REVEAL™ Rapid B Cell Linear Epitope Mapping Service | ProImmune

REVEAL™ B Cell Epitope Discovery Services

Rapid B Cell Linear Epitope Mapping

Mapping linear epitopes can be done quickly with well-defined, easily controllable, high-throughput technologies. Although not all conformational epitopes are resolved, mapping linear B cell epitopes can be a quick way of getting a relative ranking of antigenic regions within proteins and between different proteins in a group.

Our service identifies the specific linear peptide segments from any larger protein that bind to antibodies in a given sera or plasma sample submitted by the client, using rapid synthesis of peptide libraries and immunoassay technology.

Application notes for B cell epitope mapping

Peptide libraries for epitope mapping

Peptide arrays for epitope mapping

Protocol for the preparation of blood plasma and serum (PDF)

B cell epitope prediction service

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B Cell Epitopes

There are two main classifications of B cell epitopes:

Linear, or continuous, epitopes are defined by the primary amino acid sequence of a particular region of a protein. The sequences that interact with the antibody are situated next to each other sequentially on the protein.
Epitopes that are defined by the conformational structure 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.

Conformational epitopes are thought to form the majority of strong antibody binding epitopes on most proteins. However numerous conformational epitopes may also be recognizable as linear epitopes, although with lower affinity. Linear B cell epitopes typically vary from 5 to 20 amino acids in length. If the antibody-binding region of the protein is unknown, an overlapping peptide library can be generated using a specific peptide length and offset, for example, 15mers offset by 5 amino acids, i.e. overlapping by 10.

Mapping linear epitopes in a protein has the following benefits:

It can be done quickly with well-defined, easily controllable high throughput technologies, which include standard rapid synthesis of overlapping peptide libraries and standard immunoassays.
Although not all conformational epitopes may be mapped, linear B cell epitope mapping can be a quick way of getting a relative ranking of antigenic regions within proteins and between different proteins in a group.

Purpose of the REVEAL™ Service

Using the REVEAL™ epitope mapping service, the specific linear protein segments from any larger protein or peptide, that bind to antibodies in a given sample such as sera or plasma, can be identified. In this way, 'hot-spot' areas of the protein can be discovered. Alternatively, if these areas of the protein sequence are already defined, the optimal epitope sequence can be elucidated using REVEAL™ with a peptide library with an offset of 1, or with a truncated peptide library, or with a series of peptides with single amino acid substitutions. Peptide Library Design.

Applications

Definition and ranking of individual linear epitopes in an antigen

Although protein antigens may contain both linear and discontinuous epitopes, detailed knowledge of the linear epitopes alone may be a useful means of comparing the antigenicity of candidate proteins.

Identification of peptides for immunization

If a strong candidate linear epitope can be found in a protein antigen, this short peptide sequence can be used to produce polyclonal or monoclonal antibodies against the target protein, with the advantage that the binding sequence in the protein will already be known.

Discovery of cross-reactive determinants between related proteins

Determining a linear epitope that occurs on several related proteins gives useful information about the potential cross-reactivity of immune responses between these proteins.

Definition of strongly antigenic regions in proteins

Clusters of linear epitopes may indicate regions of elevated antigenicity and activity in a protein and may be useful in identifying protein sub-units relevant to the research application. For example, a sub-unit of a protein may confer most of the antigenicity of the whole protein and be easier to manufacture than the whole protein. In addition, to reduce the risk of unwanted side effects in vaccination it may be of benefit to use a sub-unit of a protein that has fewer epitopes that are cross-reactive with self-proteins.

Comparison of different serovars or subtypes in terms of their relevant antigenicity

Comparative results from linear mapping using peptides with sequence variation can shed light on the antigenicity of proteins from different strains of an organism. This application may also show whether vaccination produces an immune response that is relevant across a number of serovars or subtypes.

Combining linear epitope mapping with epitope prediction

Comprehensive B cell epitope prediction and linear epitope mapping can be carried out when both the 3D structure and the protein sequence are known. However, the 3D structure is not available for many proteins, especially transmembrane proteins, which are particularly difficult to crystallize. In vaccine development, transmembrane proteins are of special interest when they have a domain on the surface of the pathogen that could be bound by antibodies. Linear epitope mapping alone, or combined with epitope prediction, can give an insight into the antigenicity of transmembrane proteins.

The REVEAL™ B Cell Linear Epitope Mapping Service In Detail

The REVEAL™ B Cell Epitope Mapping Service uses standard immunoassay methodologies to screen plasma or sera samples submitted by the customer. Based on the proteins of interest, ProImmune can design and synthesize an appropriate peptide library for the project. Our services include cost-effective solutions for a wide range of project requirements: e.g. from a handful of peptides screened against a similar number of antibody samples, to hundreds or even thousands of peptides screened against hundreds of antisera using ProArray™ Custom Peptide Microarrays.

Biotinylated custom peptides are bound to a microtiter plate and plasma or sera samples are added at dilutions of 1:200 and 1:400, along with controls to determine background signal. Peptides that are bound by antibodies in the samples are detected using a horseradish peroxidase-conjugated polyclonal antibody, and visualized by chemiluminescence using a standard plate-reader.

The customer receives a report outlining the methods used and all results from the REVEAL™ assay, proposing, ranking and discussing any predicted epitopes.

Data generated from the REVEAL™ assay will depend upon the quality of the antibody being tested. It should be noted that sera and plasma samples sometimes contain multiple antibodies, which may give weak signals and/or bind to several different peptides. The data generated from such samples can still provide good information about which regions of a protein sequence are most antigenic, and highlight areas on which to carry out further investigation.

Discontinuous epitopes that depend on the correct 3D conformation of the protein, may not be detected by linear epitope mapping, or may only give a weak signal. If the 3D structure of the protein is known, it may be possible to synthesize reconstructed discontinuous epitopes as linear peptides to be included in the REVEAL™ assay.

Example of B cell epitope mapping experimental data

Figure 1: Example of B cell epitope mapping experimental data. A library of overlapping peptides derived from the sequence for Hepatitis B surface antigen was screened for binding with plasma samples with and without reactivity to whole antigen. Red bars indicate samples with positive reactivity to whole antigen and blue bars indicate samples with negative reactivity to whole antigen. Strong binding was detected with positive samples in the regions represented by peptides 58, 59 and 63, suggesting that these sequences contain linear epitopes. The sequence corresponding to peptides 58 and 59 overlaps with epitopes published by Ijaz et al. (2003, J Gen Virol 84:269-75) and Cousaget et al. (1991, Res Virol 142:461-7). Additionally, both sequences overlap with epitopes published by Chen et al. (2003, World J Gastroenterol 9:304-8) and Heijtink et al. (2001, Vaccine 19:3671-80) and are listed in the Immune Epitope Database.