the first word in a sentence and only capitalize appropriate words in the text (T cell, M. tuberculosis). Sloppy writing reduces the credibility of the database. Do not use contractions.
  • Do not describe routine details of the assay. These fields are not meant to describe the experimental protocol, but rather to clarify what is present in the other fields and are to be used only when needed.
  • Do not repeat the information already captured in other fields.
  • Avoid interpretation of the manuscript. It is preferable to paraphrase author’s interpretations or conclusions from article text.

Special Note:
Do not hit the enter key when entering data into any free text field!

Add Epitope

This section captures the molecular structure of an epitope, which is defined as the structure interacting with receptors of the immune system (T cell, B cell/ antibody, MHC).

The fields utilized to capture epitope information are as follows:

Epitope location This is a free text field used to identify where the exact sequence or structure of the epitope was provided, whether within the manuscript or if it was found outside of the primary reference. This field is for internal use to validate the structures curated. Tables, figures, text, or text sections, such as materials and methods, where the actual structure was provided should be entered. If the structure was provided by the author through email contact or a cited reference, enter “author provided” or “cited reference” and attach the author’s email with the sequences or the relevant page from the cited reference to the curation print out. You must cite the reference used in this format Sette et al, J Immunol:25(4) 2006 [PMID:1234567].

Epitope Name This field should reflect how the author refers to the epitope being curated (p22, FLU 307-319, etc). If no name was provided by the author, the antigen name followed by the residues is acceptable (HA 307-319).

Reference Starting Position and Reference Ending Position The author specified positions are always recorded in the Reference Starting Position and Reference Ending Position fields exactly as the authors indicated them. Do not edit what the authors published.

Important Note: Repeating Epitopes
When exactly the same epitope sequence occurs at multiple positions in the same source antigen and reactivity is not demonstrated to be site specific, the positions of the first occurrence will be entered in the position fields with comments regarding the repetitive nature of the sequence entered in the epitope Comments field.

Reference Region For discontinuous epitopes, the reference region field is to be filled with the residues and positions exactly as stated by the authors.

Epitope Object The epitope structure is entered via the Object Editor #Epitope Object


Epitope Object

The epitope structure is curated via the Object Editor. Assign the epitope’s source organism based on what the authors say. If it has strain info, capture it regardless of what was used in the assays. The object editor will allow an epitope to be of the following object types and subtypes:

  • Sequence Molecule, no natural source

Subtypes: Peptide, no natural source, DNA, no natural source, and RNA, no natural source. This selection is used only for sequence molecules (peptides, RNA, DNA) that do not have a natural source. Epitopes of this type cannot have a natural source entered. They also cannot have source antigen and source organism as antigen or immunogen relations.

  • Fragment of a natural sequence molecule

Subtype: Peptide from Protein. This selection is made for peptide sequences that are derived from natural proteins. The majority of epitopes will fall into this category. Epitopes of this type must have a natural source entered into the object editor.

  • Discontinuous region on accession sequence molecule

Subtype: Discontinuous protein residues. This selection refers to discontinuous sequence epitopes. Epitopes of this type must have a natural source entered into the object editor. These epitopes are determined by loss of reactivity when the epitopic residues are mutated or differ between two proteins.

  • Region on Multi-Chain Molecule

Subtype: Region on Multi-Chain Protein. This is the subtype to select for discontinuous epitopes that are made up of residues that reside on two chains of a multi-chain molecule. If the epitope is made up of residues that are only found on one chain of a multi-chain molecule, the single chain protein should be captured as the epitope source and the epitope object type and subtype will be Fragment of a natural sequence molecule-Peptide from Protein.

  • Fragment of a Natural Non-Sequence Molecule

Subtypes: Peptidoglycan fragment, Glycolipid fragment, Carbohydrate fragment, Lipid fragment, Fatty acid fragment, Other fragment. This selection refers to natural non-sequence molecules that are derived from a larger natural molecule. Epitopes of this type must have a natural source which contains the epitope structure entered into the object editor. These epitope structures and their sources are selected from the Molecule Finder.

  • Accession Non-sequence Molecule

Subtypes: Peptidoglycan, Glycolipid, Carbohydrate, Lipid, Fatty acid, Other. This selection refers to any non-sequence molecule that is not derived from a larger natural molecule. It may be natural or artificial. Epitopes of this type may have a source organism entered into the object editor, if needed. These epitope structures are selected from the Molecule Finder.

  • Discontinuous Region on a Natural Non-Sequence Molecule

Subtype: Polysaccharide repeating unit. This type is used for discontinuous non-sequence epitopes. Currently, only polysaccharide repeating units have been curated. These epitope structures and their sources are selected from the Molecule Finder.


Object Editor

Objeditorpeptide.jpg

Once the object type and subtype have been selected, the object editor allows you to enter the peptide sequence of the epitope, select its source antigen from the molecule finder, and select its source organism from the organism finder. You can change the organism strain if needed. You cannot edit across species.

First enter the peptide sequence and then click on the Molecule Finder button. The finder will search the previously used sources for ones that contain the peptide sequence that you entered and will only return hits that match and/or IEDB created source IDs (SRCs). When you see a molecule that contains the epitope sequence and its name matches the text of the reference, you then select it. The source organism will be auto-populated with the source organism of the molecule. You may need to alter the strain information to match your reference. Do not change the organism information across species.

The molecule finder will calculate the positions of the peptide within the source you selected and will auto-fill the starting and ending positions within the editor. These positions may or may not match the Reference Positions that you enter outside of the editor.

Objedisct.jpg

In order to enter discontinuous/conformational peptide epitopes, you follow the same steps, however, you enter Swiss-Prot/GenBank numbering along with the residues utilizing standard amino acid notation in the Discontinuous Residues field within the object editor.

Important Note: It is important to always check the source cited for discontinuous epitopes to determine if the residues you are entering do indeed occur and at what positions they occur in the source.


ObjeddisP.jpg

Non-sequence epitopes and their sources are selected from the Molecule Finder. If the non-sequence molecule that is needed is not already present within the Molecule Finder, see a senior curator to add this molecule along with its SMILES structure.

How to add an accession molecule

If no accession molecules match your sequence in the Molecule Finder, go to NCBI’s BLAST page and enter your sequence there.

Add.jpg


What if there are no BLAST matches?

When there are no BLAST matches for a peptide sequence stated to be natural:

If cited references discuss the peptide’s source, you must look them up. Many typos can be caught that way. Attach refs.
If author contact is needed, you must do this first. Attach author communication
As always, curate with a filler value as if you had the correct value in place.
See the section on IEDB IDs for complete details #When and How to use an IEDB Source ID (SRC)

Epitope Fields

After completing the epitope object, additional information regarding the epitope is captured.

Epitope Evidence Code

This drop down menu is used to describe how certain the curator is of the source to which he/she assigned the epitope. In some cases authors will provide exactly the accession ID for the epitope's source, while in other cases, the exact source organism may be vague.


  • Author provided Identifier-Select this when the authors provide a Uniprot/Genbank ID & the epitope sequence is found within that source.


  • Exact match to reference information-Use this selection when the source name and organism name exactly match the text and there is no doubt regarding the accession being the one intended by the authors.


  • Representative selection (based on incomplete info)-Use this selection when there are several potential matches to the author provided source name and/or organism name and you must select among them.


  • Imperfect match-Use this evidence code when the epitope sequence is found only within a source and/or organism strain that differs from exactly the source that the manuscript refers to. Do not assign sources across organism species. Because internal identifiers can be created, this is only to be used when the SRC would only be used for this one case, and therefore will not be generated. An example is when a very specific lab strain was used.


  • Internal Identifier-no external match available-This choice is to be made every time an internal IEDB source (SRC source) is selected.


Epitope Structure Defines


This is a drop down menu used to describe to what level the epitope structure was defined. Define the epitope as an exact epitope if the authors state that it is defined, optimal, or minimal. If the authors do not specify whether the epitope is minimal or not, then use the guidelines below.

The choices are:

  • exact epitope-For linear Antibody/B Cell and Class I epitopes, if the sequence is 11 residues or less in length (with a minimum of 7 aa for class I epitopes), the epitope should be designated as an Exact Epitope. For linear Class II epitopes, select Exact Epitope if the sequence is 15 residues or less in length.
  • epitope containing region/antigenic site-For linear Antibody/B Cell and Class I epitopes, if the sequence is 12 residues or greater, Epitope Containing Region/Antigenic Site should be selected. For linear Class II epitopes, select Epitope Containing Region/Antigenic Site if the epitope sequence is 16 residues or greater in length.
  • partial epitope-For nonlinear/discontinuous epitopes and the curation of key residues, select Partial, Epitope Containing Region/Antigenic Site or Exact Epitope as applicable. Haptens are curated as partial epitopes because they are recognized as a modification of another structure.
  • deduced epitope-Use this selection to describe epitope structures that were never actually tested in experimental assays, but were instead deduced via overlapping peptide scans, deletions, or mutations, etc. If an epitope is deduced and then tested in an assay, do not select this choice.See #Deduced Epitopes for more information.

Important Note: The size criteria used to determine epitopic region designations apply regardless of the qualitative data present for the epitope. For example, if only negative data is present in the reference for a particular structure, the designation as Exact Epitope will still be used if the size criteria are met.

Important Note: Partial epitopes are not captured when they are involved in the structural features of the recognition. That is, if a mutation results in a significant structural change, this mutation cannot be assumed to be involved in recognition by the antibody or TCR and is not captured as an epitope.


Related Object Type This is a drop down menu used to relate that the epitope has a relevant relationship with another molecule. The two choices are analog and mimotope. Select the appropriate choice and enter as much information as you can regarding the object. One may enter an organism name, an accession molecule, and/or a peptide sequence.

The epitope is an analog of:-See the guidelines on #Analogs before curating an epitope which is an analog. In most cases, analogs are not captured as epitopes.
The epitope is a mimotope of:-Mimotopes are functional mimics of natural molecular structures which bear little or no sequence homology to their biological counterparts. Mimotopes should be captured as separate epitope records. See #Mimotopes for more information.


Epitope Comments As with all comments fields, these fields should only be used when necessary. The comments should be a complete “stand alone” concept written in proper American English and must provide information of value that would otherwise be lost given the current IEDB fields. Any information derived from an assay belongs in the Comments on Assay field rather than the Epitope Comments field. Standardized comments may be utilized to provide examples of the type of information this field is meant to convey.

Important Note: If the epitope structure was never tested in any assays, there must be an epitope comment that states "The epitope was deduced."

Amino Acid Configuration

The amino acids recorded in Linear Sequence and Conformation Sequence fields are assumed to be in the L-amino acid configuration by default. If a D-amino acid configuration is reported, record the corresponding D-amino acids in "lower" case (SIINFeKL instead of SIINFEKL for a D-glutamate residue) and mention in the Epitope Comments field that the amino acids in the lower case are in the D-amino acid configuration.

X in sequence

Whenever the modification actually means that there is no longer an amino acid present, we will use an X in the sequence at that position and identify what is at that position with the modification field. This includes the I/L uncertainty in MS sequencing and the introduction of a non-amino acid.

Add MHC

Experimental data characterizing the interaction between an epitope and an MHC molecule is entered under the tab labeled MHC binding.

Important Note: All experimental MHC binding data given in a reference will be entered in the database irrespective of whether the qualitative assessment is positive or negative. This may not be bulk curated under any circumstances. TCR antagonism used in an MHC binding inhibition assay will be captured as an MHC binding context.

Important Note: In the curation of MHC binding assays, the exact peptide sequence that is tested MUST be entered as the epitope. There are no carriers possible in these assay types.

MHC Fields

The fields utilized to capture MHC binding contexts are as follows:

Location of Data This field is used to identify where the MHC binding data appears. All tables, figures, or text where the actual binding data is provided should be entered. If bulking different figures or tables, enter all of them here.

Assay Information

Assay

The Assay Finder is used to select the Assay Type, Assay Type Group, and Assay Type Units. The units available are EC50 nM, IC50 nM, t1/2 (min), Other (see comments)/None, KD nM, Koff (s^-1), Tm (degree C), and Angstroms. Be sure to utilize the correct units for the assay you are entering.

Qualitative Measurement The Qualitative Measurement field is a required field . Select the appropriate response based upon the figures and text provided in the manuscript. All assay contexts in the database must be labeled as either positive or negative. At times, this can be controversial. The overriding rule is to follow what the author states, however, data may be present without comment from the authors regarding the result. For truly confusing data, the author may be contacted for clarification. For data that can be reasonably considered negative or positive based upon other information provided in the manuscript, the curator may make a judgment. In most cases, the presence of a precise numerical value for MHC binding contexts is curated as a positive outcome.

It consist of a drop down menu of:

  • Positive
  • Positive-Low
  • Positive-Intermediate
  • Positive-High
  • Negative


Rule 1: When authors specify a qualitative assessment in the reference as either positive or negative, their assessment will be recorded in the database.

Rule 2: When a qualitative assessment can be inferred from the information in the manuscript (threshold is provided), this assessment will be entered into the database.

Rule 3: When no qualitative assessment is provided by the authors, this data will not be entered into the database.


Measurement Inequality These selections may be used in order to add additional information regarding the quantitative value.

Quantitative Measurement Whenever quantitative data are available, they must be captured. Always capture quantitative values, when provided, for MHC binding assays. Be sure to select the appropriate units and check your math if conversions are required.

Always use author statements to determine which values are considered positive or negative. Different assays and different authors use different criteria for positive or negative binding values.

  • MHC Binding data for select authors:Alex, John Sidney, Buus, Lund.

Class I

IC50 < = 50nM - Positive-High

51 to <= 500nM- Positive -Intermediate

501 <= 5000nM - Positive-Low

>5000nM  - Negative


Class II

IC50 < = 100nM - Positive-High

101 to <= 1000nM - Positive -Intermediate

1001 <= 10000nM - Positive-Low

>10000nM  - Negative


Important Reviewer Note: Be sure to check the quantitative values for errors in conversion.


Assay Comments This field is used to enter comments relevant to the binding assay such as comparisons made between the binding of an analog or mutant epitope to the binding of the native epitope. As with all comments fields, this field should only be used when necessary. The comments should be a complete “stand alone” concept written in proper English and must provide information of value that would otherwise be lost given the current IEDB fields.

MHC Allele Name

The Allele name is selected from the Allele Finder application. This table includes Allele Name, Class, Organism, Restriction level, Haplotype, Locus, Serotype, and Molecule. Each of the table headings can be used to order the table. The search window allows one to search by organism, class, and allele. Synonyms will also be found so if you enter an allele name into the finder and a different allele name is returned, be sure to note that they are synonyms.

Important Note: When a specified MHC Allele is not available for selection through the Allele Finder, please contact a senior curator to add an allele to the finder.

Add MHC Ligand Elution

Only experimental data in which the authors elute peptides or ligands from a cell or purified receptors such as MHC is entered here. The peptide or ligand is then detected in the eluate through sequencing (Mass Spectrometry/Edman Degradation) or by a specific T cell with a known recognition of that peptide. These methods are used in order to demonstrate that cells will naturally process an antigen and present the epitope on the cell surface or bound to a relevant receptor. This specifically excludes epitopes given directly in the assay.

The processing of artificial antigens created in order to study processing are not curated. The processing of analogs created in order to study processing of the wild type sequence is also not captured. Processing of natural antigens should always be curated.


MHC Ligand Elution Fields

The fields utilized to describe MHC Elution Assays are as follows:

Location of Data This field is used to identify where the MHC elution data appears. All tables, figures, or text where the actual data is provided should be entered. If bulking different figures or tables, enter all of them here.

Host Organism Name Use the organism finder to select both the species and the strain of the host organism. Multiple hosts with the same MHC may be bulked. If so, then a comment must be made. See the section on #Host Organism for more details.

Organism Geolocation Name Use the Geolocation Finder to specify the country where the hosts were exposed to the immunogen, as stated by the authors. If the authors do not state where hosts were at the time of exposure, do not use the author affiliations to enter this field. Instead, leave blank. If the exposure occurs in a territory of a country that is located a far distance from that country, select the closest country by distance rather than the country that territory belongs to. If the exposure occurs in a country that cannot be found, please request the country be added to the finder.

This finder utilizes the Gazetteer geographic location ontology with each term having a GAZ ID and linking out to the ontological definitions, synonyms, and hierarchical structure. Within the curation finder, synonyms are searchable.

Sex Choices are M, F, or blank. Always capture the sex if possible.

Age This is a free text field to enter the age(s) or range of the hosts. Always capture when provided.

MHC Types Present In cases where the peptide is eluted from an indistict MHC group (HLA-DR), this field may be used to further describe the MHC present (HLA-DRB1).

In vivo Process

In order to capture an in vivo process, the organism from which the APC were derived must have been exposed to a relevant immunogen in vivo prior to harvesting the APC from the animal. If the APC were instead exposed to the immunogen in vitro, do not capture an in vivo process, but do select the in vivo process type of "No immunization". Describe the in vitro exposure of the APC to the immunogen in an in vitro process. If the organism is exposed in vivo and after harvest, the APC are also exposed in vitro, curate both in vivo and in vitro processes.

1st In Vivo Process Type

Select from the drop down menu the most applicable type. See the section on #Process Types for further details.

Disease State If the host organism has/had a disease that is/was the result of exposure to the immunogen being curated, use the Disease Finder to select the appropriate disease from those listed by ICD10 or generated by the IEDB. Be sure to follow the guidelines for Disease State for the in vivo Process type which was selected #Process Types. Refer to the section on #Disease State and Stage for further details.

Disease Stage If disease state is filled, stage must also be filled and vice versa. The choices are:

  • Acute
  • Chronic
  • Other
  • Post
  • Unknown

Refer to the section on #Disease State and Stage for further details.

Immunogen Reference Name This field is used to describe situations where the reference provides additional information regarding the immunogen such as life stage, fixation state, etc, than the object name as it appears in the object fields.

Immunogen The immunogen is entered via the object editor. First the relationship of the immunogen to the epitope must be selected. This drop down list is generated based upon the object type of the epitope to which the assay is associated. For example, if the epitope is of the object type Sequence molecule, no natural source, then the Immunogen Epitope Relation drop down list will not contain "Source Organism" as a choice because the epitope is not natural and therefore, it does not have a source organism. See the sections on #Object Types and Subtypes and #Epitope Relations for full explanations.

Important Note: The immunogen is never the epitope. When curating MHC Ligand Elution contexts in which the organism is exposed to or the antigen presenting cells are incubated with the source organism of the epitope, the protein from which the epitope was derived, or a fragment of the source antigen, the immunogen should not be <epitope>, but rather the larger antigen from which the cells derived the epitope should be selected (source protein or source organism that was processed).

When the origin of the eluted peptide is not specifically known, the following guidelines apply:
-epitope of viral origin is eluted, the antigen epitope relation is <source organism>
-epitope of known self origin is eluted, the antigen epitope relation is <source antigen>
-epitope of unknown origin is eluted, the antigen epitope relation is <other>


Immunogen Evidence Code

This drop down menu is used to describe how certain the curator is of the object he/she assigned as the immunogen. In some cases authors will provide exactly the accession ID for the immunogen, while in other cases, the exact immunogen may be vague.


  • Author provided Identifier-Select this when the authors provide a Uniprot/Genbank ID or NCBI Taaxonomy ID for the immunogen.


  • Exact match to reference information-Use this selection when the source name and organism name exactly match the text and there is no doubt regarding the immunogen object being the one intended by the authors.


  • Representative selection (based on incomplete info)-Use this selection when there are several potential matches to the author provided source name and/or organism name and you must select among them.


  • Imperfect match-Use this evidence code when the closest match to the immunogen name in the reference is a source and/or organism strain that differs from exactly what the manuscript refers to. Do not assign sources across organism species. Because internal identifiers can be created, this is only to be used when the SRC would only be used for this one case, and therefore will not be generated. An example is when a very specific lab strain was used.


  • Internal Identifier-no external match available-This choice is to be made every time an internal IEDB source (SRC source) is selected.


Immunogen Containing Object

The object editor is used to create the object that acts as a carrier, a delivery mechanism, or an expression mechanism for the immunogen. There is no need to repeat information regarding the immunogen unless it helps explain the carrier construct. For example, when the immunogen appears in the middle of a longer peptide construct, you may want to enter the entire peptide sequence.

The possible object types for container objects are as follows:

  • Recombinant Organism-Enter both the organism which is recombinant and the insert that has been engineered into it.
  • Plasmid-Only enter information regarding the insert that is expressed by the plasmid.
  • Infected Cell-Select the cell type which is infected.
  • Transfected Cell-Select the cell type and describe what is transfected into it.
  • Pulsed Cell-Select the cell type and describe what is pulsed onto it.
  • Display Library-Select from the subtypes of Phage display, Yeast display, Bacterial display, and Ribosomal display.
  • Complexed Molecules-Select from a list of subtypes which allows the entry of peptide sequences, proteins, and all nonsequence molecule types. Peptide conjugate, Protein conjugate, and Multi-Antigenic Peptide (MAP) are commonly used subtypes.


Adjuvants Select from an alphabetical drop down list that contains commonly used adjuvants. For rare adjuvants, select "Other" and use the Immunization Comments field to explain what was used.


Route Select from an alphabetical list of commonly used routes. For rare routes, select "Other" and use the Immunization Comments field to explain how the immunization was performed.


Dose Schedule This field should be filled with a very short description of dosage and number of doses. See standardized text for appropriate text, but be sure to remove the brackets that are contained in the standardized text.

In vitro Process

Use these fields to describe exposure of APC to an immunogen in vitro.

In Vitro Administration Process Type

Currently the two choices are:

  • Primary induction in vitro -select this choice when naive APC are exposed in vitro
  • Restimulation in vitro -select this choice when the organism which was the source of the APC was previously exposed to an immunogen in vivo prior to the harvest of the APC and the APC were also exposed to an immunogen in vitro.


Responder Cell Type and Stimulator Cell Type

Do not use these two fields. They will be removed shortly. Describe the APC used in the assay in the APC fields below.


Immunogen Reference Name This field is used to describe situations where the reference provides additional information regarding the immunogen such as life stage, fixation state, etc, than the object name as it appears in the object fields.

Immunogen The immunogen is entered via the object editor. First the relationship of the immunogen to the epitope must be selected. This drop down list is generated based upon the object type of the epitope to which the assay is associated. For example, if the epitope is of the object type Sequence molecule, no natural source, then the Immunogen Epitope Relation drop down list will not contain "Source Organism" as a choice because the epitope is not natural and therefore, it does not have a source organism. See the sections on #Object Types and Subtypes and #Epitope Relations for full explanations.

Important Note: The immunogen is never the epitope. When curating MHC Ligand Elution contexts in which the organism is exposed to or the antigen presenting cells are incubated with the source organism of the epitope, the protein from which the epitope was derived, or a fragment of the source antigen, the immunogen should not be <epitope>, but rather the larger antigen from which the cells derived the epitope should be selected (source antigen or source organism that was processed).

When the origin of the eluted peptide is not specifically known, the following guidelines apply:
-epitope of viral origin is eluted, the antigen epitope relation is <source organism>
-epitope of known self origin is eluted, the antigen epitope relation is <source antigen>
-epitope of unknown origin is eluted, the antigen epitope relation is <other>


Immunogen Evidence Code

This drop down menu is used to describe how certain the curator is of the object he/she assigned as the immunogen. In some cases authors will provide exactly the accession ID for the immunogen, while in other cases, the exact immunogen may be vague.


  • Author provided Identifier-Select this when the authors provide a Uniprot/Genbank ID or NCBI Taaxonomy ID for the immunogen.


  • Exact match to reference information-Use this selection when the source name and organism name exactly match the text and there is no doubt regarding the immunogen object being the one intended by the authors.


  • Representative selection (based on incomplete info)-Use this selection when there are several potential matches to the author provided source name and/or organism name and you must select among them.


  • Imperfect match-Use this evidence code when the closest match to the immunogen name in the reference is a source and/or organism strain that differs from exactly what the manuscript refers to. Do not assign sources across organism species. Because internal identifiers can be created, this is only to be used when the SRC would only be used for this one case, and therefore will not be generated. An example is when a very specific lab strain was used.


  • Internal Identifier-no external match available-This choice is to be made every time an internal IEDB source (SRC source) is selected.


Immunogen Containing Object

The object editor is used to create the object that acts as a carrier, a delivery mechanism, or an expression mechanism for the immunogen. There is no need to repeat information regarding the immunogen unless it helps explain the carrier construct. For example, when the immunogen appears in the middle of a longer peptide construct, you may want to enter the entire peptide sequence.

The possible object types for container objects are as follows:

  • Recombinant Organism-Enter both the organism which is recombinant and the insert that has been engineered into it.
  • Plasmid-Only enter information regarding the insert that is expressed by the plasmid.
  • Infected Cell-Select the cell type which is infected.
  • Transfected Cell-Select the cell type and describe what is transfected into it.
  • Pulsed Cell-Select the cell type and describe what is pulsed onto it.
  • Display Library-Select from the subtypes of Phage display, Yeast display, Bacterial display, and Ribosomal display.
  • Complexed Molecules-Select from a list of subtypes which allows the entry of peptide sequences, proteins, and all nonsequence molecule types. Peptide conjugate, Protein conjugate, and Multi-Antigenic Peptide (MAP) are commonly used subtypes.


Immunization Comments Use this field to explain the host or host population and to explain to immunization procedures. If populations, routes, adjuvants, or disease stages were bulked, a comment is mandatory.

Assay Information

Assay


The Assay Finder is used to select the Assay Type, Assay Type Group, and Assay Type Units.

Qualitative Measurement The Qualitative Measurement field is a required field . Select the appropriate response based upon the figures and text provided in the manuscript. All assay contexts in the database must be labeled as either positive or negative. MHC Ligand elution assays are generally only curated when positive.

Measurement Inequality These selections may be used in order to add additional information regarding the quantitative value. This field does not apply for Ligand Elution assays.

Quantitative Measurement This field also does not apply as these assays do not have units.


Antigen Presenting Cells

The APC that processed the immunogen and from which the MHC was derived are described here.

Cell Tissue Type Select from the drop down menu of tissue types. Use "Other" if the appropriate tissue is not present, add a comment describing the tissue, and consult a senior curator about potentially adding to the list.

Cell Type Select from the drop down menu of cell types. Use "Other" if the appropriate cell type is not present, add a comment describing the tissue, and consult a senior curator about potentially adding to the list.

Cell Culture Conditions This field is used to describe the conditions of the APC at the time of assay. The potential choices are:

  • Direct Ex Vivo

This is used to describe cells that were not exposed to any antigen in vitro prior to assay.

  • Short Term Restimulated

This is used for cells stimulated with an antigen one or more times in vitro which is then washed out prior to the addition of the assay antigen. If cells are restimulated many rounds in vitro, select Cell Line/Clone instead.

  • Cell Line / Clone

Use this selection for cell lines or clones that do not fall into the two following categories (B-LCL and hybridoma)

  • Cell Line / Clone (EBV tranformed, B-LCL)

This is used to describe EBV transformed cell lines

  • Cell Line / Clone (Hybridoma)

This is used to describe cell lines that are fused to create hybridomas

  • Blast Activated

This is used to describe cells which are blast activated. Common examples are PHA and LPS stimulated cells.

  • In Vivo

This selection is only used when the assay is performed in vivo

  • Other / Unknown

Only chose this selection when the conditions of the cells is not described. In the event, the above categories do not apply, see a senior curator.


MHC Allele

MHC Allele Name The Allele name is selected from the Allele Finder application. This table includes Allele Name, Class, Organism, Restriction level, Haplotype, Locus, Serotype, and Molecule. Each of the table headings can be used to order the table. The search window allows one to search by organism, class, and allele. Synonyms will also be found so if you enter an allele name into the finder and a different allele name is returned, be sure to note that they are synonyms.

Important Note:When a specified MHC Allele is not available for selection through the Allele Finder, please contact a senior curator to add an allele to the finder.

MHC Evidence Code Currently the evidence codes present in MHC Ligand Elution assays are identical to those of T cell assays. These will be replaced with MHC ligand elution appropriate evidence codes shortly.


Antigen

Antigen Reference Name This field is used to describe situations where the reference provides additional information regarding the antigen.

Antigen The antigen is entered via the object editor. First the relationship of the immunogen to the epitope must be selected. The antigen in these assay is always the epitope. "Epitope" is the only available choice.


Assay Comments This field is used to enter comments relevant to the elution assay such as conditions that altered processing of the epitope. As with all comments fields, this field should only be used when necessary. The comments should be a complete “stand alone” concept written in proper English and must provide information of value that would otherwise be lost given the current IEDB fields.

Add T Cell

T Cell Assay Fields

The fields utilized to describe T Cell Assays are as follows:

Location of Data This field is used to identify where the T cell data being described by this context appears in the manuscript. All tables, figures, or text where the actual data is provided should be entered. If bulking different figures or tables, enter all of them here.

Host Organism Name Use the organism finder to select both the species and the strain of the host organism. Multiple hosts with the same MHC may be bulked. If so, then a comment must be made. See the section on #Host Organism for more details.

Sex Choices are M, F, or blank. Always capture the sex if possible.

Age This is a free text field to enter the age(s) or range of the hosts. Always capture when provided.

MHC Types Present In cases where the peptide is recognized in the context of an indistict MHC group (HLA-DR), this field may be used to further describe the MHC present (HLA-DRB1). Additionally, when restriction is not known, the MHC Types present field should be used to describe the alleles present among the responding hosts.

In vivo Process

In order to capture an in vivo process, the organism from which the effector cells were derived must have been exposed to a relevant immunogen in vivo prior to harvesting the effector cells from the animal. If the effectors were instead exposed to the immunogen in vitro, do not capture an in vivo process, but do select the in vivo process type of "No immunization". If the organism is exposed in vivo and after harvest, the effectors are also exposed in vitro, curate both in vivo and in vitro processes.

1st In Vivo Process Type

Select from the drop down menu the most applicable type. See the section on #Process Types for further details.

Disease State If the host organism has/had a disease that is/was the result of exposure to the immunogen being curated, use the Disease Finder to select the appropriate disease from those listed by ICD10 or generated by the IEDB. Be sure to follow the guidelines for Disease State for the in vivo Process type which was selected #Process Types. Refer to the section on #Disease State and Stage for further details.

Disease Stage If disease state is filled, stage must also be filled and vice versa. The choices are:

  • Acute
  • Chronic
  • Other
  • Post
  • Unknown

Refer to the section on #Disease State and Stage for further details.

Immunogen Reference Name This field is used to describe situations where the reference provides additional information regarding the immunogen such as life stage, fixation state, etc, than the object name as it appears in the object fields.

Immunogen The immunogen is entered via the object editor. First the relationship of the immunogen to the epitope must be selected. This drop down list is generated based upon the object type of the epitope to which the assay is associated. For example, if the epitope is of the object type Sequence molecule, no natural source, then the Immunogen Epitope Relation drop down list will not contain "Source Organism" as a choice because the epitope is not natural and therefore, it does not have a source organism. See the sections on #Object Types and Subtypes and #Epitope Relations for full explanations.

Immunogen Evidence Code

This drop down menu is used to describe how certain the curator is of the object he/she assigned as the immunogen. In some cases authors will provide exactly the accession ID for the immunogen, while in other cases, the exact immunogen may be vague.


  • Author provided Identifier-Select this when the authors provide a Uniprot/Genbank ID or NCBI Taaxonomy ID for the immunogen.


  • Exact match to reference information-Use this selection when the source name and organism name exactly match the text and there is no doubt regarding the immunogen object being the one intended by the authors.


  • Representative selection (based on incomplete info)-Use this selection when there are several potential matches to the author provided source name and/or organism name and you must select among them.


  • Imperfect match-Use this evidence code when the closest match to the immunogen name in the reference is a source and/or organism strain that differs from exactly what the manuscript refers to. Do not assign sources across organism species. Because internal identifiers can be created, this is only to be used when the SRC would only be used for this one case, and therefore will not be generated. An example is when a very specific lab strain was used.


  • Internal Identifier-no external match available-This choice is to be made every time an internal IEDB source (SRC source) is selected.


Immunogen Containing Object

The object editor is used to create the object that acts as a carrier, a delivery mechanism, or an expression mechanism for the immunogen. There is no need to repeat information regarding the immunogen unless it helps explain the carrier construct. For example, when the immunogen appears in the middle of a longer peptide construct, you may want to enter the entire peptide sequence.

The possible object types for container objects are as follows:

  • Recombinant Organism-Enter both the organism which is recombinant and the insert that has been engineered into it.
  • Plasmid-Only enter information regarding the insert that is expressed by the plasmid.
  • Infected Cell-Select the cell type which is infected.
  • Transfected Cell-Select the cell type and describe what is transfected into it.
  • Pulsed Cell-Select the cell type and describe what is pulsed onto it.
  • Display Library-Select from the subtypes of Phage display, Yeast display, Bacterial display, and Ribosomal display.
  • Complexed Molecules-Select from a list of subtypes which allows the entry of peptide sequences, proteins, and all nonsequence molecule types. Peptide conjugate, Protein conjugate, and Multi-Antigenic Peptide (MAP) are commonly used subtypes.


Adjuvants Select from an alphabetical drop down list that contains commonly used adjuvants. For rare adjuvants, select "Other" and use the Immunization Comments field to explain what was used.


Route Select from an alphabetical list of commonly used routes. For rare routes, select "Other" and use the Immunization Comments field to explain how the immunization was performed.


Dose Schedule This field should be filled with a very short description of dosage and number of doses. See standardized text for appropriate text, but be sure to remove the brackets that are contained in the standardized text.


2nd In Vivo Process Type

In cases where the organism was immunized with a second, different, but relevant immunogen in vivo prior to assay, utilize the second in vivo process fields. These fields are identical to the fields for 1st in vivo process. See the section on #Process Types for further details.


In vitro Process

Use these fields to describe exposure of effector cells to an immunogen in vitro.

In Vitro Administration Process Type

The choices are:

  • Primary induction in vitro -select this choice when naive effector cells are exposed in vitro
  • Restimulation in vitro -select this choice when the organism which was the source of the effector cells was previously exposed to an immunogen in vivo prior to the harvest of the effectors and the effectors were also exposed to an immunogen in vitro.


Responder Cell Type This field describes the cell population which is exposed in vitro prior to the assay and from which the effector cells will eventually be generated. For example, if PBMC are incubated with an immunogen and then CD8+ cells are subsequently isolated for an assay, this field should reflect "PBMC".

Stimulator Cell Type This field describes the cells which present the in vito immunogen to the responder population prior to assay. These are APC.


Immunogen Reference Name This field is used to describe situations where the reference provides additional information regarding the immunogen such as life stage, fixation state, etc, than the object name as it appears in the object fields.

Immunogen The immunogen is entered via the object editor. First the relationship of the immunogen to the epitope must be selected. This drop down list is generated based upon the object type of the epitope to which the assay is associated. For example, if the epitope is of the object type Sequence molecule, no natural source, then the Immunogen Epitope Relation drop down list will not contain "Source Organism" as a choice because the epitope is not natural and therefore, it does not have a source organism. See the sections on #Object Types and Subtypes and #Epitope Relations for full explanations.


Immunogen Evidence Code

This drop down menu is used to describe how certain the curator is of the object he/she assigned as the immunogen. In some cases authors will provide exactly the accession ID for the immunogen, while in other cases, the exact immunogen may be vague.


  • Author provided Identifier-Select this when the authors provide a Uniprot/Genbank ID or NCBI Taaxonomy ID for the immunogen.


  • Exact match to reference information-Use this selection when the source name and organism name exactly match the text and there is no doubt regarding the immunogen object being the one intended by the authors.


  • Representative selection (based on incomplete info)-Use this selection when there are several potential matches to the author provided source name and/or organism name and you must select among them.


  • Imperfect match-Use this evidence code when the closest match to the immunogen name in the reference is a source and/or organism strain that differs from exactly what the manuscript refers to. Do not assign sources across organism species. Because internal identifiers can be created, this is only to be used when the SRC would only be used for this one case, and therefore will not be generated. An example is when a very specific lab strain was used.


  • Internal Identifier-no external match available-This choice is to be made every time an internal IEDB source (SRC source) is selected.


Immunogen Containing Object

The object editor is used to create the object that acts as a carrier, a delivery mechanism, or an expression mechanism for the immunogen. There is no need to repeat information regarding the immunogen unless it helps explain the carrier construct. For example, when the immunogen appears in the middle of a longer peptide construct, you may want to enter the entire peptide sequence.

The possible object types for container objects are as follows:

  • Recombinant Organism-Enter both the organism which is recombinant and the insert that has been engineered into it.
  • Plasmid-Only enter information regarding the insert that is expressed by the plasmid.
  • Infected Cell-Select the cell type which is infected.
  • Transfected Cell-Select the cell type and describe what is transfected into it.
  • Pulsed Cell-Select the cell type and describe what is pulsed onto it.
  • Display Library-Select from the subtypes of Phage display, Yeast display, Bacterial display, and Ribosomal display.
  • Complexed Molecules-Select from a list of subtypes which allows the entry of peptide sequences, proteins, and all nonsequence molecule types. Peptide conjugate, Protein conjugate, and Multi-Antigenic Peptide (MAP) are commonly used subtypes.


Immunization Comments Use this field to explain the host or host population and to explain to immunization procedures. If populations, routes, adjuvants, or disease stages were bulked, a comment is mandatory.

Adoptive Transfer

These fields describe procedures where immune reactivity (T cells) is transferred from one animal into another. In cases where adoptive transfer was performed in a curatable T cell assay, utilize these fields to capture the details of what material was transferred and any procedures performed on the recipient animal.

  • In order to be curated, this assay must be epitope relevant, that is, the transferred material must be epitope specific.
  • For details on these fields, see the section #Guidelines on Adoptive Transfer.

Recipient Organism

Organism Name

Sex

Age

MHC Types Present


Transferred Effector Material

In order to be curated, the transferred material must contain T cells. If only tissue is transferred, select the tissue type and leave the other effector fields blank. Utilizes the same fields usually used to describe effector cells:

Transferred Effector Cell Tissue Type

Transferred Effector Cell Type

Transferred Effector Cell Culture Conditions

Transferred TCR Name

TCR Chain Types

TCR Molecule (Object Editor)


In vivo Process in Recipient

These fields describe any procedures performed upon the recipient animal after the immune material was transferred. Utilize the exact same fields as in vivo process 1 or 2

Disease State & Stage

Immunogen Ref name

Immunogen Epitope Relation

Immunogen

Immunogen Evidence

Immunogen Containing Object

Adjuvants

Route

Dose schedule


Adoptive Transfer Comments

Explain the adoptive transfer procedure in complete concise sentences.

Assay Information

Assay

The Assay Finder is used to select the Assay Type, Assay Type Group, and Assay Type Units. Be sure to utilize the correct units for the assay you are entering.

Qualitative Measurement The Qualitative Measurement field is a required field . Select the appropriate response based upon the figures and text provided in the manuscript. All assay contexts in the database must be labeled as either positive or negative. At times, this can be controversial. The overriding rule is to follow what the author states, however, data may be present without comment from the authors regarding the result. For truly confusing data, the author may be contacted for clarification. For data that can be reasonably considered negative or positive based upon other information provided in the manuscript, the curator may make a judgment.

It consist of a drop down menu of:

  • Positive
  • Positive-Low
  • Positive-Intermediate
  • Positive-High
  • Negative


Measurement Inequality These selections may be used in order to add additional information regarding the quantitative value.

Quantitative Measurement T cell assays do not usually have quantitative values. Quantitative values in FACs assays may be misleading due to gating of specific populations. Be sure to select the appropriate units and check your math if conversions are required.

Number of Subjects Tested Use this field to enter the number of subjects that were tested or the number of distinct subjects that effector cells were assayed from.

Number of Subjects Responded Of the number tested, enter the number that responded by recognition of the assay antigen.

Response Frequency (%) This field will be calculated for you if you enter the above two values. If the percentage of subjects that responded is known, but the number tested and number responded is not known, enter the response frequency here.

Effector Cells

Effector cell assignment is made following these guidelines:

  • Phenotype identification- Direct demonstration of the effector cell phenotype will be used to assign effector cell type. For example, CD8+ staining of the population producing IFNg.
  • Cell Isolation –Isolation or purification procedures will be used to identify the cell type of the effector cells present in the assay. For example, the use of a cell population after CD8 depletion would be identified as CD4+ T cells.
  • Biological process measured-The response measured by the assay type will NOT be used to identify the cell type of the effector cells. For example, measurement of proliferation may be an indicator of CD4+ T cells, however, if splenocytes were used in the assay, the effector cell type should be entered as splenocytes.
  • MHC Restriction –MHC restriction of the epitope will NOT be used to assign the cell type of the effector cells used in the assay. That is, if PBMC are used in an assay utilizing a Class II epitope, PBMC will be entered into the effector cell field. However, assignment of specific MHC restriction or restriction to the level of Class I or Class II should be performed whenever possible and may be attributed to the assay type when the authors state or imply such.


The effector cells that recognized the assay antigen are described via the following fields:

Effector Cell Tissue Type Select from the drop down menu of tissue types. Use "Other" if the appropriate tissue is not present, add a comment describing the tissue, and consult a senior curator about potentially adding to the list.

Effector Cell Type Select from the drop down menu of cell types. Use "Other" if the appropriate cell type is not present, add a comment describing the tissue, and consult a senior curator about potentially adding to the list. When different effector cells are used in the same assay type, they may be bulked. For multiple effector cell types, different T cell lines and clones may be bulked as well as PBMC and select T cell populations (CD4+, for example). When bulking effector cell types, be sure to capture the most relevant data and never bulk different immunogens or different immunized host species.

Effector Cell Culture Conditions This field is used to describe the conditions of the effector cells at the time of assay. The potential choices are:

  • Direct Ex Vivo

This is used to describe cells that were not exposed to any antigen in vitro prior to assay.

  • Short Term Restimulated

This is used for cells stimulated with an antigen one or more times in vitro which is then washed out prior to the addition of the assay antigen. If cells are restimulated many rounds in vitro, select Cell Line/Clone instead.

  • Cell Line / Clone

Use this selection for cell lines or clones that do not fall into the two following categories (B-LCL and hybridoma)

  • Cell Line / Clone (EBV tranformed, B-LCL)

This is used to describe EBV transformed cell lines

  • Cell Line / Clone (Hybridoma)

This is used to describe cell lines that are fused to create hybridomas

  • Blast Activated

This is used to describe cells which are blast activated. Common examples are PHA and LPS stimulated cells.

  • In Vivo

This selection is only used when the assay is performed in vivo

  • Other / Unknown

Only chose this selection when the conditions of the cells is not described. In the event, the above categories do not apply, see a senior curator.


Assayed TCR Molecule

The TCR of the effector cells may be described in detail. This is only done when the TCR is chimeric, the author has provided an external identifier (Uni-prot or GenBank ID), or there is additional pertinent information to capture.

Assayed TCR Molecule Name This is a free text field to capture the name of the TCR. The name of T cell clones, when specified by the authors, is entered only when there is a clear reason to do so. For example, the authors describe recognition or lack thereof by certain clones and specifically discuss the unusual reactivity of the clones. Differing fine specificity of clones does not warrant entering the clone names. All clones generated through immunization with the same immunogen may be bulked, regardless of the restimulating antigen, carriers, or adjuvants.

Assayed TCR Molecule Chain 1 Type This is a drop down menu of chain types.

Assayed TCR Molecule Chain 2 Type This is a drop down menu of chain types.

Assayed TCR Molecule The TCR molecule is entered via the object editor application. Complete the relevant information for a multi-chain molecule in order to describe the TCR molecule studied in the assay.

Antigen Presenting Cells

When different APC are used in the same assay type, they may be bulked. Curate the APC giving the best response or the APC type that was used most often in the reference. The APC that presented the antigen to the effector cells are described in the same manner as the effector cells above utilizing the following fields:

Cell Tissue Type

Cell Type

Cell Culture Conditions


Autologous or Syngeneic

The relationship of the source organism of the APC to the source organism of the epitope is described in this field. The choices are:

  • Yes
  • No
  • None

The default value is set on None so be sure to select Yes or No when entering APC. Autologous is when the APC are derived from the same organism from whom the effector cells were derived. Syngeneic means genetically identical members of the same species.

When the APC are not autologous or syngeneic, further describe their source with the following fields:

Cell Source Organism

Sex

Age


MHC Allele

MHC Allele Name

When restriction of the epitope is known or demonstrated, always capture it here.

  • Different APC with the same MHC should be bulked.
  • Assays performed to demonstrate restriction should be bulked.
  • Positive responses with APC of different MHC should never be bulked. All assays for an epitope with more than one restriction should be duplicated for each positive allele.
  • If restriction is demonstrated only to a class level(Class I or Class II), be sure to capture that here. In such a case, you may also use MHC Types present to further describe the alleles present.

The Allele name is selected from the Allele Finder application. This table includes Allele Name, Class, Organism, Restriction level, Haplotype, Locus, Serotype, and Molecule. Each of the table headings can be used to order the table. The search window allows one to search by organism, class, and allele. Synonyms will also be found so if you enter an allele name into the finder and a different allele name is returned, be sure to note that they are synonyms.

Important Note: When a specified MHC Allele is not available for selection through the Allele Finder, please contact a senior curator to add an allele to the finder.


MHC Evidence Code

Further explain how the restriction was demonstrated via the evidence codes. Only one evidence code may be selected at a time, therefore the code referring to the assay type which most narrowly defined the restriction of the epitope should be used. Select the highest one available in each reference and apply it to all assays for that epitope. Bulk restriction determining assays under other assay by the use of the evidence code. The evidence code is meant to replace the need for comments.

The choices are:

  • Cited reference

Use this one when the authors state that the restriction of the epitope was already known prior to their publication.

  • MHC binding assay

Use this one when authors perform MHC binding assays in the same reference to demonstrate restriction.

  • T cell assay -Single MHC type present

This code is to be used when the APC (or tetramer/multimer) utilized in a T cell assay expresses only a single allele and the assay is positive, thus demonstrating the restriction.

  • T cell assay -MHC subset identification

Select this code when authors isolate certain subsets of MHC bearing APC (as with an antibody) to demonstrate restriction.

  • T cell assay -Mismatched MHC molecules

This code is used when a T cell assay is performed utilizing different APC with different alleles are used to show restriction.

  • T cell assay -T cell subset identification

This code refers to the isolation of T cell subsets to show restriction. Blocking antibodies or purification of subsets (CD4/CD8) are commonly used.

  • T cell assay -Biological process measured

Use this code when the authors consider the type of response they measured to be indicative of a certain T cell subset, such as CTL assays being Class I restricted.

  • MHC binding prediction

This code is to be used when predictive analysis of the epitope-MHC interaction, based upon the sequence or structure of the epitope without experimental assay, is used to infer a restriction.

  • Statistical association

This is the lowest evidence code and refers to the association of a positive response with the presence of certain alleles within the responding population.

Antigen

Antigen Reference Name This field is used to describe situations where the reference provides additional information regarding the antigen such as life stage, fixation state, etc, other than the object name as it appears in the object fields.

Antigen The antigen is entered via the object editor. First the relationship of the antigen to the epitope must be selected. This drop down list is generated based upon the object type of the epitope to which the assay is associated. For example, if the epitope is of the object type Sequence molecule, no natural source, then the Antigen Epitope Relation drop down list will not contain "Source Organism" as a choice because the epitope is not natural and therefore, it does not have a source organism. See the sections on #Object Types and Subtypes and #Epitope Relations for full explanations.


Antigen Evidence Code

This drop down menu is used to describe how certain the curator is of the object he/she assigned as the antigen. In some cases authors will provide exactly the accession ID for the antigen, while in other cases, the exact antigen may be vague.


  • Author provided Identifier-Select this when the authors provide a Uniprot/Genbank ID or NCBI Taaxonomy ID for the antigen.


  • Exact match to reference information-Use this selection when the source name and organism name exactly match the text and there is no doubt regarding the antigen object being the one intended by the authors.


  • Representative selection (based on incomplete info)-Use this selection when there are several potential matches to the author provided source name and/or organism name and you must select among them.


  • Imperfect match-Use this evidence code when the closest match to the antigen name in the reference is a source and/or organism strain that differs from exactly what the manuscript refers to. Do not assign sources across organism species. Because internal identifiers can be created, this is only to be used when the SRC would only be used for this one case, and therefore will not be generated. An example is when a very specific lab strain was used.


  • Internal Identifier-no external match available-This choice is to be made every time an internal IEDB source (SRC source) is selected.


Antigen Containing Object

The object editor is used to create the object that acts as a carrier, a delivery mechanism, or an expression mechanism for the antigen. There is no need to repeat information regarding the antigen unless it helps explain the carrier construct. For example, when the antigen appears in the middle of a longer peptide construct, you may want to enter the entire peptide sequence.

The possible object types for container objects are as follows:

  • Recombinant Organism-Enter both the organism which is recombinant and the insert that has been engineered into it.
  • Plasmid-Only enter information regarding the insert that is expressed by the plasmid.
  • Infected Cell-Select the cell type which is infected.
  • Transfected Cell-Select the cell type and describe what is transfected into it.
  • Pulsed Cell-Select the cell type and describe what is pulsed onto it.
  • Display Library-Select from the subtypes of Phage display, Yeast display, Bacterial display, and Ribosomal display.
  • Complexed Molecules-Select from a list of subtypes which allows the entry of peptide sequences, proteins, and all nonsequence molecule types. Peptide conjugate, Protein conjugate, and Multi-Antigenic Peptide (MAP) are commonly used subtypes.

Assay Comments

This field is used to enter comments relevant to the T cell assay. If bulking different assays, there should be a comment. If neither the immunogen nor the antigen are the epitope, there should be a comment. As with all comments fields, this field should only be used when necessary. The comments should be a complete “stand alone” concept written in proper English and must provide information of value that would otherwise be lost given the current IEDB fields.

T Cell Topics

Tetramer/Multimer Assays

In MHC Tetramer/Multimer staining assays, there are no Antigen-Presenting Cells. Therefore, leave the APC fields blank. However, the MHC restriction of the tetramer must be entered in the MHC Allele field. The information describing the effector cells is entered as usual along with the details of immunization.


MHC Fields

The following guidelines below are used to complete the MHC fields present under the T Cell Response and MHC Ligand Elution assays.

  • If restriction is demonstrated for a T cell context, each restriction that is shown is curated separately as a new context with each allele listed once as the restricting allele. For example, if a CTL assay is done and restriction is known to be both HLA-A2 and HLA-A4, then two separate CTL assays will be curated, one with restriction of A2 and one with restriction of A4.
  • Promiscuous binders are captured as multiple contexts if restriction is demonstrated in a manner to warrant the use of an evidence code. If the epitope is recognized by six alleles, for example, six contexts will be curated. When potential restriction is discussed without experimental evidence, restriction is not captured. The author's comments should be captured under assay comments and the MHC Types present field may be used.
  • In cases where restriction is not fully demonstrated, but the MHC types of the responding population are known, these may be captured under MHC types present. The MHC types present field captures the common alleles shared by the responders among the group. Do not enter all MHC types tested, but rather, all MHC types shared by the population demonstrated to recognize the epitope. The purpose of this field is to add potential restriction information in cases where exact restriction is not yet known. In a population in which no allele(s) are shared by the population-leave the MHC Allele(s) Present field BLANK.
  • If a restriction is not specified but the response is shown to be either a Class I or Class II response, then enter only the Class I alleles or the Class II alleles in the MHC Types Present field under the most appropriate category (Immunized Organism or Assay: APC Organism). If additional information is known about the MHC Types present among the responding population, BOTH the MHC Allele and MHC Types Present fields may be utilized.
  • MHC restriction should be captured when the epitope has a known MHC binding motif and the antigen presenting cells express that allele.

Important Note: If a restriction is determined for a set of assay types, MHC classes, or species, do not apply that restriction to different assay types, MHC classes, or species. For example, if an epitope is determined to be H-2b restricted, do not curate an assay with a human host as having this murine restriction. If an epitope is shown to have a class II restriction, do not apply that restriction to an assay that measures a Class I type response.


Important Note: If human population data without restriction is present, always capture this data. In addition, curate any other assays that contain restriction data. This situation arises when a mixed population of unknown or unclear MHC types is first tested.

For example, 10/15 human subject's PBMC proliferate in response to the epitope. Later, the authors determine specific restriction of cell clones to be HLA-DR4, however, of the 15 people tested and the 10 who responded, it is not known how many actually express HLA-DR4. In this example, curate 2 assays:

1. One with the # tested/responded = 15/10 and no MHC restriction assigned

2. One with no # tested/responded and MHC restriction = HLA-DR4

Enhancement Assays

In enhancement assays, a T cell epitope is used to enhance either a T cell response or an antibody response. There are two assay types: T cell Assay-Enhancement of antibody response and T Cell Assay-Enhancement of T cell response. An enhancement assay is curated under the T cell epitope which is responsible for the enhancement, ONLY when the manuscript demonstrates that the T cell epitope is responsible for the response, that is, the response MUST be tested with and without the presence of the T cell epitope.

Use the following guidelines to curate these assays:

The immunogen is always the T cell epitope. Immunogen Epitope Relation =<Epitope>.

The antigen is the entity to which the response is enhanced. For example, with enhancement of the antibody response to the B epitope, the antigen is the B epitope. Antigen Epitope Relation =<Other>, <Other Structure from Source Organism>, or <Taxonomic Related>. For example, with enhancement of the antibody response to HBV core protein, the antigen is HBV core protein. With enhancement of the T cell response to the T2 epitope, T2 epitope is the antigen. Antigen Epitope Relation =<Other>.

Important Note: When a T cell epitope enhances the response to a number of B cell epitopes, that data may be bulked with the antigen entered as a representative B cell epitope and the other epitopes commented upon in assay comments. Enhancement of the response to multiple T cell epitopes may also be bulked.

Enhancement assay.jpg

Challenge Assays

In accordance with rule (#Presence of Epitope), challenge assays in which the immunization is performed with the epitope as the immunogen are the only challenge assays included in the database. The assay will be recorded as either a T cell or B cell context, depending on other information available for the epitope.

Challenge with Tumor Assays

In these assays, immunization with the epitope prevents establishment, growth, etc of tumors expressing the source antigen. Note that the tumor cells may be administered before or after the epitope. Use comments to describe order of events.

IV1 = admin in vivo to prevent/reduce

IV1 Imm = epitope

Assay Type = Challenge Assay

Antigen = Source Antigen

Carrier = transfected cells (usually, but not always)

Important Note: Must have assay comment: Administration of the epitope prevented growth of tumor cells expressing the X protein.

Competition/Inhibition Assays

When curating inhibition/competition assays, the antigen is always the competitor. However, when the entity whose binding is being inhibited is not captured in any other context, this interaction should also be curated in a separate assay as a direct binding/recognition assay when that interaction is curatable. Thus, the competitor will be the antigen in the competition assay and the inhibited antigen will be captured as the antigen in a binding assay. Note that this only applies if that binding is not captured elsewhere and is curatable.

Direct binding and competition assays with the same antigen, immunogen, and outcome should be bulk curated under the competition assay.


TCR

TCR rearrangements Vβ chain type, etc) are not captured in the current database structure. However, such data should be described in the Comments on Assay field under the appropriate T cell response.

T Cell Phenotype

T cell phenotype data (upregulation of co-stimulatory molecules like CD45) are not captured in the current database structure, but can be mentioned in the "Comments on Assay" field under the appropriate T cell response.

Tolerization Data

Data related to tolerization is not entered into the database. TCR antagonism is also not captured in the current database structure. This feature will be considered for future releases.

Add B Cell

B Cell Assay Fields

In vivo Process

In order to capture an in vivo process, the organism from which the antibody(ies) were derived must have been exposed to a relevant immunogen in vivo prior to harvesting the antibody(ies) from the animal.

1st In Vivo Process Type

Select from the drop down menu the most applicable type. See the section on #Process Types for further details.

Disease State If the host organism has/had a disease that is/was the result of exposure to the immunogen being curated, use the Disease Finder to select the appropriate disease from those listed by ICD10 or generated by the IEDB. Be sure to follow the guidelines for Disease State for the in vivo Process type which was selected #Process Types. Refer to the section on #Disease State and Stage for further details.

Disease Stage If disease state is filled, stage must also be filled and vice versa. The choices are:

  • Acute
  • Chronic
  • Other
  • Post
  • Unknown

Refer to the section on #Disease State and Stage for further details.

Immunogen Reference Name This field is used to describe situations where the reference provides additional information regarding the immunogen such as life stage, fixation state, etc, than the object name as it appears in the object fields.

Immunogen The immunogen is entered via the object editor. First the relationship of the immunogen to the epitope must be selected. This drop down list is generated based upon the object type of the epitope to which the assay is associated. For example, if the epitope is of the object type Sequence molecule, no natural source, then the Immunogen Epitope Relation drop down list will not contain "Source Organism" as a choice because the epitope is not natural and therefore, it does not have a source organism. See the sections on #Object Types and Subtypes and #Epitope Relations for full explanations.

Immunogen Evidence Code

This drop down menu is used to describe how certain the curator is of the object he/she assigned as the immunogen. In some cases authors will provide exactly the accession ID for the immunogen, while in other cases, the exact immunogen may be vague.


  • Author provided Identifier-Select this when the authors provide a Uniprot/Genbank ID or NCBI Taaxonomy ID for the immunogen.


  • Exact match to reference information-Use this selection when the source name and organism name exactly match the text and there is no doubt regarding the immunogen object being the one intended by the authors.


  • Representative selection (based on incomplete info)-Use this selection when there are several potential matches to the author provided source name and/or organism name and you must select among them.


  • Imperfect match-Use this evidence code when the closest match to the immunogen name in the reference is a source and/or organism strain that differs from exactly what the manuscript refers to. Do not assign sources across organism species. Because internal identifiers can be created, this is only to be used when the SRC would only be used for this one case, and therefore will not be generated. An example is when a very specific lab strain was used.


  • Internal Identifier-no external match available-This choice is to be made every time an internal IEDB source (SRC source) is selected.


Immunogen Containing Object

The object editor is used to create the object that acts as a carrier, a delivery mechanism, or an expression mechanism for the immunogen. There is no need to repeat information regarding the immunogen unless it helps explain the carrier construct. For example, when the immunogen appears in the middle of a longer peptide construct, you may want to enter the entire peptide sequence.

The possible object types for container objects are as follows:

  • Recombinant Organism-Enter both the organism which is recombinant and the insert that has been engineered into it.
  • Plasmid-Only enter information regarding the insert that is expressed by the plasmid.
  • Infected Cell-Select the cell type which is infected.
  • Transfected Cell-Select the cell type and describe what is transfected into it.
  • Pulsed Cell-Select the cell type and describe what is pulsed onto it.
  • Display Library-Select from the subtypes of Phage display, Yeast display, Bacterial display, and Ribosomal display.
  • Complexed Molecules-Select from a list of subtypes which allows the entry of peptide sequences, proteins, and all nonsequence molecule types. Peptide conjugate, Protein conjugate, and Multi-Antigenic Peptide (MAP) are commonly used subtypes.


Adjuvants Select from an alphabetical drop down list that contains commonly used adjuvants. For rare adjuvants, select "Other" and use the Immunization Comments field to explain what was used.


Route Select from an alphabetical list of commonly used routes. For rare routes, select "Other" and use the Immunization Comments field to explain how the immunization was performed.


Dose Schedule This field should be filled with a very short description of dosage and number of doses. See standardized text for appropriate text, but be sure to remove the brackets that are contained in the standardized text.


2nd In Vivo Process Type

In cases where the organism was immunized with a second, different, but relevant immunogen in vivo prior to assay, utilize the second in vivo process fields. These fields are identical to the fields for 1st in vivo process. See the section on #Process Types for further details.

Adoptive Transfer

This set of fields is used to describe procedures where immune reactivity (B cells or antibodies) is transferred from one animal into another.

  • In order to be curated, this assay must be epitope relevant, that is, the transferred material must be epitope specific.
  • For details on these fields, see the section #Guidelines on Adoptive Transfer.

Recipient Organism

Organism Name

Sex

Age

MHC Types Present


Transferred antibody material

Describe the transferred material, which must contain antibodies or B cells, as you normally capture the assayed antibody

Transferred Antibody Molecule Source Material (If you encounter the need for additional transferred materials, please bring up the curation issue.)

Transferred Antibody Molecule Source Material (materials assayed)

Transferred Antibody Molecule

Transferred Antibody Molecule immunoglobulin domain

Transferred Antibody Molecule purification status

Transferred Antibody Molecule name

Transferred Antibody heavy chain type

Transferred Antibody light chain type

Transferred Antibody Molecule (Object Editor)


In vivo Process in Recipient

These fields describe any procedures performed upon the recipient animal after the immune material was transferred. Utilize the exact same fields as in vivo process 1 or 2

Disease State & Stage

Immunogen Ref name

Immunogen Epitope Relation

Immunogen

Immunogen Evidence

Immunogen Containing Object

Adjuvants

Route

Dose schedule


Adoptive Transfer Comments

Explain the adoptive transfer procedure in complete concise sentences.

Assay Information

Assay

The Assay Finder is used to select the Assay Type, Assay Type Group, and Assay Type Units. Be sure to utilize the correct units for the assay you are entering.

Qualitative Measurement The Qualitative Measurement field is a required field . Select the appropriate response based upon the figures and text provided in the manuscript. All assay contexts in the database must be labeled as either positive or negative. At times, this can be controversial. The overriding rule is to follow what the author states, however, data may be present without comment from the authors regarding the result. For truly confusing data, the author may be contacted for clarification. For data that can be reasonably considered negative or positive based upon other information provided in the manuscript, the curator may make a judgment.

It consist of a drop down menu of:

  • Positive
  • Positive-Low
  • Positive-Intermediate
  • Positive-High
  • Negative


Measurement Inequality These selections may be used in order to add additional information regarding the quantitative value.

Quantitative Measurement Certain B cell assays usually have quantitative values that should be captured. Be sure to select the appropriate units and check your math if conversions are required.

Special Note: All assays generating K on (Ka [M^-1 s^-1]), K off (Kd [s^-1]), and KD values (Enzyme-Linked Immuno Sorbent Assay (ELISA) or Surface Plasmon Resonance (SPR)) are always to be curated as separate contexts.


Number of Subjects Tested Use this field to enter the number of subjects that were tested or the number of distinct subjects that effector cells were assayed from.

Number of Subjects Responded Of the number tested, enter the number that responded by recognition of the assay antigen.

Response Frequency (%) This field will be calculated for you if you enter the above two values. If the percentage of subjects that responded is known, but the number tested and number responded is not known, enter the response frequency here.


Assayed Antibody

Assayed Antibody Source Material

This field clarifies the origin and purification status of the antibody used. Values such as serum, purified immunoglobulin, etc are available through a pull down menu.

In the event the antibody binding domain (Fab, Fv, VHH, etc) is artificially displayed on a construct or phage, <Displayed Ab(s)> is selected.

Special Note: With antibody originally selected from a library and subsequently expressed without a carrier, the Materials assayed will be <purified immunoglobulin> and the library origin will be reflected in the Antibody type field as <Display library>. See the Appendix (section (#Displayed Antibodies)) for more details on display libraries.


Assayed Antibody Immunoglobulin Domain

  • When the isotype of the antibody is not specified in the reference, but a known secondary antibody is used, the isotype of the primary antibody may be inferred from the secondary antibody used in the assay. In most of the cases, the Chain 1 Isotype field will capture the heavy chain and the Chain 2 Isotype field will capture the light chain.
  • Different isotypes may be bulked when the outcome is the same and the distinction between them is not significant. Always comment on all of the isotypes studied. If the authors discuss the different isotypes and/or it is a significant point of the paper, curate the isotypes as separate assays.

Assayed Antibody Purification Status This field captures if the antibody(ies) tested is monoclonal or polyclonal. Polyclonal monospecific is to be used when the antibody was selected by binding to a specific peptide or when the immunization was performed with a single epitope.


Assayed Antibody Name

Assayed Antibody Heavy Chain Type Assayed Antibody Light Chain Type Assayed Antibody

Chimeric antibodies are generated from more than one species. A description of the multiple species the antibody was derived from is included in the Comments on Assay field.


Antigen

Antigen Reference Name This field is used to describe situations where the reference provides additional information regarding the antigen such as life stage, fixation state, etc, other than the object name as it appears in the object fields.


Antigen Conformation Definition Field This field describes the conformational type of the antigen used in a B cell assay as either native or non-native.

Native Select <Native> when the no alteration to the tertiary structure of the tested antigen has been made. Native conformation is commonly accepted as the biologically active form of the protein (or other chemical type). This also includes synthetic or recombinant peptides identified by the authors as having native conformation.

The following will be considered enough evidence for choosing Antigen Conformation Recognized = Native:

Antigen = Source Antigen

OR

Antigen = Source Organism

AND

Assay Type = Neutralization Antibody dependent Cytotoxicity Assay Challenge Assay Cytopathic Effect Assay (CPE) Hemagglutination-Inhibition Calorimetry Colony Immunoblot Inhibition Assay

Non-native/unknown Select <Non-native/unknown> when the tested antigen is not in its native conformation. This would include short synthetic peptides and proteins that are deliberately denatured or denatured in their preparation. This value also includes antigens for which the physical nature of the antigen is neither stated by the author, nor decipherable from the paper (unknown).

Important Note: When authors’ state the conformation of the antigen is native, and it is reasonable to believe so, the conformation should be entered as Native, even when the above criteria are not met. Examples of such situations are when assembled viral particles or sporozoites are used in an ELISA and the authors’ specify recognition of native antigen.

Important Note: Assays having different antigen conformations may be bulk curated if the outcome is the same. Bulk curate under the native conformation.


Antigen The antigen is entered via the object editor. First the relationship of the antigen to the epitope must be selected. This drop down list is generated based upon the object type of the epitope to which the assay is associated. For example, if the epitope is of the object type Sequence molecule, no natural source, then the Antigen Epitope Relation drop down list will not contain "Source Organism" as a choice because the epitope is not natural and therefore, it does not have a source organism. See the sections on #Object Types and Subtypes and #Epitope Relations for full explanations.


Antigen Evidence Code

This drop down menu is used to describe how certain the curator is of the object he/she assigned as the antigen. In some cases authors will provide exactly the accession ID for the antigen, while in other cases, the exact antigen may be vague.


  • Author provided Identifier-Select this when the authors provide a Uniprot/Genbank ID or NCBI Taaxonomy ID for the antigen.


  • Exact match to reference information-Use this selection when the source name and organism name exactly match the text and there is no doubt regarding the antigen object being the one intended by the authors.


  • Representative selection (based on incomplete info)-Use this selection when there are several potential matches to the author provided source name and/or organism name and you must select among them.


  • Imperfect match-Use this evidence code when the closest match to the antigen name in the reference is a source and/or organism strain that differs from exactly what the manuscript refers to. Do not assign sources across organism species. Because internal identifiers can be created, this is only to be used when the SRC would only be used for this one case, and therefore will not be generated. An example is when a very specific lab strain was used.


  • Internal Identifier-no external match available-This choice is to be made every time an internal IEDB source (SRC source) is selected.


Antigen Containing Object

The object editor is used to create the object that acts as a carrier, a delivery mechanism, or an expression mechanism for the antigen. There is no need to repeat information regarding the antigen unless it helps explain the carrier construct. For example, when the antigen appears in the middle of a longer peptide construct, you may want to enter the entire peptide sequence.

The possible object types for container objects are as follows:

  • Recombinant Organism-Enter both the organism which is recombinant and the insert that has been engineered into it.
  • Plasmid-Only enter information regarding the insert that is expressed by the plasmid.
  • Infected Cell-Select the cell type which is infected.
  • Transfected Cell-Select the cell type and describe what is transfected into it.
  • Pulsed Cell-Select the cell type and describe what is pulsed onto it.
  • Display Library-Select from the subtypes of Phage display, Yeast display, Bacterial display, and Ribosomal display.
  • Complexed Molecules-Select from a list of subtypes which allows the entry of peptide sequences, proteins, and all nonsequence molecule types. Peptide conjugate, Protein conjugate, and Multi-Antigenic Peptide (MAP) are commonly used subtypes.

B Cell Topics

Western Blots

When a whole cell lysate is studied by western blot, the Antigen selected is according to the level of purification or separation performed. The concept is that pools of protein are captured as protein while heterogeneous pools of surface and cytoplasmic molecules are captured as species. Refer to a senior curator or EC member for difficult scenarios.

  • Whole cell lysate is to be captured as Source organism rather than source protein. Assay comments should be used to express any specific reactivity to particular proteins/bands observed on the gel and author statements regarding reactivity.
  • If further separations or purifications are performed such as immunoprecipitation or fractionation studies prior to being run on the gel, the Antigen will be source antigen.


Important Note: In the event a cell or organism is constructed to express the protein of interest and the whole cell lysate of the engineered cell/organism is run on the gel, the Antigen will be source antigen.

Important Note: Prion proteins studied by western blot will be captured as source antigen.


Antibody Display Libraries

When antibodies originate from an antibody display library (bacteriophage or other construct), the Immunization field should reflect the status of the individuals from which the library was derived. <No immunization> is selected with the use of naïve or synthetic libraries. See Appendix (section (#Displayed Antibodies)) for more details on Displayed libraries.

Challenge Assays

In accordance with rule (#Presence of Epitope), challenge assays in which the immunization is performed with the epitope as the immunogen are the only challenge assays included in the database. The challenge will be recorded as the antigen under either a T cell or B cell context, depending on other information available for the epitope.

Competition/Inhibition Assays

When curating antigen competition of antibody binding inhibition/competition assays, the antigen is always the competitor. However, when the entity whose binding is being inhibited is not captured in any other context, this interaction should also be curated in a separate assay as a direct binding/recognition assay when that interaction is curatable. Thus, the competitor will be the antigen in the competition assay and the inhibited antigen will be captured as the antigen in a binding assay. Note that this only applies if that binding is not captured elsewhere and is curatable.

  • Direct binding and competition assays with the same antigen, immunogen, and outcome should be bulk curated under the competition assay.

See the figure for details:

Comp1.jpg


When curating antibody competition of antibody binding inhibition/competition assays, the antigen is the object being shown to bind the antibody of unknown specificity. The immunogen is the object used to generate the antibody of unknown specificity. In other words, there must be one antibody of known epitope specificity (1). This antibody(1) is used to compete for binding to the assay antigen with an antibody of unknown specificity(2). By competing with each other, the unknown antibody(2) is demonstrated to share the same specificity as the epitope specific antibody(1). In the Ab comp Ab bind assay, you only capture antibody 2 information with the immunogen being the immunogen used to generate this antibody. You capture the assay antigen as what the antibody was shown to bind. For the epitope specific mAb (1), there should already be previous assays captured for this mAb or you can capture a direct binding assay for it.

  • Antibody competition of antibody binding is not a very good assay and should be bulk curated under any other type of assay demonstrating binding of the antibody to the same antigen.

See the figure for details:

Comp2.jpg



Object Types and Subtypes

Epitope Objects

The following object types are valid choices for epitopes:

  • Sequence Molecule, no natural source

Subtypes: Peptide, no natural source, DNA, no natural source, and RNA, no natural source. This selection is used only for sequence molecules (peptides, RNA, DNA) that do not have a natural source. Epitopes of this type cannot have a natural source entered. They also cannot have source antigen and source organism as antigen or immunogen relations.

  • Fragment of a natural sequence molecule

Subtype: Peptide from Protein. This selection is made for peptide sequences that are derived from natural proteins. The majority of epitopes will fall into this category. Epitopes of this type must have a natural source entered into the object editor.

  • Discontinuous region on accession sequence molecule

Subtype: Discontinuous protein residues. This selection refers to discontinuous sequence epitopes. Epitopes of this type must have a natural source entered into the object editor.

  • Region on Multi-Chain Molecule

Subtype: Region on Multi-Chain Protein. This is the subtype to select for discontinuous epitopes that are made up of residues that reside on two chains of a multi-chain molecule. If the epitope is made up of residues that are only found on one chain of a multi-chain molecule, the single chain protein should be captured as the epitope source and the epitope object type and subtype will be Fragment of a natural sequence molecule-Peptide from Protein.

  • Fragment of a Natural Non-Sequence Molecule

Subtypes: Peptidoglycan fragment, Glycolipid fragment, Carbohydrate fragment, Lipid fragment, Fatty acid fragment, Other fragment. This selection refers to natural non-sequence molecules that are derived from a larger natural molecule. Epitopes of this type must have a natural source which contains the epitope structure entered into the object editor. These epitope structures and their sources are selected from the Molecule Finder.

  • Accession Non-sequence Molecule

Subtypes: Peptidoglycan, Glycolipid, Carbohydrate, Lipid, Fatty acid, Other. This selection refers to any non-sequence molecule that is not derived from a larger natural molecule. It may be natural or artificial. Epitopes of this type may have a source organism entered into the object editor, if needed. These epitope structures are selected from the Molecule Finder.

  • Discontinuous Region on a Natural Non-Sequence Molecule

Subtype: Polysaccharide repeating unit. This type is used for discontinuous non-sequence epitopes. Currently, only polysaccharide repeating units have been curated. These epitope structures and their sources are selected from the Molecule Finder.

Antigen and Immunogen Objects

In addition to the above object types, additional object type may be used as antigens and immunogens.

  • Accession Sequence Molecule-Proteins and genes. These are selected by the molecule finder and all have accession IDs.

Subtypes=protein

  • Multi-Chain Molecule-Multi-chain proteins such as receptors, Ab, TCR, etc.

Subtypes= Multi-Chain Protein

  • Derivative of Organism-Drop down list of substances that are derived from organisms (milk, egg, seed, pollen, etc) typically used for allergy curations.

Subtypes= Derivative of Organism

  • Organism-all organisms present in NCBI taxonomy tree plus IEDB created organism strains (derived from the literature). These are selected via the organism finder. All have an identifier.

Subtypes=Organism

Special Note: Discontinuous regions (Discontinuous region on accession sequence molecule, Discontinuous region on accession nonsequence molecule) can NEVER be immunogens or antigens.

Container/Carrier Objects

It is common to have vague info regarding container objects. If you do not know details or do not have sequences or source organism, just select the object type & subtype & do not enter the unknown/vague info.

Special Note: Remember that container objects are to bulk curated under the optimal/most relevant.

Allowed Object Types

  • Object Library –used for display libraries. Only select object type and subtype.

Subtypes= Phage display, Yeast display, Bacterial display, Ribosomal display

  • Recombinant Organism- Select the organism that is recombinant from the finder. You can further describe what the organism expresses ONLY if it is not just the antigen/immunogen. Describe peptide inserts by entering the peptide sequence and source molecule (if any). Describe protein inserts by selecting from the molecule and organism finders.
  • Plasmid- You can further describe what the plasmid encodes ONLY if it is not just the antigen/immunogen. Describe peptide inserts by entering the peptide sequence, source molecule, and organism (if any). Describe protein inserts by selecting from the molecule and organism finders
  • Transfected Cell and Pulsed Cell- You should further describe what the cell expresses/is pulsed with ONLY if it is not just the antigen/immunogen. Select the cell type from the drop down menu. You can select the cell’s source organism as well, however, currently the field is labeled as “Peptide source organism”. This will be changed in the next internal site build. Describe peptides by entering the peptide sequence and source molecule (if any). Describe protein inserts by selecting from the molecule and organism finders.
  • Infected Cell- Select the cell type from the drop down menu. You can select the cell’s source organism as well, however, currently the field is labeled as “Peptide source organism”. This will be changed in the next internal site build.
  • Complexed Molecules- These are used to describe combinations of molecules with the antigen and immunogen that are not naturally occurring. If the antigen or immunogen object is linked to a protein, peptide, or non-sequence molecule, use these subtypes to enter any relevant information. Describe peptide inserts by entering the peptide sequence, source molecule, and organism (if any). Describe protein inserts by selecting from the molecule and organism finders. You do not need to repeat the immunogen or antigen, but in some cases you may want to in order to explain the orientation of a peptide immunogen/antigen within a larger peptide construct. Use the Comments fields to explain complex carrier objects
Subtypes= peptide conjugate (used to describe a peptide), protein conjugate (used to describe a protein +/- additional peptide), fatty acid conjugate, glycolipid conjugate, carbohydrate conjugate, Multi-Antigenic Peptide (MAP), multichain protein conjugate, other complexed molecule


How to Curate Container/carriers

How to enter what the container (carrier) has in addition to the Antigen/Immunogen:

1. Select the object type and subtype=start with the larger object (organism, plasmid, cell).

2. Within the editor, you can describe peptides or proteins/nonseq molecules (that are connected to the Imm/Ag)= capture what was expressed/transfected/etc by/in it. See the figure below.

3. Use comments to describe complicated container objects Complexmol.jpg

Peptide Constructs For all container objects: Enter as much info as you can. You do not need to repeat the Ag/Imm. If you do not know a peptide sequence, select the appropriate obj type & subtype & use comments to explain the construct. If you do know the peptide sequence, you can enter it if you choose to. However, the database allows you to only enter one peptide sequence (with one source) per each container object. You can select a natural source antigen and organism for the peptide if it is natural.

In this Example, The epitope being curated is the B cell epitope: Peptconstruct.jpg

Treat all other container objects involving peptide sequences similarly. Enter the peptide sequence into the only peptide sequence field available. Plasmid.jpg


Important Note: When a peptide linker is added to an epitope in order to link it to a carrier, the antigen or immunogen is to be curated as <Epitope>, the linker residues should be entered into the Comments on Assay or Immunization Comments fields.

Linker: A linker is a few residues used to LINK the epitope to another functional unit which may be another epitope, a MAP construct, a vector, etc. The linker is not entered as carrier. If the epitope is not linked to anything else, additional residues added to a natural epitope sequence are captured according to the above figure in either comments or captured as part of a Peptide Containing the Epitope.

Important Note: Regardless of the Imm/Ag Type selected and the particular use of the Carrier, Comments, and Adjuvant fields, no single component of the Imm/Ag should ever be repeated. That is, if certain residues are captured under the sequence of PeptContEpitope as selected for Imm/Ag, then those residues should not also be entered in the Carrier field. Likewise, residues captured in the Carrier field should not be entered in the Imm/Ag field. The same concept applies to the use of the Adjuvant field.


Important Note: In the curation of MHC binding assays, the exact peptide sequence that is tested MUST be entered as the epitope. There are no carriers possible in these assay types.

Guidelines on Epitope Structure

Epitope Source Molecule

The natural source from which the epitope was derived is always entered as the Source Molecule for the epitope. For example, if a gene encoding for a peptide in the Hepatitis A Virus envelope protein is inserted into a Vaccinia virus vector, the Source Molecule of the epitope is the Hepatitis A Virus envelope protein rather than the Vaccinia virus vector.

The protein ID provided by the authors will be entered into the database. When the authors do not specify a sequence ID, these guidelines will be followed:

  1. Enter the peptide sequence into the object editor.
  2. Use the Molecule Finder to see if the IEDB Accession table already has a source that contains the peptide sequence.
  3. If no match is found or none match the source name mentioned in the text, use NCBI’s Protein BLAST to identify a GenBank or UniProt match and add that source to the IEDB Molecule Finder.
  4. If a match cannot be identified by BLAST, see a senior curator to obtain an IEDB (SRC) identifier.

Important Note: Remember that the exact sequence of all epitopes must be found within the source antigen to which they are assigned. In the event that the epitopic sequence cannot be found within the source the authors describe, an IEDB Source ID may be assigned. Follow the guidelines below to obtain and use IEDB Source IDs.


How to curate epitopes from polypeptides and multimers

In the example below, there are two acceptable ways to curate the epitope's source molecule:

  • Senario 1 (ideal)
Epitope source antigen = Accession ID4-polyprotein
Antigen =Source Antigen
  • Senario 2 (acceptable)
Epitope source antigen = Accession ID1
Antigen =Other Structure from Source Organism (Accession ID4 -polyprotein)


Multi1.jpg


In the following example of an epitope from a multi-chain protein, the epitope's source is just the chain that it is derived from.

Epitope source antigen = Accession ID 1
Antigen = Source Antigen
Use Ag ref name/comments to elaborate on exactly what was tested.
Logic follows capturing "the most epitopy thing" and the carrier rules.

Multi2.jpg


How to curate peptide epitopes that repeat in the source antigen

When determining the epitope structure of a repeating peptide sequence, such as NANPNANPNANP from P. falciparum, the smallest tested peptide should be curated as the epitope.

For example, if the immunogen = NANPNANPNANP and the antigen = NANP, then the epitope is NANP. The immunogen in this example would be a Fragment of Source Antigen.

When and How to use an IEDB Source ID (SRC)

Natural sequence or non-natural sequence?

First it is important to identify if the epitope is natural or unnatural according to the authors. If the authors’ state the sequence was derived from a naturally occurring source (for ex, sequenced from a patient), then whatever source (GenBank, SwiProt, or IEDB) is used, it should have a natural source. If the authors state that the sequence was artificially created or is a lab induced mutation or analog of a wild type sequence, then the epitope does not need an SRC, it will be captured as a Sequence Molecule, no natural source.

Important Note: When a natural sequence repeated an unnatural number of times is to be entered as the epitope sequence (because the natural sequence is not used in the curated assays), it should be entered as artificial & thus have no natural source.

What if a sequence is not found in SwissProt/Genebank databases?

If the epitope is natural, first try to BLAST the sequence using NCBI's BLAST. Note that the species of the BLAST match must match what the author's state as the source of the epitope. For example, a bovine BLAST match cannot be used for a human peptide.

Important Note: if the manuscript provides a long peptide sequence that does not match by BLAST, be sure to BLAST only the epitope sequence rather than the entire peptide sequence.

If still no match can be found by BLAST, then scrutinize where the mismatch occurs and confirm that the sequence you entered in the BLAST is the exact sequence specified in the manuscript. If there were no errors on your part, then determine if a typo was likely. Sometimes when a scanned doc is converted to a pdf, errors occur in sequences, placing unusual characters in the place of residues. An author may repeat a single aa unintentionally, for ex, the author states SIIINFEKL instead of SIINFEKL. With very obvious typos, for ex, the sequence is correct in several places in the manuscript, but entered with a typo in one location, it is ok to enter the correct, BLAST-matching sequence as the epitope. However, if there is any uncertainty, the author MUST be contacted in order to clarify the EXACT sequence used in the assays. Follow the guidelines for author contact and CTS use as applicable. If the authors fail to respond within 2 weeks and/or you cannot obtain a reliable source for the corrected sequences, proceed to assign a IEDB source ID.

IEDB Source IDs

IEDB source IDs are a list of molecules that are used as the source antigen when the UniProt/GenBank databases do not have a matching source for a given epitope or when the source is non-proteinaceous.

Natural IEDB Source ID

If you are certain your sequence does not match anything by BLAST AND the author states the ENTIRE sequence being entered into the epitope sequence field is natural, then you should obtain or use an IEDB Source ID with a natural source. First check if an IEDB Source ID exists with the correct source antigen name for your case and that your species is part of the higher taxonomical category group the IEDB source is assigned to. If so, use this ID, and in YOUR epitope source field select the appropriate species and strain as provided in the reference. All uses of IEDB Source IDs are subject to review so be sure that you are using it correctly. If the particular ID you need is not present, then contact Laura Z. to obtain an IEDB Source ID and continue your curation with a place holder source ID and a note on your cover sheet stating that the ID must be changed before promoting the reference.

Obtain an IEDB Source ID

To obtain an IEDB ID:

  • Email Randi (rvita@liai.org) with PMID, epitope name and/or epitope sequence, location, & details regarding if & when the author was contacted.
  • Write on the cover sheet what you need added. Write on the PDF where something will be changed. Have the curation assigned to RV by circling RV on the cover sheet in the PR section.
  • If cited references discuss the peptide’s source, you must look them up. Many typos can be caught that way. Attach refs.
  • If author contact is needed, you must do this first. Attach author communication
  • As always, curate with a filler value as if you had the correct value in place.

Epitopes from Display Libraries

When an epitope is identified through the use of a bacteriophage, baculovirus, or other randomized peptide library, it must be determined whether the phage-derived epitope sequence is homologous to a sequence from a biological source.

  • If a natural homolog has been identified, then its sequence will be captured as the epitope.
  • If a corresponding natural homolog was not identified in the reference, then capture the epitope as the object type Sequence Molecule, no natural source.

Epitope Source Organism

The Organism name from which the epitope sequence originates must be entered into the Source Organism field. Often the epitope sequence originates from an organism such as a virus, bacterium, eukaryotic organism, or, less often, a plant. In order to enter the organism into this field the Organism Finder is used.

Use the Organism Finder button to access the Organism table and type the name of the organism into the blank box followed by clicking on the Search button. Click through the entries until reaching the appropriate organism species and strain. Click on the name in order to enter it. If you cannot find the organism name that is needed, see a senior curator about having the organism species and strain added to the organism table.

When the strain of the source organism is not specifically mentioned in the reference and all uses of the source organsim are performed with a particular strain, the epitope may be attributed to that strain, provided the exact epitopic sequence is found in that strain. For example, when the authors describe a Clostridium botulinum epitope and do not mention which type it was derived from, but always immunize and assay with Type A, the epitope may be assigned the source organism of Clostridium botulinum Type A.

Quasispecies/Minor Species

In the event the epitopic sequence is demonstrated to mutate over time, each new sequence represents a new epitope, antigen, or immunogen. If the new sequence matches a SwissProt/Genbank entry, that source may be used as the epitope source antigen. If the variant sequence does not match an existing sequence, an IEDB source should be assigned.

Carbohydrate Epitopes

The source of carbohydrate epitopes can be vague or confusing at times as carbohydrates may be present across many species. Always capture the source as described by the authors. If the authors mention multiple potential sources, assign the most generic source which captures all of the mentioned species. For example, if multiple strains of the same species are mentioned, curate the source for the generic species. If different species of the same family are mentioned, curate the source as the generic family.

Analogs

Analogs are synthetic constructs of peptide sequences or chemical compounds that share some structural features in common with another sequence or compound. They are often used to determine the role of specific amino acids in the binding or immunogenicity of an epitope. The source of an analog is always artificial.

If authors state peptide is from X protein, X org, no matter what the sequence is, the peptide is natural (“Peptide from protein”). Use SRC if it does not blast. If authors state they altered a natural seq, the peptide is “Peptide, no natural source.” Be sure to enter the “Epitope related object” that the epitope is an analog of.

Analogs are not captured as separate epitope entries, unless one of the following criteria are met. If an analog is not curated as an epitope, use the Comments on Assay field to mention the details of the analogs that were tested. See sections (#Key Residues) or (#Linear Epitopes) for further instructions on strategies for curation of many commonly encountered analogs.

Only curate analogs as epitopes in specific circumstances:

1. Both the immunogen and the antigen in the assay is the analog

2. The paper only uses the analog throughout

3. For that particular assay type, the authors only use the analog. For ex, they only show that the analog treats the disease and never show that the wild type does.

4. All analog sequences accompanied by MHC binding data or sequences that are designed to improve upon the immunogenicity of an epitope must be entered as separate epitope entries. Additionally, all contexts that do not use the natural epitope as either the immunogen or antigen, but instead use only the analog sequence will also require the analog be entered as a separate epitope entry. An example of such an assay would be the use of a CTL line that was raised to the analog lysing APC presenting the analog.

5. Quantitative SPR binding data is present

6. Tetramer assay is present.

7. The analog’s altered properties compared to the wild type sequence make it a biologically important reagent, for clinical applications or assay development according to the authors’ intent.


Clarifying note: Analogs created as single amino acid substitutions of an epitope with the intent of establishing critical residues for Antibody or TCR binding are not curated as epitopes. The results are summarized in the assay comment for the parent epitope.


Be sure to enter the natural object that the analog is derived from in the Epitope Related Object fields. Enter as much information as you have. An epitope related object can be a peptide, protein, non-sequence molecule, or organism.

How to curate analogs used in assays

  • Example 1

Assay 1: Imm = Source organism, Ag = wild type Assay 2: Imm = Source organism, Ag = series of analogs

Bulk all antigens together under the wild type. We are primarily concerned with key residues = those mutations that abolish immune recognition.

Curate 1 assay: Imm = Source organism, Ag = wild type Assay comments = “Recognition of the epitope was abolished when residues 3 or 4 were mutated.”

  • Example 2

Assay 1: Imm =wild type, Ag = wild type Assay 2: Imm = series of analogs, Ag =wild type

Bulk all immunogens together. Curate 1 assay: Imm =wild type, Ag = wild type Include immunization comment “Mice were administered the epitope or alanine substituted analogs of the epitope.” Include assay comment “Immunogenicity of the epitope was reduced by alanine substitutions of residues 4 or 5.”

  • Example 3

Assay 1: Imm =wild type, Ag = Source antigen Assay 2: Imm = series of analogs, Ag = Source antigen

Outcome: wild type protects and some analogs also protect = Bulk all immunogens together. Curate 1 assay: Imm =wild type, Ag = Source antigen with appropriate comments (as before)

  • Example 4

Assay 1: Imm =wild type, Ag = Source antigen negative Assay 2: Imm = 1 analog, Ag = Source antigen positive

Curate both assays separately with the positive outcome under analog = epitope

  • Example 5

Assay 1: Imm 1=wild type, Imm 2= wild type Ag = Source antigen Assay 2: Imm 1= series of analogs, Imm 2 = wild type, Ag = Source antigen

Outcome: Wild type and some analogs induce tolerance, some analogs do not induce tolerance. Bulk all under wild type with appropriate comments.

Outcome: Wild type does not induce tolerance, one analog does. Curate tolerance assay under wild type (negative) and under analog (positive).

Mimotopes

Mimotopes are functional mimics of natural molecular structures which bear little or no sequence homology to their biological counterparts. Mimotopes should be captured as separate epitope records. Follow the guidelines below to capture mimotopes. If a biological homolog is identified from the mimotope structure and is tested, the naturally occurring sequence should also be captured as a separate epitope context.

Enter the object that the epitope mimics in the Epitope Related Object fields.

  • If the mimotope has a natural source antigen, specify those details using the [Source Antigen] fields.
  • If the mimotope is not natural, it will be captured as a Sequence Molecule, no natural scource.


Minimal Epitopes vs Optimal Epitopes

The minimal epitope is the shortest length or smallest structure that produces a cellular or humoral response (is immunogenic) or can serve as an antigen (is antigenic). For MHC restricted responses, a minimum of 7aa are required for a sequence to be considered an epitope (see guidelines in section (#Truncation)).

The authors may describe the epitope with the greatest response as the optimal epitope. There are cases where the minimal epitope does not necessarily give the optimal response. This situation is common with Class II and Antibody/B cell epitopes.

In the event the minimal epitope is not the optimal epitope, the optimal epitope will be captured instead of the minimal epitope. For example, in Figure 5 (PubMed ID: 15048720) the optimal responses in panels a and c are produced by the longer peptides (represented by diamond symbols) and thus are not the minimal epitopes. In this instance, the optimal epitopes in panels a and c will be captured instead of the minimal epitopes.

Minimal Epitope vs Fine Specificity

Fine specificity refers to the detailed pattern of reactivity of different T cell clones or monoclonal antibodies when recognizing the same epitope due to changes of the components such as individual amino acids or sugars within an epitope. Typically, multiple T cell clones and monoclonal antibodies will recognize different amino acids as key residues within the same stretch of the protein sequence. Follow the guidelines below when there are fine specificities reported for a group of T cell clone or monoclonal antibodies.

  • Class I epitopes: if T cell clones respond optimally to amino acid sequences of different lengths in a truncation analysis, each of these sequences will be entered as separate epitopes, even though they may contain the same core sequence.
  • Class II epitopes: if multiple T cell clones respond to an amino acid sequence that satisfies the criteria of an epitope (e.g. 15 amino acids or less), but differ in their individual reactivities within that stretch of amino acids (e.g. clone 1 recognizes aa 1-13 and clone 2 recognizes aa 3-15), then the longer sequence containing all of the residues recognized by all of the clones will be entered as a single epitope (e.g .aa 1-15). This is considered a single epitope with different fine specificities.
  • B cell epitopes: Linear Antibody/B cell epitopes will be entered in the database similarly to Class II epitopes as above. For conformational Antibody/B cell epitopes, assume that each mAb defines its own epitope unless otherwise indicated by data or stated by the author.

These guidelines will be overruled when the authors consider the epitopes distinct, in which case they will be entered as individual epitopes.

Figure 5. Example for curating minimal epitope vs optimal epitope.


Ambiguous Cases for Designating a Sequence as an Epitope

Typically the assignment of a sequence as an epitope is unambiguous. However there are a number of scenarios in which more than one structure may be assigned as the epitope within the context of a single assay. Accordingly there may be ambiguity in deciding which structure is to be considered the epitope.

  • The sequence or molecular structure of an epitope may be shown to be conserved in several different antigens and in different species. The database only allows reference to one source antigen/source organism per epitope entry. In order to enter multiple source organisms, multiple epitope entries are required.
  • Cross-reactivity studies may utilize an epitope molecular structure conserved across multiple source antigens or organisms; however, only reference to one source may be used per epitope entry in ICS.
  • Experimental data might reflect the use of an immunogen and an antigen derived from different source antigens or organisms, creating confusion regarding whether the immunogen or antigen should be designated as the epitope under which the experimental data will be curated.

Use the following guidelines to determine the structure to be entered as the epitope:

Case 1: If both the immunogen and the antigen are naturally existing molecular structures or both are artificial and one of them is an epitopic region while the other is a minimal epitope, the minimal epitope will be the captured epitope and is entered in the [Epitope Structure] category.

Case 2: If the immunogen and the antigen are both naturally existing molecular structures or both are artificial and both of them are minimal epitopes, then both sequences will get captured in separate entries as epitopes. The end result of this is that each epitope will receive an identical copy of the assay context. Duplication of assay contexts is necessary to insure that both epitopes receive equal priority in the database.

Case 3: If either the immunogen or antigen is artificial, that is, it does not exist in nature while the other is a naturally existing molecular structure, the natural structure will be designated as the epitope and entered in the [Epitope Structure] category. The artificial antigen or immunogen will be specified in the context (as assay antigen or immunogen) of the curated natural epitope.


Important Note: When an epitope sequence is present in more than one source and the other potential sources of the epitope are not specifically tested, but rather this information is only commented upon, the epitope is assigned to the source which was tested in the reference. In contrast, if several natural sources containing the epitopic sequence are studied in the reference, the epitope should be curated under separate entries for each source.

Viral Escape Mutations

When entering viral escape studies into the database, the emphasis should be on antibody binding to residues of the wild-type sequence.

Binding of a monoclonal antibody (mAb) or antisera to the wild-type sequence must be demonstrated along with a loss of binding to escape mutants in order for the data to be included in the database. The negative binding data of the mAb or antisera to escape mutants is not curated.

Each mAb should be treated as though it defines a separate epitope, unless explicitly stated otherwise by the authors. If the panel of monoclonal antibodies is used to characterize binding to an antigen, follow the guidelines described in section (#Panel of Monoclonal Antibodies).

Multiple amino acid residues identified by a series of binding experiments may be captured as a single entry in the Conformational Sequence field if identified as belonging to a single epitope by the authors. Individual binding experiments (inhibition, neutralization, ELISA) will be captured as separate contexts of the conformational epitope.

Information regarding the method used to determine the epitope sequence should be summarized in the [Epitope structure] Comments field.


Discontinuous Epitopes

These epitopes are curated similarly to escape mutants. The wild type residues that when mutated result in loss of recognition are curated as discontinuous epitopes.

Pool of Peptides

When the antigen or immunogen is comprised of a pool of peptides and the result of the assay is negative, each peptide of the pool will be entered in the database (when provided by the reference). When an epitope is tested both as part of a pool and alone, those contexts may be bulked under the epitope used alone with a comment stating the same results were demonstrated through the use of a peptide pool.

When the response to a pool of peptides is positive, this data is generally not entered in the database but the de-convolution (testing of individual peptides within the pool) experiments are curatable, if available. However, there may be cases in which pools of overlapping peptides are used to define epitopic regions/domains without further de-convolution. If the peptide pool defines a continuous epitopic region/domain ≤ 50 amino acids, the data will be curated under the entire amino acid sequence encompassed by the peptide pool. The Epitope Comments field should explain that the epitope is actually comprised of a peptide pool and the Antigen should be captured as "Epitope".

Key Residues

When critical residues for the recognition of a linear epitope by T cell or B cell receptors, antibody, or MHC are described in a reference, this information is context dependent. Use the Assay Comments field to describe the key residues. The experiments identifying key residues of linear epitopes are not captured as separate contexts.

Important Exception: When discontinuous epitopes are defined in a reference by only key residues, the key residues should be entered in the Reference Region and Discontinuous Residues fields.

Sequence field. Thus, in assays identifying a single amino acid as a key residue, that amino acid will be entered in the Conformational Sequence field unless there is evidence that it is part of a linear epitope.

We assume that a mAb recognizing a conformational epitope defines the epitope, unless otherwise specifically stated by the authors.

Linear Epitopes

When alanine-scanning mutagenesis or other residue substitutions are used to determine key residues within a linear epitope sequence, the key residues should be captured in the Comments on Assay field.

The sequences utilized to deduce key residues are not captured in separate contexts. Rather, in the Comments on Assay field, capture in one or two short and concise statements how the residues were determined to be key. Use standard amino acid notation to denote key residues (Amino acid one-letter code and its residue number e.g. L107). The principal prerequisite for capturing epitopic determinant data is the demonstration of antibody or TCR binding to the native epitope sequence with appropriate controls. All MHC binding data and all naturally processed data are always curated, even when used to deduce key residues.

Figure 3 (pasted from reference with PubMed ID: 15213134) shows an example of alanine substitution data that should not be curated as separate contexts, but may be summarized as a comment. The Epitope Comments field should state "Alanine substitutions for residues F295, T300, Y301, and Y302 eliminated T cell activation, identifying these as critical amino acid residues in the epitope".


Figure 3. Experimental data demonstrating critical amino acid residues of an epitope are not curated as separate contexts.
Conformational Epitopes

The primary data to capture is a demonstration of antibody binding to the native sequence in non-denaturing assays (ELISA, X-Ray Crystallography, NMR).

As with linear epitopic determinants, the peptide sequences of all mutations or variants tested are not captured as individual epitopes. Enter relevant details regarding the variants or mutants in the Comments on Assay field and information regarding the determination of the epitope structure in the [Epitope Structure] Comments field.

Deduced Epitopes

A deduced epitope is one that is not directly tested as an isolated structure , but rather deduced by the authors by various methods such as the use overlapping peptide scans. Deduced epitopes may be curated, however, those epitopes defined only by computer prediction algorithms in the absence of validating experimental data will not be included in the database. Additionally, when curating the use of multiple peptides (as antigen) in order to deduce an epitope, only one context should be curated using the optimal or minimal peptide as the antigen with a comment regarding the use of multiple peptides.

Conservancy/Cross-Reactivity

These guidelines determine the process used to curate data relating to conservancy and cross-reactivity.


Rule 1: When an epitope is analyzed for conservancy or cross-reactivity among different natural proteins or pathogens with experimental data presented in the reference for each of the different sources, the different structures should be entered in the database as separate epitopes for each of the different natural proteins or pathogens according to section (#Ambiguous Cases for Designating a Sequence as an Epitope).

Rule 2: When sequence or homology analysis of an epitope and/or source protein data is present, the significance of the data should be mentioned in the Epitope Comments field.

Rule 3: When an artificial peptide is used for conservancy analysis among multiple proteins / source organism, only ONE [Epitope Structure]/[Source Antigen] pair is curated with all others recorded in the Comments field. The significance of the data will be used to determine the chosen epitope-source antigen pair.

Important Note: These cross-reactivity and conservancy rules are applied only to different species and NOT to different strains. Different strains are curated according to the guidelines specified in section (#Decision Scheme for Bulk Curation).

Host Organsim

The species and strain or ethnicity of the host organism is captured using the Organism Finder. Different species cannot be bulked. Each new immunized organism is curated as a new context. Different strains may be bulked in certain situations. When the outcomes of the assays are the same for B cell assays, different strains of mice may be bulked. All of the different strains should be entered in this field, separated by commas. Different strains may be bulked in T cell contexts if the outcome is the same and the MHC alleles are the same. The curator should use discretion and author statements to determine when different strains of animals should not be bulk curated.

Transgenic Mice

Transgenic mouse strains can be found in the organism finder. If you need a new transgenic strain, see Laura Z to have it added to the finder.

Ethnicity

Ethnicity can be defined as "of or relating to large groups of people classed according to common racial, national, tribal, religious, linguistic, or cultural origin or background". Ethnicity is entered via the organism finder. All previously curated ethnicities and/or geographical origins that have been curated are now present on the organism finder. If you need a new ethnicity, see Laura Z to have it added.

http://www.ncbi.nlm.nih.gov/projects/mhc/ihwg.cgi?cmd=PRJOV&ID=9

Information regarding the ethnicity will be entered in the Comments on Immunization field when the ethnicity is not clearly specified in the text, does not appear in the "Population" list, or a geographic region is used to describe the origin of the patients involved (geographic location does not necessarily correlate with ethnicity).

Process Types

In vivo process types

Important Note: Every assay requires either one in vivo process or an in vitro process “primary induction in vitro”.

  • In vivo processes are sequential
Up to two in vivo processes can be recorded in the sequential order in which they occurred.
Use in vivo 1 for the first relevant immunogen to which the host was exposed and in vivo-2 for subsequent exposures to a different immunogen.
In cases where the sequence of events is unknown, the process most relevant to the paper will be entered in in vivo process 1.
  • Relevancy is determined by the relationship of the immunogen to the antigen

For example, if the assay is measuring the response to an influenza epitope, prior exposure of the host to EBV is deemed irrelevant and is not captured (except in comments). Prior exposure to influenza is captured instead. With multiple immunizations, select the most relevant and distinct immunogens to capture.

Example:A human was naturally exposed to a pathogen and acquired a disease as a result and was then immunized multiple times with a peptide vaccine. in vivo process 1 would be the exposure to the pathogen with the disease states and stage filled in. The in vivo 1 immunogen would be the Organism (Pathogen). In vivo-2 would be immunization with the peptide vaccine and the immunogen 2 (peptide) would reflect the first vaccination with subsequent vaccinations being commented upon.


Important Note: Immunogen 1 and 2 should not be the same. Do not use in vivo process 2 to capture boosts or repetitions of in vivo process 1. Do not capture different adjuvants, routes, or carriers with the same immunogen as separate processes. Those fields should be bulked under one in vivo process.

Important Note: When several immunization procedures, schedules, and doses are used, these may be bulked into one assay context. Curate the procedure giving the best results or the one most often used and comment on the other procedures.

In Vivo Process 2

To warrant the additional use of a second in vivo process, the second process must do one of the following: Be of a different process type than in vivo 1. Have a different immunogen and thus different epitope relation Result in a change in the disease status of the host Alter the outcome of the assay –this does not include boosts.

  • Please do not confuse a challenge assay with an IVP2. See Challenge assay rules (*). This is to only be used if the assay type is NOT challenge assay, but instead is the measurement of a different response to an antigen.In IVP2, used for immunotreatments aimed at reducing a preexisting disease (which is recorded in IVP1). The disease name and stage then must be blank.
  • Heterologous infections

Infections irrelevant to the outcome of the assay should not be captured even though they have a different immunogen. For example, if authors note that malaria patients also had exposure to dengue, but no further studies related to dengue were performed, there would be no need for a second in vivo process.

Administration Group

Administration is to be used for planned processes such as the experimental administration of a substance in the course of an experiment. In vivo processes:

Administration in vivo- to cause disease

To be used in cases of experimental induction of a disease (or sensitization) by inoculation of an immunogen. The immunogen will be the pathogen/allergen causing the disease.

  • The disease name and stage then must be filled.
  • To be used only in those assays that do not measure immune recognition directly but that, instead, record the existence of disease: “Reduction of disease after treatment” (B cell) and “Treatment” (T cell).
  • The assay has a hypothesis that the role of the immunogen is to cause a disease.


Administration in vivo- to prevent or reduce disease

To be used when the intent of the immunization protocol is to prevent or reduce the establishment of a disease.

  • To be used only in those assays that do not measure immune recognition directly, but that, instead, record the existence of disease:
“Reduction of disease after treatment” (B cell) and “Treatment” (T cell)-for the administration of the immuno-treatment (preventive/therapeutic) of the disease
“Challenge” assays (both B and T) -for the administration of immunogens which are tested for their protective capacity in a challenge assay
  • Sensitizations to allergens, with a pre or post-treatment to reduce the ‘allergic response’ that are curated in a “reduction of disease after treatment” or “treatment” assay are to be considered analogous to the induction of allergy (disease), and recorded this way. This is not optimal but in general will go along the thrust of the papers, where the authors are trying to mimic experimentally (a part of) an allergic syndrome and test its treatment.
  • The assay has a hypothesis that the role of the immunogen is to prevent a disease (increase survival or decrease symptoms).


Administration in vivo

To be used when there is an experimental inoculation of an immunogen and either it does not cause a disease or the outcome is unknown

  • This category will account for most of the curation cases
  • If the administration induces a disease, as stated by the authors, the disease name and stage should then be recorded in the disease fields.
  • To be used for humans vaccinated with established vaccines, even when this was not administered in relation to the experiment being performed.

Administration in vivo Decision Tree: Admin.jpg

Occurrence with Disease Group

A disease is only captured if there is a relevant immunogen. This group involves unplanned processes that led to current or past disease as determined by:

a) symptomatic diagnosis or

b) presence of pathogen.


  • Disease Name and Stage are required fields
  • Existing immune reactivity is NOT evidence for disease. Many subjects can and will be seropositive without clinical infection or disease. Beware as author may state that donors are/were infected or diseased based only on the presence of antibodies or other immune response to a pathogen.
  • If the data points to the existence of disease AND the authors refer to the donors as patients, you can sometimes infer they have a disease (usually the stage will be then unknown).


Important Note: Do not to overstate diseases. It is safer to err on the side of not recording an existing disease than to include a non-existing one. Do not use the Natural Occurrence Group and do not select an ICD10 classification of “infection” when the hosts are only described as seropositive with no other information provided on their disease state. However, sustained seropositivity to pathogens that induce a chronic disease does warrant classification of “infection”.


Occurrence of Infectious Disease

Use when an infectious disease is established as a result of an encounter with an immunogen. The causative agent is an infectious pathogen (virus, bacteria, unicellular or multicellular parasites, fungi). Infectious diseases include parasites. This process type is also to be used for elective (e.g. ingestion) and accidental exposures.

  • The disease name and stage then must be filled. Given proven infection (past or present), the infection itself can be recorded as the disease if no other description of the disease is available.


Occurrence of Allergy

Use when an allergic syndrome is established as the result of an encounter with an allergen (recorded as the immunogen). It will also be used for elective and accidental exposures.

  • The disease name and stage then must be filled. For allergic patients without specification of the allergen involved (i.e., atopic controls) the immunogen fields are left blank, and the disease is captured as “Allergy, unspecified” or with an ICD-10 code, if a match is available.


Occurrence of Autoimmune Disease

To be used when there is an autoimmune process in the host.

  • For now, only used when autoimmunity is linked to an infectious process and the epitopes are from the infectious agent. IVP1 will then be ‘Natural occurrence of infectious disease’ with infectious disease filled and Immunogen = infectious agent, IVP2 will be ‘Natural occurrence of autoimmune disease’ with Immunogen = Blank.
  • Autoimmune status after infection will be recorded only if the patients all suffer from it or if there is a differential response to the epitope in diseased and non-diseased infected individuals.
  • If infected patients with and without the autoimmune disease are tested and the outcome is the same, they will be bulked under the infectious agent exposure alone.
  • For self-epitopes, rules have not been devised yet = avoid these curations and park them. Please note these rules do not apply to epitopes from self-antigens in autoimmunity.


Occurrence of Cancer

To be used when the host has a form of cancer.

  • For now, only used when cancer is a result of an infectious process and the epitopes are from the infectious agent. IVP1 will then be ‘Natural occurrence of infectious disease’ with infectious disease filled and Immunogen = infectious agent, IVP2 will be ‘Natural occurrence of cancer’ with Immunogen = Blank.
  • Cancer status after infection will be recorded only if the patients all suffer from it or if there is a differential response to the epitope in diseased and non-diseased infected individuals.
  • If infected patients with and without the cancer are tested and the outcome is the same, they will be bulked under the infectious agent exposure alone.
  • For self-epitopes, rules have not been devised yet = avoid these curations and park them. Please note these rules do not apply to epitopes from self-antigens in cancer.


Occurrence of Disease

This process type is used for diseases that do not correspond to any of the above process types. Examples are toxic syndromes caused by toxins, prion-based diseases, and spontaneous diseases.

  • Spontaneous diseases (immunogen blank) will be recorded only if the patients are also given an epitope-related immunogen to treat that disease.


Important Note:A disease only gets curated if there is a relevant immunogen. If the disease is not caused by an epitope-related agent nor is there a treatment for it with an epitope-related agent, the disease is not recorded, nor its causative agent. Exceptions to this may be made when we curate Cancer and Autoimmunity self-epitopes.

Important Note:Immunogen = BLANK can occur for ‘Occurrence of Disease’, Autoimmune Disease, Allergy, and Cancer. Curate only if the patients are also given an epitope-related immunogen to treat that disease:

IVP1: Occurrence of X disease. Disease Name and Stage are filled. Immunogen is left blank
IVP2: Follow the Administration in vivo guidelines for recording IVP2.


Examples for the Occurrence Group:

1. If an epitope-related agent causes the disease, the disease gets curated in the same IVP as the disease. Epitope is from HCV 1a and donors are hepatitis C patients.

IVP1: Occurrence of Infectious Disease. Immunogen is HCV (taxonomical parent).


2. Patients with dystonia, and given Botox for this condition, are tested for Botox peptide recognition.

IVP1: Occurrence of Disease. Disease Name is Dystonia. Immunogen is left blank.
IVP2: Administration in vivo. Immunogen is Botox (source antigen).


3. TB patients are tested for TB peptide recognition, but are also stated to be HIV positive.

IVP1: Occurrence of Infectious Disease. Disease is TB. Immunogen is M. tuberculosis. The HIV status is recorded in the Immunization comments.


Exposure without Evidence for Disease Group

This group is used to describe unplanned processes (natural and accidental exposures) that did not lead to the existence of a disease. To be used when there is reason to believe that the subjects have been exposed, but there is no evidence that that exposure led to a disease.

  • The disease name and stage must be left blank or may be put as ‘Healthy’ (code U6) when that state is explicitly mentioned.
  • The categories of ‘Exposure (…) without disease’ are hierarchical. The first category that is applicable to the donors should be used.


Exposure with Immune Reactivity

This process type is to be used when the exposure is assumed because there is immunological recognition (antibody or T cell mediated recognition of the pathogen/allergen or parts of it). As when there is seropositivity for a pathogenic agent, but there is no explicit information regarding a disease outcome.

  • The disease name and stage should be left blank.


Exposure without Evidence for Disease-Documented Exposure

This selection is used when there is a known event of exposure to a pathogenic agent, but there is no explicit information regarding a disease outcome of seropositivity. Examples include household contacts of diseased individuals, hospital staff, and accidental needle prick without any information provided on seropositivity.

  • The disease name and stage should be left blank.


Exposure without Evidence for Disease-Environmental Exposure to Endemic / Ubiquitious Agent

This selection is to be used when there is inferred exposure to a pathogenic agent, by merit of its endemicity or ubiquity, but no information on the disease outcome, seropositivity, or actual events of exposure. This includes pathogens that have a high incidence in the population (Influenza, CMV, EBV, Candida, etc). Examples are EBV exposure in healthy subjects and P.falciparum exposure in an endemic geographic area.

  • The disease name and stage should be left blank.


Exposure without Evidence for Disease

This is to be used sparingly when there is the need to record an exposure and the above process types do not apply and “No immunization” is inappropriate.

  • The disease name and stage should be left blank.


Other in vivo Process Types

No immunization

Use this process type when the host has no prior exposure to an immunizing agent relevant to the assay performed. Use this for naïve hosts.

  • The immunogen and disease fields must be blank.

Important Note: In cases with no relevant in vivo immunogen such as cancer, autoimmunity, or unknown natural exposure with additional in vitro restimulation with a relevant immunogen, be sure to capture the in vitro process as primary induction in vitro.


Unknown

Only use this selection when the route or mode of exposure is unknown. Examples are with mAbs and T cell lines where the cited reference cannot be found.

  • The immunogen may be filled if known.


In vivo Process Types-Curatability

Before curating any in vivo process type, first be certain that the assay is curatable. Every assay requires either one in vivo process or an in vitro process of “primary induction in vitro”.

Polyclonal responses

In order to be curatable, one of the following must apply:

  • In vivo process -1 immunogen is the epitope or the antigen is the epitope.
  • Primary induction in vitro immunogen is the epitope or the antigen is the epitope.
  • For certain allergy & epitope challenge/treatment assays, either in-vivo process 1 or in vivo process 2 immunogen is the epitope or the antigen is the epitope.


Monoclonal responses

All are curatable under the epitope as long as the response is epitope specific, i.e., the immune receptor was shown to bind the epitope.

Disease State and Stage

Disease states and stages are captured as part of the in vivo process. The Disease State field reflects the disease state of the individual(s) used in the context. When multiple diseases are reported in the reference, the one disease which is most appropriate to the context will be entered while the others will be noted in the comments.

Important Note:Same Process

Disease state and stage are entered only in the in same vivo process that is utilized to capture the exposure to the immunogen which resulted in that disease state.

Disease State

If exposure to the immunogen resulted in a disease state, then also complete the disease state and stage fields. Different disease states generated by exposure to the same immunogen and having the same outcome may be bulk curated.

  • The “Z” ICD-10 codes cover some previously uncovered infections that have no disease description. Be sure to check them out.
  • For allergic diseases, if the particular syndrome is available as an ICD-10 code, use it. If not, comment on any details regarding the particular syndrome and use “Allergy to immunogen” (U36).
  • Diseases which are the result of an administered immunogen are only to be captured in the Disease State field when evidence of disease is demonstrated (clinical symptoms, pathological evidence, author stated, etc) and/or are relevant to the assay being captured.


Disease Stage

If the disease state is filled, the stage must also be filled and vice versa.

For similar outcomes, curators may decide to bulk curate different disease stages. The most positive or most relevant stage should be selected.

Capture the stage of the disease at the time the experimental data was generated. The choices are:

Acute A short-term infection or disease characterized by a dramatic onset and rapid recovery is recorded as <Acute>. Primary infections fall under this category.

Chronic A long-term infection or illness and partial remission is recorded as <Chronic>. Post An illness a subject has recovered from is recorded as <Post>. Latent (potentially existing but not presently evident or realized) and remission (a period during which symptoms of disease disappear [complete remission]) are recorded as <Post>. Note that partial remission will be recorded as <Chronic>.

Other Any disease stage that cannot be classified under <Acute>, <Chronic> or <Post> will be classified under <Other>. Cancer stages and household contacts will be recorded as <Other>.

Unknown When the disease stage is not clearly specified or unavailable in the reference, it will be recorded as <Unknown>. The main distinction between <Other> and <Unknown> is that <Other> specifies a disease stage mentioned in the reference, but not classifiable as <Acute>, <Chronic>, or <Post> while <Unknown> specifies a disease stage which is not explicitly mentioned.


In vitro Process Types

Any exposure of effector cells in vitro to an immunogen prior to the addition of the assay antigen is captured here. The two choices are:

Primary induction in vitro

This type is to be used when the effector cells were taken from a naive animal and exposed to an immunogen prior to assay.


Restimulation in vitro

This type refers to effector cells taken from a host which was exposed to a relevant immunogen in vivo prior to and restimulated in vitro prior to the assay. When cells from an immunized (exposed) subject are washed after restimulation and before the assay antigen is added to measure a T cell or B cell/antibody response, this is considered an in vitro restimulation.

Important Note: When cells are stimulated in vitro by means other than exposure to an immunogen (as with mitogens or other cell culture supplements) prior to use, this is not considered an in vitro restimulation.


Antigen/Immunogen Epitope Relations

Curate the Ag/Imm as it was tested in the assay. Capture the strain information provided for the Ag/Imm regardless of the epitope’s source organism.

Only use Epitope, Fragment of Source Antigen, Source Antigen, and Source Organism is the Ag/Imm should have the exact accession ID for the protein and the exact strain information (I.e.Tax ID) as the epitope’s source.

Taxonomic relations are used for peptides, protein, organisms, or other non-peptidic structures (carbs, lipids, etc). The exact relationship is determined by comparing the Ag/Imm organism strain to the epitope’s organism strain.

Follow these guidelines:

Agimmr.jpg

Autofillable Epitope Relations

These are relationships of the immunogen or the antigen to the epitope that are identical to the epitope structure or to the source of the epitope as entered on the epitope tab.

Epitope This is to be used when the exact epitope structure is being used as the antigen or immunogen. When a carrier or vector is used with the epitope structure, "Epitope" is still selected. When the antigen or immunogen is the epitope presented in a different chemical type, for example as DNA rather than peptide, "Epitope" is also selected.

Fragment of Source Antigen This is to be used when any naturally occurring fragment of the source antigen (exactly the same accession ID and the same Tax ID) that completely contains the epitopic sequence and is larger than the epitope structure is used. This selection is also used to capture the study of recognition of degradation analysis and enzyme or chemically treated fragments of the source antigen.

Important Note: When a number of fragments of the source antigen are tested, these fragments may be bulked into one assay context. Curate the fragment giving the best result and comment on the other fragments that were used.


Source Antigen This selection is made when the same molecule as entered as the source antigen of the epitope is used as either the immunogen or the antigen. If mutations are made in the molecule or if carrier/container objects are utilized, still select Source Antigen. If the molecule used as the immunogen or antigen is from a different strain than the epitope, Do not select Source Antigen.

Source Organism This is selected when the exact species and strain from which the epitope is derived is used in the assay.


New Epitope Relations

In addition to the autofillable epitope relations (old Ag/Imm Type) of Epitope, Source Antigen, Frag SAg, Derivative of Source Organism, and Source Organism, there are a few new values. These were devised to further refine “Other” by analysis of previously curated Ag and Imm that were assigned to “Other”. The choice of “Other” can still be made when none of the following apply, but you should first determine that none apply before selecting “Other”.


Taxonomically Related (parent)

Select if the imm/Ag is either an organism or is derived from an organism which is a higher order than the epitope's source organism as selected on the epitope tab. An example is if the epitope is from influenza A/PR/8 and the host was exposed to influenza of unknown strain (infuenza A).


Taxonomically Related (sibling)

Select if the imm/Ag is either an organism or is derived from an organism which is within the same family as the epitope's source organism selected on the epitope tab. An example is if the epitope is from influenza A/PR/8 and the host was exposed to influenza of a different strain (A/Wilson-Smith/1933).


Taxonomically Related (child)

  • Select if the imm/Ag is either an organism or is derived from an organism which is a lower order than the epitope's source organism selected on the epitope tab. An example is if the epitope is from influenza A of unspecified strain and the host was exposed to influenza of a specific strain (A/PR/8).


Structurally Related

This selection is to be used for homologous, but taxonomically unrelated structures. A percentage of homology requirement has not yet been decided.

  • This applies to analogs even if authors call homologous entity a mimotope.
  • This applies to all object types (organisms, proteins, peptides, etc) that are or contain an object that is structurally similar to the epitope.


Other Structure from Source Organism

Use for structures that are derived from the epitope’s source organism, but do not fully contain the epitope sequence.


Other

Any immunogen or antigen that cannot be described by any of the previous choices will be labeled as "Other". Common examples of this type include peptides originating from species unrelated to the epitope's source organism and mimotope related assays where the immunogen or antigen have no structural similarity to the epitope.


BLANK

The Immunogen Epitope Relation may be left blank in certain circumstances. These will occur ONLY when the process type is selected as: Unknown, Occurrence of Cancer, Occurrence of Autoimmune Disease, and No Immunization.

Important Note: Remember that when the immunization procedure is unknown or uncertain, the in vivo process type is selected as Unknown and the Immunogen Epitope Relation is left BLANK.


Examples For all, peptides were synthesized utilizing the XXX accession ID of RSV A2


  • Epitope:
Source antigen = XXX accession ID
Source Organism = RSV A2
Epitope Evidence = Author provided identifier


  • Mice were immunized with RSV Strain Long
Immunogen Epitope relation = Taxonomic sibling
Organism = RSV Strain Long
Immunogen Evidence = exact match


  • Assay antigen was a protein from bovine RSV (monoclonal response)
Antigen Epitope Relation = Taxonomic sibling
Organism = Bovine RSV
Antigen Evidence = exact match

Taxr.jpg

Evidence Codes

Epitope Evidence Codes

These evidence codes are used to reflect the certainty of the accession ID, organism species, and strain that you are assigning as the epitope’s source.

  • These codes are hierarchical.


Author Provided Identifier

Use this one when the authors provide (or cite) a Uniprot/Genbank ID & the epitope sequence is found within that source.

Eev2.jpg


Exact Match to Reference Information

Use this selection when the accession assigned as the epitope’s source was the only possible match to the text.

Eev5.jpg

Representative Selection (based on incomplete info)

Use this selection when the accession assigned to the epitope is one of many matches to the text.

Eev4.jpg Eev6.jpg

Imperfect Match

Use this evidence code when the epitope sequence is found only within a source and/or organism strain that differs from exactly the source that the manuscript refers to. Do not assign sources across organism species. Can create needed strains so do so if needed.

Eev3.jpg

Internal Identifier-no external match available

This choice is to be made every time an internal IEDB source (SRC source) is selected.

Eev1.jpg


Antigen/Immunogen Evidence Codes

Ag/Imm evidence is based on how certain you are of what was tested as the antigen or what was used as the immunogen. There are cases where the authors will provide an accession ID or an exact strain of an organism as the antigen or immunogen. There are other cases where the exact details of the antigen or immunogen are unknown as with natural exposure to pathogens.

  • Relates to how certain you are of the entire object that you are saying was used in the assay.
  • These codes are hierarchical.


Author provided Identifier

Use this one when the authors provide a Uniprot/Genbank ID or Tax ID for the antigen or immungen.


Exact Match to Reference Information

Use this code when the accession ID or Tax ID assigned to the Ag/Imm are the only possible choices which match the text exactly.


Representative Selection (based on incomplete info)

Use this code when the manuscript does not provide the exact source name and/or organism name or when there are many potential matches to the text.


Imperfect Match

Use this code when the Ag/Imm sequence is found only within a source and/or organism strain that differs from exactly the source that the manuscript refers to or when the exact strain information cannot be captured. Do not assign sources across organism species.


Internal Identifier-no external match available

This choice is to be made every time an internal IEDB source (SRC source) is selected.


Selecting an Ag/Imm evidence code:

If the Ag/Imm is a peptide

Are you certain about the sequence? For example, Imm= “Epitope” Evidence = “exact match”

Are you uncertain about the sequence? For example, when a fragment of source Ag is used and the authors only stated the positions, or when there is an extra amino-acid added for conjugation that is left out of the sequence. Evidence = “Representative” or “Imperfect match”


If the Ag/Imm is a Protein

Usually is “Representative” b/c selecting from a number of potential similar proteins present within GenBank/Uni-Prot

Were you provided with an accession ID? If so, Evidence = “Author provided identifier”

Are you using an SRC for the Ag or Imm? Evidence = “Internal identifier”


If the Ag/Imm is an Organism

Do you know the exact strain used? Evidence = “Exact”

Is it a generic strain because you do not know the strain? Evidence = “Representative”

Are you using a different strain (with comments) because the one used was a rare clinical isolate? Evidence = “Imperfect match”


Follow these guidelines: Evi.jpg

Guidelines on MHC Alleles

Mutations Alleles that are mutated in the binding region in order to study the importance of residues in binding are not curated, but are commented on under the natural allele context (if present). Alleles that are mutated outside of the binding region are curated as the wild type allele. For example, a chimeric allele will be captured as the wild type of the binding portion.

Naturally occurring mutations in alleles are curated and should be added to the Allele finder if not present.

Chains When both α- and β- chains of HLA class II alleles are specified in the reference, both of them will be recorded.


Guidelines on Adoptive Transfer

The adoptive transfer fields are used to curate assays performed on effector material or antibody material derived from an animal which received an adoptive transfer of immune material.

Adt1.jpg Adt2.jpg Adt3.jpg Adt4.jpg Adt5.jpg


Bulk Curation

Bulk Curation refers to capturing multiple data points under one context record. Obviously this strategy is not ideal for all context records, but applies only to certain scenarios. When performing bulk curation, the overriding goal is to capture the authors’ conclusions. Different antigens, immunogens, and host organisms are never bulk curated. Assays giving different outcomes are never bulk curated. Different assays which measure the same type of response may be bulked curated. For example, different assay types measuring the binding of a mAb to the same antigen (WB & ELISA) can be bulk curated. Do not bulk curate assays which measure different responses such as the expression of different cytokines.

Bulking similar data is meant to capture the most relevant data while not creating redundant entries.

  • Bulking can be subjective.
  • Each reference should be treated as a whole.
  • Respect the authors intent.
  • Do not get caught up in assay optimization
  • The best way to bulk is to plan ahead!


Bulking by Fields

Fields to Always Bulk

Capture the most representative or the best protocol of those tested. Note the difference between assay optimization and truly different outcome.

  • Immunogen/Antigen containing object
Curate under the most positive carrier or vector and comment on all versions that were studied.
  • Adjuvant
Curate as the most positive or most frequently used. Comment on other procedures used.
  • Route
Curate as the most positive or most frequently used. Comment on other procedures used.
  • Dose schedule
Curate as the most positive or most frequently used. Comment on other procedures used.
  • Tissue type of effector and APC
Curate under the most positive response or under the most frequently used and comment on all APC/effectors studied.
  • Cell type of effector and APC
Curate under the most positive response or under the most frequently used and comment on all APC/effectors studied.
  • Restimulation immunogen
Curate the most positive or most frequently used. Comment on others. Note that immunization procedures with and without restimulation may be bulked
  • Sex
Curate the most positive or most frequently used. Comment on others.
  • Age
Curate the most positive or most frequently used. Comment on others.
  • Evidence codes
Curate the most positive or most frequently used. Comment on others.

Fields to Sometimes Bulk

These fields are more difficult to bulk, however, in some cases they should be bulked curated. They are also the most controversial fields to bulk. If you decide to bulk them, use comments to explain what assays were performed. Use the cover sheet to explain your bulking scheme. If you have doubts, ask other curators for their opinions before curating.

Special Note: If the only difference between potential assays is going to be a free text field, those assays should always be bulked.


  • Antibody Isotypes

Bulk when the response is the same. Always comment on the isotypes studied. If the authors specifically discuss the different isotypes and/or it is a significant point of the paper, curate isotypes separately. If different isotypes are equally studied and have the same response, bulk under the higher level (IgG) and comment on the specific ones studied (IgG1, IgG2a, etc). If a particular isotype was studied much more frequently, then curate that one and comment on the others.


  • Antigen Conformation

If antigen conformation is not the same, bulk under native. If antigen conformation is not the same and outcome is not the same, do not bulk. Fields to Never Bulk Different Immunogen always = new assay.


  • Different strains/Cross-reactivity

It can be difficult to decide when and how to bulk reactivity to different strains of the same organism. See the guidelines on cross-reactivity (##).


Fields to Never Bulk

  • Immunogen (different epitope relation)
Different immunogens should be curated separately. Note that sometimes strains of the same species and multiple peptide fragments can be bulked as they are considered essentially the same immunogen.
  • Antigen (different epitope relation)
Different antigens should be curated separately. Note that sometimes strains of the same species and multiple peptide fragments can be bulked as they are considered essentially the same antigen.
  • Host Organism species
Different species cannot be bulked. Different strains of the same species can be bulked in B cell assays and in T cell assays if they have the same MHC.
  • Different MHC in T cell assays
Be sure not to capture assays that are performed to determine restriction
  • Immunization in vivo and primary induction in vitro
Initial exposure to the immunogen in vivo and in vitro cannot be bulked.

Bulking of Assay Types

For all assay types:

  • Do not bulk different assays with different outcomes unless the difference is due to carrier, adjuvant, dosing ,etc.
  • In most cases, in vivo outcome and in vitro outcome should not be bulked.


T cell assays:

  • Do not bulk different cytokines. ICS, ELISA, bioassay all measure cytokine release and can be bulked if measuring the same cytokine.
  • Do not bulk assays that reflect differing responses may not be bulked-such as tetramer staining, CTL response, and proliferation.
  • Do not bulk in vitro assays with biological outcomes such as protection or exacerbation assays.


B cell assays

  • Bulk different assays measuring the same outcome (binding for ex) if antigen configuration is the same.
  • If bulking competition and direct binding assays with the same outcome, curate under competition. ELISA and western blot to show antibody binding may be bulked together under ELISA, both may be bulked under competition assay.
  • B cell protection assays and binding assays cannot be bulked.
  • Do not bulk biological outcomes such as neutralization, protection, etc with in vitro binding assays.


MHC Binding Assays:

  • MHC binding and MHC inhibition of binding (T cell response) cannot be bulked.
  • Separate MHC binding assays using either radioactivity or fluorescence could be bulked.

Cross-Reactivity

Before deciding whether to bulk reactivity across different strains of the same species, answer these questions:

Is the data meaningful or interesting? Is the epitope sequence present (exactly) in the different strains? What was the purpose of the studies?


Some useful guidelines:

1. If studies across strains with known differences in sequence were performed in order to map the epitope sequence or determine key residues, DO NOT CURATE the cross reactivity. Comment on the key residues or how the epitope sequence was mapped.

2. When the epitope sequence is known or inferred to be present within the other strains, you SHOULD BULK all negatives and all positives under one representative strain (one for negative and one for positive) with comments regarding the other strains that were studied.

3. In general, different strains and isolates with micro-variations in sequence SHOULD BE BULKED.

4. In general, with strains having macro-variations in sequence, DO NOT BULK.

Common examples: HPV strains Dengue serotypes Influenza subtypes HCV genotypes

5. Do not curate negative monoclonal or monospecific responses across different strains.


Cross-Reactivity Examples:

1. Epitope from Influenza A (no strain info),Assay antigens = Influenza A/PR8 (H1N1) and Influenza A/HK (H1N1)

Bulk both assay antigens under one context because they are of the same subtype. Curate one assay with comments.

2. Epitope from Influenza A (no strain info),Assay antigens = Influenza A/PR8 (H1N1) and Influenza Victoria (H3N2)

Do not bulk H1N1 and H3N2 because they are different subtypes. Curate two assays.

3. Epitope from Influenza A/PR8 (H1N1), Assay antigens = Influenza A unknown strain and Influenza A/PR8 (H1N1)

Bulk both under the known information as H1N1. Curate one assay with comments.

4. Epitope from Dengue 1, Assay antigens from DEN-1, DEN-2, DEN-3, DEN-4

Do not bulk because assay antigens are different serotypes. Curate 4 assays.

5. This example is from PMID 2578661

Anti-virus sera (FMDV type) O1 reacts with peptide O1, but does not cross react with the homologous from A10 or C1 serotypes (which have different sequences).

Example.jpg Example2.jpg

In this example, anti-peptide sera are considered to be monospecific and the study was performed in order to study cross-reactivity.

For each peptide as immunogen and antigen, curate every positive anti-peptide response. For example, with Imm= Peptide O1, there will be one assay with antigen =Peptide O1, one with antigen=Peptide A10, and one with antigen =Peptide C1.

For each peptide as immunogen and virus as antigen, curate every positive anti-virus response. For example, with Imm=Peptide O1, there will be three assays, one each for virus O1, virus A10, and virus C1.

For virus as immunogen and peptide as antigen, curate every positive anti-peptide response. For Imm=virus O1, there will be one assay with antigen=Peptide O1.


6. This example is from PMID 8178574

In this example, all reactivity is monoclonal and the assay was performed in order to map the epitope.

Bulk curate all of Table 1 under the wild-type sequence from which the epitope was derived as one curated assay (one antigen = wild type virus). Use comments to explain how the epitope sequence was mapped and what strains were recognized.

Example3.jpg

Common Cases for Bulk Curation

The following scenarios are commonly encountered cases for bulk curation and should always be curated in the same manner.

Mapping an Epitope

Truncation

When peptides are tested in the context of epitope mapping or minimal epitope analysis (residues are truncated to discover the minimal epitope) only the minimal epitope is entered. The other peptides that were part of the minimal epitope analysis are not curated. For class I epitopes only sequences of greater than or equal to 7aa are considered epitopes. Shorter sequences deduced from truncation analysis and not assayed directly as a peptide are considered "core residues" of an epitope of length defined by the shortest peptide used in the truncation experiment. In the figure below, only the epitope designated as D4-NS3 221-232 is curated.


CurationManual 07.gif
Protein Scan/Overlapping Peptides

Overlapping peptides tested as a part of a protein scan are curated differently depending on if the response is polyclonal or monoclonal.

Polyclonal responses All negative peptides are curated as separate epitopes. All positive peptides are curated under the minimal/optimal or deduced epitope whenever this is clear. If authors do not assert any particular epitope and there is no clear minimal/optimal epitope, then all positive peptides should be captured.


Monoclonal responses Negative peptides are not captured for monoclonal responses. All positive peptides are curated under the minimal/optimal or deduced epitope for the clone, line, or mAb whenever this is clear. If authors do not assert any particular epitope and there is no clear minimal/optimal epitope, then all positive peptides should be captured.

An example of this type of situation is shown in the figure below pasted from reference with PubMed ID 15356154. The regions I, II, and II would be curated under the optimal peptides deemed to be "the epitopes" for these regions. If, for example, the authors claim any value under 25% to be negative, every peptide with a value less than 25% would be curated for a polyclonal response. None of the negative peptides would be captured for a monoclonal response.


CurationManual 08.gif

Curating Contexts with Multiple Subjects

When the same assay is performed on samples derived from various subjects, the data will be entered as a single record. The main conclusion of the entry should be focused on an epitope-specific result rather than on the differences between various samples. In these cases the [Immunization] and [Assay] fields will capture the common information for all of the subjects, such as common MHC alleles present in the donor population. The Number of Subjects Immunized and Number of Subjects Responded fields are used to reflect the use of various subjects.

Mapping the MHC Restriction of an Epitope

Often the exact MHC restriction of an epitope is determined by using various APC/target cells with different alleles present. This type of data will not be captured as separate contexts; instead the MHC restriction will be inferred and entered for other contexts in the same reference.

For example, in the figure below pasted from reference with PubMed ID: 12023403), MHC restriction of a cell line is determined by testing multiple cell lines expressing different MHC alleles. In the same reference, the specificity of this dengue virus CD8+ CTL line was identified as a linear class I epitope. Other data in the reference is presented for the epitope along with a MHC restriction analysis. The figure below is not curated as a separate context, rather the MHC restriction will be inferred for other contexts and curated for the epitope identified from the dengue virus CD8+ CTL line.

CurationManual 09.gif

The same applies to all other assays solely performed in order to determine MHC restriction, such as the use of MHC specific antibodies.


Important Note: If a restriction is determined for a set of assay types, MHC classes, or species, do not apply that restriction to different assay types, MHC classes, or species. For example, if an epitope is determined to be H-2b restricted, do not curate an assay with a human host as having this murine restriction. If an epitope is shown to have a class II restriction, do not apply that restriction to an assay that measures a Class I type response.

Dose-Response Curves

Whenever a series of assays varies by only one variable, such as the dose of a peptide or adjuvant or the time of exposure, only the conditions giving the highest response will be curated with the other conditions noted in a comment.

T Cell Clones

Responses from multiple T cell clones will be bulk curated as one context when the T cell clones recognize the same epitope. For example, data in the figure below (pasted from reference with PubMed ID: 15033572) will be bulk curated and entered as one context for each epitope.

CurationManual 10.gif

Panel of Monoclonal Antibodies

When a panel of monoclonal antibodies is used to characterize binding to an antigen, the antibodies demonstrating comparable reactivities in an assay are bulk curated by entering all antibody names (comma delimited) in the Antibody Name field. A separate assay entry must be used for antibodies showing differing reactivities. When multiple classes or isotypes of antibody are encountered during bulk curation, the most relevant or most common antibody class/isotype used in the assay should captured in the drop-down menus for Antibody: Chain 1 isotype (and Chain 2 isotype, if it is known). The remaining information may be captured in the Comments on Assay field.

Decision Scheme for Bulk Curation

Use cross reactivity guidelines and relevance to the reference when applying novelty points.

When section (#Common Cases for Bulk Curation) (common cases for bulk curation) does not apply with multiple contexts that can be either curated separately or bulked together, follow the guidelines below:

Once one of the contexts is entered into the database a determination is made regarding the novelty of the remaining data. Points are given based upon the table below. When the sum of novelty points is greater than 2, the data will be entered as separate contexts. When the sum is 2 or less, the data will be bulk curated with a comment to capture the remaining information. Use Table 6 to determine the novelty points.

Table 6. Determining contexts to curate – Novelty Points
Difference between contexts Novelty Points
New Epitope Molecular Structure, Analog or Mimotope 3
New Epitope Source Organism / Protein 2
New immunized organism (host) 3
Antigen Epitope Relation / Immunogen Epitope Relation switches between peptide and "Source Antigen" or "Source Organism" 3
New immunized strain for T cell response 3
New immunized strain for B cell response 1
New qualitative outcome 2
New natural antigen / immunogen 2
New type of response measured 2
Difference in any other field except comment field 1

Analog data is bulked under the wild type sequence. Continue to bulk according to the analog guidelines. If the epitope is tested in a pool of peptides and tested alone, these assays may be bulked.

Negative Data

Monoclonal Receptors

Negative Immunogenicity data is not curated when the effector receptor (T cell or B cell/antibody) tested is monoclonal and there is positive data for the same receptor available. This is due to the implication that monoclonal receptors are highly specific.

Polyclonal monospecific

When a polyclonal response is deemed to be monospecific (specific to one epitope), the data will be curated as monoclonal. This occurs when the immunogen consists of one epitope or the polyclonal antibody is further purified by binding to one epitope prior to assay. Additionally, tetramer sorting or other selection by response to a single epitope can be treated as polyclonal monospecific.

Important Exception: Negative monoclonal data will be captured when the response is expected to be positive, but is negative and this lack of response is of interest.

Important Note: Negative data from T cell lines believed to be monoclonal will not be curated. Multiple passages and restimulation procedures are necessary to imply clonality of T cell lines. Short term cell lines will not be treated as monoclonal (unless otherwise demonstrated by the authors) and thus negative data generated by their use will be curated.

MHC Binding Data

Both positive and negative MHC binding data is always recorded. All MHC binding data is curated even when it is used to determine a minimal epitope.

Testing a Pool of Peptides

Section (#Pool of Peptides) applies. Negative data generated from pools of peptides is curated. Data from submissions like epitope discovery contracts should be encouraged to include quantitative information from testing pools of peptides, while for literature and patent curation, quantitative information is usually not recorded except for binding constants.

Special Issues Concerning Autoimmunity

Autoimmune Diseases

Diseases captured by the IEDB include, but are not limited to diabetes, rheumatoid arthritis, lupus, and multiple sclerosis.

In vivo Process Type

Ocurrence of Autoimmune Disease is the In vivo Process Type that is used when human study subjects have an autoimmune disease or are in the pre-disease state. Leave the immunogen field blank for diseases that occur spontaneously. In these cases, any in vitro process will be curated as "Primary Induction in vitro."

If an animal model of disease is induced by injection of an immunogen, capture the administration process and the immunogen as Administration in vivo. If the animal strain spontaneously acquires autoimmune disease, then use Ocurrence of Autoimmune Disease as the in vivo process type.


Important Note: Use the algorithm based upon the assay type to determine whether or not to use "Administration in vivo to cause disease".


Important Note: In autoimmune diseases, always curate the healthy controls as "No Immunization". In infectious diseases, do not curate unexposed controls, but in autoimmune contexts, the healthy controls express the same self antigens and are therefore relevant.


Human subjects

When curating human subjects, select the ICD-10 code that best reflects the disease state and its details. For example, use rheumatoid arthritis, unspecified M06.9 if authors are unclear, but always select seropositive rheumatoid arthritis M05 if authors specify. Always use immunization comments to fully describe the study population. Add comments such as if the donors are Anticyclic Citrullinated Peptide postive, C peptide positive, the time since diagnosis, and any criteria used to determine diagnosis. If authors use terms to describe the population that differs from the ICD-10 code, be sure to explain that in the Immunization Comments Field.


Animal models of Autoimmune Disease

When curating animal models, select the ICD-10 code of the human equivalent disease. For example, EAE is a model of human multiple sclerosis, so select the ICD10 code for MS. Be sure to always provide an immunization or assay comment that clearly explains what disease the animals have.

Important Note: Prediabetic mice (and humans) are to be curated as ICD 10 R73.0 Abnormal glucose tolerance test.

Tolerance and Treatment Assays

Tolerance Assays

This assay type is only to be used to capture a change in in vitro readouts such as ELISA, proliferation, cytokine production, etc. The typical situation is administration of one immunogen results in a positive in vitro response to the antigen = curate as "normal" assay (positive ELISA, for example). The study of tolerance is introduced when additional immunizations result in a loss of the previously positive in vitro readout.

Example: Administration of E1 results in proliferation in response to E1

Curate assay = IV1 Imm = E1, Assay type = Proliferation, Assay Ag = E1 (curate under E1)

Repeated administration of E1 results in loss of proliferative response to E1

Curate Assay = IV1 Imm = E1, IV2 Imm = E1 Assay type = Induction of Tolerance, Assay Ag = E1 (curate under E1)

Important Note: Must have assay comment: "Repeated administration of the epitope induced tolerance to the epitope as measured by loss of proliferation." Explain what caused the tolerance and how the authors concluded that tolerance was induced (loss of proliferation). Be sure to explain what the change was (either an increase or decrease).


Important Note: Only curate a tolerance assay for an epitope that was tested for the induction of tolerance. The assay comment should always be "The epitope did/did not induce tolerance.."

Example: Administration of E1 results in proliferation in response to E1

Curate assay = IV1 Imm = E1, Assay type = Proliferation, Assay Ag = E1 (curate under E1)

Additional administration of E2 results in loss of proliferative response to E1

Curate Assay = IV1 Imm = E2, IV2 Imm = E1 Assay type = Induction of Tolerance, Assay Ag = E1 (curate under E2)

The tolerance assay must be curated under E2 only. The assay comment will be "Administration of the epitope induced tolerance to the peptide as measured by loss of proliferation."


Treatment Assays

Treatment assays are curated when administration of the epitope in vivo prevents a disease from occurring, lessens the symptoms of a disease, or abolishes the symptoms of an established disease. Only curate this assay for the epitope responsible for the reduced symptoms. Use the in vivo process type of Administration in vivo to prevent or reduce disease with the immunogen being the epitope. Do not enter the disease state in the in vivo process where the epitope is administered. Only enter the disease state in the in vivo process that describes administration of the disease causing immunogen.

For example:

IV1 = Administration in vivo to cause disease IV1 Imm = hAChR IV1 Disease = Myasthenia gravis

IV2 = Administration in vivo to prevent or reduce disease IV2 Imm = epitope no disease state

Assay comment = "Administration of the epitope significantly reduced the symptoms of experimental autoimmune myasthenia gravis induced by hAChR".


Curate the prevention of disease and the treatment of disease separately.


For Example:

If Epitope is tested for the prevention of of a disease:

IV1 = Administration in vivo to prevent or reduce disease

IV1 Imm = epitope

IV2 = Administration in vivo to cause disease

IV2 Imm = hAChR

IV2 Disease = Myasthenia gravis

vs

If Epitope is tested for the treatment of an existing disease:

IV1 = Administration in vivo to cause disease

IV1 Imm = hAChR

IV1 Disease = Myasthenia gravis

IV2 = Administration in vivo to prevent or reduce disease

IV2 Imm = epitope

Curate those two situations as two separate assays. Do not bulk.

Important Note: If the epitope is administered to both cause a disease and treat a disease, capture both in vivo processes separately and in the order in which they occurred, even though the two immunogens will be the same. This is an exception to the rule that IV1 and IV2 must have different immunogens.

For example:

IV1 = Administration in vivo to cause disease IV1 Imm = epitope IV1 Disease = Myasthenia gravis

IV2 = Administration in vivo to prevent or reduce disease IV2 Imm = epitope no disease state

Assay comment = "Administration of the epitope significantly reduced the symptoms of experimental autoimmune myasthenia gravis induced by the epitope".

The above experiment will have two assays curated for the epitope: 1.Exacerbation assay and 2. Treatment assay.


Assay Antigen In autoimmune treatment and exacerbation assays, there is no clear assay antigen because the immune response occurs in vivo with the readout being either more or less disease. In these cases, the assay antigen will be curated as the host's equivalent of the epitope's source antigen. This means if the epitope is from insulin and the host is a mouse, the assay antigen is mouse insulin. If the epitope is from AChR and the host is a human, the assay antigen is human AChR.


Important Note: These assays must always have an assay comment to explain what happened. The assay type and the assay antigen will not be clear to end users and must be explained. Always state what was administered and why and what was the outcome of the administration. For example, "The epitope was administered to lessen the severity of EAE. Decrease in disease was measured by decreased paralysis, increased body weight, and overall decrease in disease score."

Exacerbation Assays

Only curate an exacerbation assay for an epitope if that epitope causes a disease, induces symptoms, or increases symptoms, when administered in vivo. Use Administration to cause disease as the in vivo process type. Enter the disease state and stage of the disease induced by the epitope in the same in vivo process where the epitope is administered. If more than one immunogen causes disease, use two separate in vivo process types. For example;

IV1 = Administration to cause disease IV1 Imm = human AChR IV1 Disease = Myasthenia Gravis

IV2 = Administration to cause disease IV2 Imm = epitope IV2 Disease = Myasthenia Gravis

Assay comment = "Administration of the epitope increased the symptoms of hAChR induced experimental autoimmune myasthenia gravis as measured by clinical score."


Important Note: In these assays, there must always be an assay comment explaining the effects of the epitope and what was measured.

Special Issues Concerning Allergy

Allergic Diseases

Subjects with existing allergy are curated using the in vivo process type of Occurrence of Allergy with the Disease State of Allergy to Allergen. Enter the Allergen that the subjects are allergic to in the Immunogen fields. When the allergen is unknown or vague, a less specific Disease State may be used such as Allergy, Unspecified [U37] and the Immunogen field is left blank.


For Celiac disease patients, use in vivo process type of Occurrence of allergy and enter the disease state of Celiac disease. For the immunogen, select Derivative or Organism = gluten.

Other disease encountered include: J45.0 Allergic asthma and L20 Atopic dermatitis.

Allergens

Many allergens are not single proteins, but are instead complexes or mixtures such as pollens, milk, venom etc. In order to capture allergens such as these, use the object type Derivative of Source Organism and select the correct allergen type from the drop down menu. For cross reactive allergens, use Derivative of Organism after selecting the taxonomic or structural relationship between the allergen and the epitope.


Allergy Assays

Tolerance, Treatment, and Exacerbation Assays are very common. See the section on autoimmune diseases #Special Issues Concerning Autoimmunity for details.


Delayed Type Hypersensitivity (DTH) assay is common. This assay is performed by skin exposure to an allergen followed by a DTH skin reaction that is measured after a second exposure to the same or a related allergen. This assay is only curated when there are two exposures. In some cases, the first exposure may have been prior to the study, as with human subjects. Do not curate a skin reaction to an irritant. The DTH assay must be reflective of a T cell immune response.

Miscellaneous

Handling Special Characters

Special characters (Greek letters, symbols) entered in database fields are recorded using special codes. A complete list of special characters and its respective code to be recorded can be found at

http://www.visibone.com/htmlref/char/ceralpha.htm

For example, if a particular word contains ã or æ or λ, you will need to type the corresponding code ã or æ or λ

Superscripts and subscripts require a special format for input. To enter a subscript, type <sub>''text''</sub>. Likewise for superscripts, type <sup>text</sup>. For more assistance in capturing special characters, please contact a senior curator.


Modified Amino Acids

The Modified Residues field is used to describe modified amino acids (naturally-occurring, post-translational modifications, or chemical/synthetic modifications). Here is an example for an N-formylated peptide:

Figure 2. Describing N-formyl Peptides (PubMed ID: 11145694).

Do not use designate the formyl-methionine residue as "fM" as shown in Figure 2. Instead, enter the N-formylated peptide sequence information as illustrated below (Table 4 ):

Table 4. Example for Entering Modified Amino Acids
Field Text
Linear Sequence MIGWII
Modification(s) <Formylation |FORM>
Modified Residue(s) M1

In the Modified Residue field, use the single letter amino acid code followed by the position of the residue in the epitope at it is captured in the Linear Sequence field. IEDB conforms to SWISSPROT when capturing post-translational modification types (http://www.expasy.org/tools/findmod/findmod_masses.html). For other modification types, it is added to the drop-down as specified in the reference after consultation with a senior curator.

Important Note: In the event a reference describes the use of an unmodified epitope and additionally that same epitope in a modified form that is unnatural (glycosylated, ubiquitinated, etc), the modified epitopes are to be curated following the rules used to curate analogs (#Analogs). They will not be captured as a separate epitope entry unless the criteria mentioned in (#Analogs) is met. If a peptide is naturally modified, it is a natural peptide (not an analog) and should be curated following normal curation rules.

Important Note: Do not guess the correct modification to use. If the exact term that the authors used is not present, refer to a senior curator regarding synonyms for modifications or the need to add new terms to the drop down list.

Crystal Structure/NMR Assays

3-D structural data is the domain of experts at SDSC. It is advised that all references containing 3D-structural data should be discussed with a senior curator to determine the curatability.