How would you map the epitope of a new monoclonal antibody?

Why

Epitopes come in two kinds. A continuous epitope is a stretch of sequence, and a discontinuous one is formed by residues brought together by folding. Most are discontinuous, and reviews note that over 70 percent of discontinuous epitopes are built from one to five short linear segments.

That shapes method choice. Peptide arrays and fragment binding find continuous epitopes cheaply but miss conformational ones, and antibodies raised against peptides often fail to bind the folded protein. Alanine scanning substitutes residues one at a time and shows which side chains contribute most to binding, though a loss of signal can also mean the mutation disturbed the fold.

Shotgun mutagenesis tests many mutants on natively folded protein in cells and can map both kinds of epitope. For definitive answers, X-ray crystallography of the complex is often regarded as the only way to define a structural epitope, limited by whether crystals can be grown. NMR works for small antigens, under about 25 kDa, and hydrogen-deuterium exchange or electron microscopy give moderate resolution on larger ones.

How to work it through

1. Write down the question: is a binding region enough, or are contact residues needed.
2. Test binding to domains or fragments to localize the region first.
3. Run competition binning against antibodies with known epitopes to group yours.
4. Use alanine or shotgun mutagenesis on the candidate region, with folding controls for every mutant.
5. Confirm with a structural method matched to the antigen's size and crystallizability.
6. Report the epitope with the method and its resolution rather than as a list of residues alone.

Where the answer stops holding

Mutagenesis can report indirect effects when a substitution destabilizes the antigen. Peptide-based methods miss conformational epitopes entirely. A crystal structure captures one conformation and a purified antigen, which may differ from the target on a living cell.