Effect of hapten heterology on thyroid hormone immunoassays

We examined the effects of hapten heterology on triiodothyronine (T3) and thyroxine (T4) immunoassays configured with protein-conjugated diiodothyronine (T2), T3 or T4, using acridinium ester (AE) as a chemiluminescent label and paramagnetic particles (PMP) as a solid phase. Assays constructed with hapten-heterologous combinations, such as immobilized anti-T4 plus labeled T3 or immunobolized anti-T3 plus labeled T2, were superior to the homologous ones in their potential for attaining higher sensitivity. This was manifested in a shift of the displacement curves to lower analyte concentrations, accompanied by unexpectedly high bound-tracer/total-tracer (B/T) ratios. This effect of hapten heterology was apparent with the use of either monoclonal or polyclonal antibodies and did not depend upon which component (antibody or protein conjugated hormone) was immobilized on the solid phase. In contrast, heterologous displacement curves with unconjugated hormone (125I-labeled) or with a labeled Fab fragment exhibited both a shift to lower analyte concentration and the expected decrease of B/T ratios. These results can be explained by the existence of positive binding cooperativity in the interaction between antibodies and haptenated proteins. Assay specificity was not adversely affected by hapten heterology when utilizing monoclonal antibodies, and hormone levels measured in serum samples correlated well with values obtained in homologous immunoassays.