How EPITOPIC’s antibody epitope fingerprinting works
The most common question of our customers is:
What is special about this approach and why do other approaches often fail?
A: A specially designed library contains a large variety of sequences. Due to the special design and care in the generation, the peptide phage library does not contain any sequence in unusual high numbers, even when the library is replicated. This is allowing the following straightforward approach, which will fail, when other starting libraries are used.
B: Only minimal amounts of antibodies are required. The selection and enrichment of binders requires theoretically not more than a few hundred antibody molecules, so even the epitopes of antibodies in a serum can be identified.
C: For each epitope up to thousand variants of 16-mer sequences containing the epitope motifs are enriched in a single step. Even a minimal enrichment is sufficient for the following steps since enrichment will not be detected on the base of single sequences but a pool of similar sequences.
D: NGS is error prone. With several specially developed algorithms, we can sort out sequencing errors. In our set up with illumina sequencers up to 30% of the obtained single sequences are expected to contain errors and are removed prior to the following analysis. This avoids unnecessary and expensive oversampling, which would multiply the project costs.
E: Data sets with up to a million peptides containing sometimes only a few hundred enriched sequences with epitope motifs are generated by translating the peptide genes.
F: Statistical fishing the epitope sequences out of these large data sets is done with the help of a proprietary software that allows to pick continuous and discontinuous peptides motifs resembling the antigen. Surrounding amino acids of the 16-mer peptides often support special peptide epitope conformations required for binding to the antibody.
G: Epitope fingerprints obtained combining the information from many peptides allow to understand not only the location of the epitope but also the individual preferences of antibodies with respect to surrounding or intermitting amino acid residues. This helps to predict specificity with respect to bound protein in comparison to related or non-related proteins from the host proteome or other species. This is unique since no additional experiments are initially required.
Header – highlighted epitope sequence (S)IYTSDnyxeE of anti-mouse CD184 antibody/CXCR4 (BD Pharmingen, Nr. 551966) in CD184 structure