ROLE OF ANTIBODY VALENCY IN HAPTEN-HETEROLOGOUS IMMUNOASSAYS

We studied the effects of hapten heterology on immunoassays of triiodo thyronine (T3), digoxin, and cortisol, in a format involving labeled m onoclonal antibodies and immobilized, protein-conjugated ligands. Repl acing the homologous conjugated ligands T3, digoxin, and cortisol with their respective analogs diiodothyronine, digitoxin, and corticostero ne led in each case to a decrease in the midpoint of displacement (ED5 0) for the same zero-dose signal. The mechanism of this phenomenon was studied by converting the bivalent anti-T3 to a monovalent whole anti body (bispecific monoclonal anti-T3 x anti-glucose-6-phosphate dehydro genase) by cell fusion. The monovalent antibody was effective as a tra cer in the homologous T3 assay, but generated a very low zero-dose sig nal with the heterologous solid phase, thus precluding sensitivity enh ancement. On the basis of these results and additional kinetic and dou ble-labeling experiments, we propose that the use of hapten heterology relies on bivalent binding of the antibody to the solid phase to comp ensate for a lower intrinsic affinity. This binding mechanism leads to lower assay concentrations of the ternary complex analyte-labeled ant ibody-immobilized hapten, thereby providing enhanced sensitivity.