Role of antibody valency in hapten-heterologous immunoassays.
We studied the effects of hapten heterology on immunoassays of triiodothyronine (T3), digoxin, and cortisol, in a format involving labeled monoclonal antibodies and immobilized, protein-conjugated ligands. Replacing the homologous conjugated ligands T3, digoxin, and cortisol with their respective analogs diiodothyronine, digitoxin, and corticosterone led in each case to a decrease in the midpoint of displacement (ED50) for the same zero-dose signal. The mechanism of this phenomenon was studied by converting the bivalent anti-T3 to a monovalent whole antibody (bispecific monoclonal anti-T3 x anti-glucose-6-phosphate dehydrogenase) by cell fusion. The monovalent antibody was effective as a tracer in the homologous T3 assay, but generated a very low zero-dose signal with the heterologous solid phase, thus precluding sensitivity enhancement. On the basis of these results and additional kinetic and double-labeling experiments, we propose that the use of hapten heterology relies on bivalent binding of the antibody to the solid phase to compensate for a lower intrinsic affinity. This binding mechanism leads to lower assay concentrations of the ternary complex analyte-labeled antibody-immobilized hapten, thereby providing enhanced sensitivity.
Piran
U
U
Ciba Corning Diagnostics Corp., Walpole, MA 02032.
Silbert-Shostek
D
D
Barlow
E H
EH
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity; D001666:Binding Sites, Antibody; D004077:Digoxin / Q000097:blood; D006241:Haptens / Q000276:immunology*; D006801:Humans; D006854:Hydrocortisone / Q000097:blood; D007118:Immunoassay; D007457:Iodine Radioisotopes; D051379:Mice; D014284:Triiodothyronine / Q000097:blood
D000911:Antibodies, Monoclonal; D006241:Haptens; D007457:Iodine Radioisotopes; D014284:Triiodothyronine; D004077:Digoxin; D006854:Hydrocortisone