Affinity characterization of monoclonal and recombinant antibodies for multianalyte detection with an optical transducer

The selectivity of immunoassay is limited by the cross-reactivity of antibodies to structurally related analytes. This becomes a drawback for applications that require discrimination of slightly different analytes. An approach to overcoming this problem is the application of antibody arrays that show differences in their affinity patterns. We have investigated this method using systematic modelling of multianalyte systems based on test-independent affinity parameters. A model system of anti-s-triazine antibodies and s-triazine derivatives has been investigated. The immunoassay is carried out in an indirect test format using an optical transducer for label-free monitoring of antibody binding at an immobilized hapten. The concentration of free antibody in equilibrium with the analyte is probed in a flow-through system. This format allows simple modelling of the response and assessment of the affinity constant from the calibration curve. The affinity patterns of five monoclonal antibodies and a recombinant single-chain fragment with respect to five s-triazine derivatives are determined by this method. An array of three antibodies is selected and the response pattern to mixtures of three analytes determined. Measured and calculated pattern correspond in principle, but systematic deviations are observed due to the perturbation of equilibrium during detection. The correlation of the true analyte concentration and the analyte concentrations predicted from the signal pattern using the affinity constants strongly depend on the selectivity and the affinity of the antibodies.