Influence of iodothyronine conjugates of bovine serum albumin and horseradish peroxidase on enzyme immunosorbent assay of thyroid hormones.

ABSTRACT

Enzyme-linked immunosorbent assays (ELISA's) reported for thyroxine (T4) and 3,5,3'-triiodothyronine (T3), involved coupling of the haptens through (i) carboxylic group to carrier protein for producing antibodies and (ii) amino group to detection labels. To improve the titer and specificity of antibodies, immunogens were prepared by coupling of carboxyl group to bovine serum albumin (BSA) either directly or through adipic acid dihydrazide (ADH), after protecting amino group through acetylation of T4 and T3. Direct coupling resulted in the incorporation of 40-50 moles of T4 and T3 per BSA molecule and helped in improving immunogenic response and specificity, especially of T4. High epitope density of immunogens evoked better antibody response, since attachement of ADH as spacer, introduced 18-27 moles of haptens into carrier protein and had less effect on antibody development, with T3 being exception. Detection labels were prepared by coupling horseradish peroxidase (HRP) to amino group of thyroid hormones directly and after preparing their methyl esters, which provided sensitive displacement curves in combination with the antibodies developed against N-acetylated-T4 and T3. Unlike methyl esters, T4-HRP and T3-HRP showed higher sensitivity and seemed to be related to the affinity of the labels for binding the antibody.