Epitope analysis using kinetic measurements of antibody binding to synthetic peptides presenting single amino acid substitutions

Abstract

The fine modulation of peptide-antibody interactions was investigated with anti-peptide monoclonal antibodies recognizing peptide 125-136 of the coat protein of tobacco mosaic virus. Nine synthetic peptides presenting single amino acid substitutions were selected for detailed analysis on the basis of their reactivity in ELISA. Kinetic measurements of the binding of four antibodies to these peptides performed with a biosensor instrument (BIAcore, Pharmacia) were used to quantify the contribution of individual residues to antibody binding. The results showed that even conservative exchanges of some residues in the epitope resulted in a small but significant decrease of the equilibrium affinity constant due mostly to a higher dissociation rate constant of the monoclonal antibodies. Two amino acid residues directly adjacent to the epitope, which appeared to play no role when tested by ELISA, were shown to influence the kinetics of binding. These data should be useful for computer modelling of the peptide-antibody interactions.

Instrumental biosensors: new perspectives for the analysis of biomolecular interactions

“…In epitope mapping studies of peptide 125-136 of the coat protein of tobacco mosaic virus, it was shown that two amino acids directly adjacent to the epitope, which appeared to play no role using ELISA, significantly influenced the binding kinetics. (59) Kinetic evaluation. Since kinetic parameters can be determined from SPR signals, this technology has been used extensively to characterise, evaluate, and select monoclonal antibodies for specific uses.…”

Antigenic mimicry of natural L-peptides with retro-inverso-peptidomimetics.

“…The ELISA procedure (direct binding and competition experiments) was as described (9). For real-time binding experiments, a BIAcore biosensor system (Pharmacia Biosensor) was used (16)(17)(18)(19). To immobilize peptides to the sensor chip, the standard procedure described (20) for cysteine-containing peptides was used.…”

“…Direct binding experiments and competition assays were performed as described (17). mAbs 4x11, 11x2, and 11x7 (200 nM) were incubated with the competitor peptides used at a molar excess of 1.75-800 over antibody.…”

“…mAbs 4x11, 11x2, and 11x7 (200 nM) were incubated with the competitor peptides used at a molar excess of 1.75-800 over antibody. Antibody kinetic constants were measured as described (17).…”

Homogeneous Two-Site Immunometric Assay Kinetics as a Theoretical Tool for Data Analysis

“…Moreover, with the recent emergence of several kiAnother possible use of kinetic information could consist in a flexible system, involving both an early netic biosensors, kinetic rates are being considered more and more in a variety of biological research applidetermination of S m and an end-point reading. Depending on some threshold values on S m , or on its preci-cations (13,(40)(41)(42). The solid-phase format of most of these methodologies hinders the interpretation of the sion of estimation, the reaction could either be stopped early if antigen concentration was determined with S m results, and precludes the extrapolation from those results to solution situations (6,13,15).…”