Antibody Epitope Mapping

Regions on the antigen recognized by the antibodies are called epitope, and the respective molecular counterpart of the epitope on the monoclonal antibodies (mAbs) is called paratope. Antibody epitope mapping is the process of identifying the binding site, or epitope on its target antigen, usually a protein. This process is crucial in the discovery and development of new therapeutics, vaccines, and diagnostics. Antibody epitope mapping can be performed using various techniques such as mass spectrometry, peptide microarrays, and phage display. The identification and characterization of epitopes can help elucidate the binding mechanism of an antibody and can strengthen intellectual property protection. Experimental epitope mapping data can also be incorporated into robust algorithms to facilitate in silico prediction of B-cell epitopes based on sequence and/or structural data.

Epitopes are generally divided into two classes: linear and conformational/ discontinuous. Linear epitopes are formed by a continuous sequence of amino acids in a protein, while discontinuous epitopes are formed by amino acids that are nearby in the folded 3D structure but distant in the protein sequence. Note that conformational epitopes can include some linear segments.

Both linear and discontinuous epitopes have their own strengths and weaknesses, and the choice of which to use depends on the specific application. Linear epitopes are easier to synthesize and more stable, while discontinuous epitopes are more complex and more immunogenic. However, discontinuous epitopes are also more prone to conformational changes, which can affect their binding affinity and specificity

Fusion Biolabs provides antibody epitope mapping using three strategies:

I. Antibody Epitope Mapping Using Random Peptide Phage Display Libraries

Small peptides (approximately 16-24 amino acids) with random sequences are displayed and used to perform panning against the studied mAb. Phage libraries displaying millions of peptides with randomized sequences are extremely useful tools for mapping antibody epitopes. Fusion Biolabs has created random peptide libraries by inserting short synthetic oligonucleotides with a completely randomized sequence usually at the engineered 5’-end of gIII gene. Please see page Antibody Phage Display Library Panning Services for details. Huge libraries with about 10¹⁰ individual phages, each containing a unique nucleotide sequence, have been generated. A single phage then provides a unique peptide sequence displayed as fusion to the engineered coat protein gIII.

In many cases, antibodies are able to select peptides with reasonable affinity for their combining sites (paratopes) from these libraries. Ideally, consensus motives can be deduced from multiple peptide sequences and matched to areas of the antigen against which the antibody was raised, and thus critical components of the antibody epitope can be defined.

However, since these peptides are random and not directly related to the actual target antigen protein, the resulting sequences show conserved properties, but have to be analyzed carefully to determine the corresponding parts on the target.

The main procedure includes:

1. Peptide phage display library preparation and functional confirmation
2. Affinity selection of Antibody-binding phage
3. Monoclonal phage ELISA
4. Binding phage sequencing
5. Raw data analysis, epitope determination (full project report)

II. Antibody Epitope Mapping Using Antigen Overlapping Peptide Phage Library

In this strategy, using libraries with sequences of a single target (called SINGLE-GENE library), can provide more reliable information regarding the recognized epitope, since parts of the antigen can be directly defined as the epitope without further complex analysis. This is very cost-effective for large molecular target/protein.

The main procedure includes:

1. Antigen library construction and packaging: gene amplification, fragmentation, end-repair, phagemid-fragment ligation, library construction and packaging;
2. Antigen panning against the studied mAb;
3. Monoclonal phage ELISA screening;
4. Selection and sequencing of positive hits;
5. Raw data analysis, epitope determination (full project report).

III. Antibody Epitope Mapping Using Overlapping Synthetic Peptide Library of Antigen

For small molecular target/protein, this strategy based on direct ELISA approach is considered a relatively straightforward, rapid, practical and cost-effective technique in identifying epitopes, specially linear epitopes.

The main procedure includes:

1. Overlapping biotin-labeled peptide library design and preparation
2. ELISA using peptide via streptavidin coated 96-well plate
3. Raw data analysis, epitope determination (full project report)

Deliverables

a) Phage peptide library panning raw data /ELISA raw data

b) Antibody epitope mapping analysis data

c) Antibody epitope mapping detail project report

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Antibody Epitope Mapping Services

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