B Cell Epitope Mapping

There are two important aspects to consider when looking for potential B cell epitopes. The first is the primary amino acid sequence of the protein and the second is the conformational structure of the protein. The surface of a protein is not a simple string of amino acids, but a complex of loops and folds determined by the interaction between side chains of the residues.

REVEAL™ B Cell Linear Epitope Mapping

ProArray™ Peptide Microarrays

Prospector™ Custom Peptide Libraries

REVEAL™ B Cell Epitope Prediction Service

Peptide Applications Overview

There are two main classifications of B cell epitopes:

• Linear, or continuous, epitopes are defined by the primary amino acid sequence of a particular area of a protein. The surfaces which interact with the antibody are situated next to each other sequentially on the protein.

• Epitopes that are defined by the conformational structiure of the native protein. These epitopes may be continuous or discontinuous, i.e. components of the epitope can be situated on disparate parts of the protein, which are brought close to each other in the folded native protein structure.

This makes B cell epitopes more difficult to identify than T cell epitopes which are determined mainly by the primary amino acid sequence.

The primary amino acid sequence is known and is readily available for a large number of proteins. The 3-dimensional crystallographic structure has been identified for relatively few proteins, and X-ray crystallography is is a difficult and time consuming method to use. To date, most B cell epitope mapping experiments have made use of linear peptide fragments from antigenic proteins. These peptides can be homologous enough to parts of the whole antigen in order to allow antibody binding.

There are two pre-requisites to identifying linear B cell epitopes. Firstly the amino acid sequence of the protein must be known. The sequences of a wide range of proteins are available on the NCBI website using the basic local alignment search tool (BLAST) (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Secondly there must be a specific antibody available for the antigen of interest. These can be monoclonal or polyclonal antibodies or sera from animals or patients.

An overlapping peptide library can be generated to isolate the antibody-binding region of the protein. B cell epitopes do not have a defined length and can vary from 5 to 20 amino acids in length. One approach would be to set a defined peptide length, e.g. 15-mer, and to generate a library of peptides with a uniform overlap e.g 3 amino acids. In this way 'hot-spot' areas of the protein can be identified and the optimal epitope can be found using further experiments with truncated peptide libraries or individual custom peptides. See Peptide Design for further information.

There are two main peptide based assays that can be used to identify linear B cell epitopes; enzyme linked immunosorbent assay (ELISA) and inhibition assays.

ELISA

ELISA is the standard method used when trying to identify linear B cell epitopes. The principle behind this assay is as follows:

• Biotinylated peptides are coated onto a streptavidin-coated plate

• The antibody (or serum>) to be tested is diluted appropriately and added to the plates

• An HRP-conjugated anti-species antibody is added to bind to the antigen-specific antibody/serum

• HRP is detected using a coloured substrate and analyzed using a standard plate-reader

The data generated from ELISA protocols depends upon the quality of the antibody that is being tested. The clearest results are obtained from monoclonal antibodies which, by their very nature, respond to only one epitope. With polyclonal antibodies, there is the possibility that antibodies that bind to specific peptides may be recognizing denatured antigen or cross-reacting with an unrelated antigen. Sera samples, which can contain multiple antibodies, often give very weak signals and may give responses to several peptides. Whilst the data generated from experiments with polyclonal antibody or serum samples may initially be confusing, this information can provide a good indication of the best areas of the antigen on which to carry out further investigations.

Inhibition Assays

When ELISA assays are carried out using serum or a polyclonal antibody, there can be responses to multiple peptides some of which may be denatured or irrelevant peptides. It may be necessary to carry out further experiments to determine the true epitope. An inhibition assay uses soluble antigen to block the binding of the antibody to the peptide. The assay is still based on the standard ELISA principle but includes the addition of a specific blocking peptide.

• Biotinylated peptides are coated onto a streptavidin-coated plate

• Serum is added to the plate with an increasing dilution of antigen (blocking peptide)

• An HRP-conjugated anti-species antibody is added to bind to the antigen-specific serum

• HRP is detected using a coloured substrate and analyzed using a standard plate-reader

An inhibition of the serum binding to plate-bound peptide with increasing concentration of blocking peptide, indicates that the correct linear epitope has been identified. An irrelevant blocking peptide will have no competitive effect on the antigen binding.

Clearly this methodology can take a great deal of time to carry out and will also use up a large amount of reagent. For this reason, the best method is to narrow down the potential epitopes using an initial ELISA assay and then confirm the correct epitope using an inhibition assay.

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