Selection of hapten structures for indirect immunosensor arrays

A multianalyte immunosensor array can be implemented by immobilization of different haptens in distinct areas of a single cavity or flow cell. In this case a mixture of different antibodies for different analytes is used in an indirect ELISA-format. The selection of the right hapten structures is very important to build up an array successfully. A system of independent hapten/antibody combinations is needed, with one immobilized hapten (coating antigen) reacting only with one antibody. If more than one antibody binds to a coating antigen no ideal calibration curves are obtained. This phenomenon is known as shared-reactivity and can lead to double-sigmoidal curves. To use monoclonal antibodies to 2,4,6-trinitrotoluene (TNT) and 2,4-dichlorophenoxyacetic acid (2,4-D), two different haptens had to be found, one only reacting with the TNT-antibody, the other only binding to the 2,4-D-antibody. 2,4-Dichlorophenoxybutyric acid was used for the 2,4-D antibody and 2,4,6-trinitrophenyl-8-aminooctanoic acid for the TNT antibody. Although 4-nitrotoluene, 2,4-dinitrotoluene and 4-amino-2,6-dinitrotoluene showed only very low cross-reactivities to the 2,4-D antibody the corresponding haptens 4-nitrophenylacetic acid, 2,4-dinitrophenyl-6-aminohexanonic acid, and 4-amino-2,6-dinitrotoluyl-(N)-glutarate are useful coating antigens for this antibody. The structure of the coating antigens had no significant influence on the mid-points (IC50) of the test for 2,4-D and even haptens with very low cross-reactivities could be used. With all haptens a test midpoint of about 0.2 μg/L for 2,4-D was achieved. For the direct assay format with immobilized antibodies the same test midpoint of 0.2 μg/L for 2,4-D was obtained. As a conclusion, the selectivity of a monoclonal antibody should not be influenced by the used tracer or coating antigen as well. It could be shown that the affinity constants of an antibody to the analytes are the main sensitivity and selectivity determining parameters for competitive immunoassays. A two-dimensional microtiter plate array was used to determine the analytes 2,4-D and TNT in parallel with a mixture of antibodies.