Epitope mapping

Epitope mapping is the process of identifying the binding sites, or ‘epitopes’, of antibodies on their target antigens (which are proteins).

Identification and characterization of the binding sites of antibodies can aid in the discovery and development of new therapeutics, vaccines, and diagnostics.^[1][2]

Epitopes (the binding sites on the protein) can be divided into linear and conformational. Linear epitopes are formed by a continuous sequence of amino acids in a protein, while conformational epitopes are composed of amino acids that are discontinuous in the protein sequence but are brought together upon three-dimensional protein folding. The vast majority of antigen-antibody interactions have conformational epitopes.^[3]

Methods for epitope mapping

Epitope mapping of complex target antigens, such as integral membrane proteins or multi-subunit proteins, is often challenging because of the difficulty in expressing and purifying these types of antigens.

There are several methods available for mapping antibody epitopes on target antigens :

• The gold standard approach is X-ray co-crystallography, which allows direct visualization of the interaction between the antigen and antibody. However, this approach is technically challenging, requires large amounts of purified protein, and can be time-consuming and expensive.

• Array-based oligo-peptide scanning (sometimes called overlapping peptide scan or pepscan analysis): This technique uses a library of oligo-peptide sequences from overlapping and non-overlapping segments of a target protein and tests for their ability to bind the antibody of interest. This method is fast and relatively inexpensive, and specifically suited to profile epitopes for large number of candidate antibodies against a defined target.^[4][5] By combining non-adjacent peptide sequences from different parts of the target protein and enforcing conformational rigidity onto this combined peptide (such as by using CLIPS scaffolds^[6]), discontinuous epitopes can be mapping with very high reliability and precision.^[7]

• Site-directed mutagenesis: Using this approach, systematic mutations of amino acids are introduced into a protein sequence followed by measurement of antibody binding in order to identify amino acids that comprise an epitope. This technique can be used to map both linear and conformational epitopes, but is labor-intensive and slow, typically limiting analysis to a small number of amino acid residues.

• Mutagenesis Mapping.^[8] This approach utilizes a comprehensive mutation library, with each clone containing a unique amino acid mutation and the entire library covering every amino acid in the target protein. Amino acids that are required for antibody binding can be identified by a loss of reactivity and mapped onto protein structures to visualize epitopes.^[9] This approach has recently been used to epitope map a panel of antibodies against human CCR5, a GPCR coreceptor for HIV entry.^[10]

• A method growing in popularity is Hydrogen–deuterium exchange which gives information about the solvent accessibility of various parts of the antigen and the antibody, demonstrating reduced solvent accessibility where protein to protein interaction occurs.

• Other methods, such as phage display, and limited proteolysis, provide high throughput but lack reliability, especially for conformational epitopes.^[3]

References

External links

• Epitope mapping at the US National Library of Medicine Medical Subject Headings (MeSH)