Epitope Mapping by Label-Free Biomolecular Interaction Analysis

Abstract

The diversity of B-cell response to a large immunogen gives rise to a series of antibodies that can be used for epitope mapping of an antigen. This is based on the relative reaction pattern for all antibodies in relation to each other and other ligands to the studied protein. With the introduction of an instrument system, BIAcore, label-free real-time biomolecular interaction analysis (BIA) was made possible. It is based on biosensor technology, with a carboxymethyl-dextran-coated gold surface and an integrated fluidics for transport of liquid. The basic idea is to measure label-free binding of an analyte from a continuous flow to an immobilized ligand in real time. With an automatic approach, quantitative analysis and sequential injection characteristic biospecific binding parameters such as affinity and kinetic constants can be measured. The instrument system was adopted at an early stage for epitope mapping. With label-free detection, antibodies from tissue culture media can be analyzed without purification. Binding of both antigen and a series of antibodies can be individually determined in molar ratio by sequential injections. The quantitative aspects of BIA offer the possibility of further refined epitope mapping. The relative binding pattern for 30 monoclonal antibodies against HIV-1 p24 core protein has been analyzed. Multideterminant analysis and peptide identification of binding sites were performed. Verification of the binding pattern has also been performed in relation to mapping with ELISA as well as the binding to peptides derived from the antigen sequence. Functional domains of proteins in relation to an epitope map have been identified forTaqpolymerase.