Formaldehyde-mediated peptide coupling for the titration of epitope-specific antibody in an ELISA format

Synthetic peptides are commonly used as an immobilised substrate in ELISA to measure antibody responses in serum. However, in addition to poor adsorption on the plastic surfaces of plates, titration of antibody response to a specific epitope of interest is challenging because the majority of antibodies in serum may be directed to other regions of the antigen. Using Ras peptides containing either the G12V or G12D mutation as a model antigen, we have tested formaldehyde as a crosslinker to immobilise Ras peptides on the plastic surface of microtitre plates in the presence of bovine serum albumin (BSA) as a carrier. Using this method, specific antibody against the G12V mutation was titrated from a simulated rabbit serum. Non-cognate Ras peptides were included in the ELISA reactions to suppress binding against non-mutated regions of the peptide. Our results showed that formaldehyde enhanced in-well peptide immobilisation of peptide approximately 3-fold and this enhancement required addition of BSA as a carrier. Wild type Ras peptide was not ideal as a non-cognate competitor as it tended to inhibit specific antibody binding against G12D or G12V. In the presence of the non-cognate peptide containing G12D, concentrations of the anti-G12V antibody in a simulated serum were determined with approximately 95% accuracy. Our method will be useful to determine a specific antibody response against a certain epitope in various peptide-based immunotherapy trials.