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

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.

Effect of different combinations of antibodies and enzyme labels on ELISA of progesterone.

In steroid immunoassays, selection of right combination of antibody and enzyme-labeled antigen determine the sensitivity and specificity of ELISA. Antibodies raised against different positions of progesterone adopting heterologous systems were reported to provide better assays for progesterone. Four different antibodies developed against progesterone-11α-hemiglutarate-BSA (P-11-HG-BSA), progesterone-11α-hemisuccinate-BSA (P-11-HS-BSA), progesterone-3-O-carboxymethyloxime-BSA (P-3-CMO-BSA), and progesterone-3-O-carboxymethyloxime-ovalbumin (P-3-CMO-ova) were tested in combination with enzyme-labeled P-11-HG, P-11-HS, progesterone-11α-carboxymethyl ether (P-11-CME), P-3-CMO, 17-hydroxyprogesterone-3-O-carboxymethyl oxime (17-P-3-CMO), and progesterone-4-carboxymethyl thioether (P-4-CMTE). These were variously labeled with penicillinase, alkaline phosphatase (ALP), and horseradish peroxidase (HRP). When antibody developed against P-11-HS-BSA was tested with P-3-CMO labeled separately with penicillinase, ALP, and HRP, the type of enzyme used had no effect on the performance of the assay. It was found that a homologous assay using P-3-CMO-ova as immunogen and P-3-CMO-HRP as label, as well as a heterologous ELISA with antibody raised against P-11-HS-BSA in combination with P-3-CMO-HRP, provided sensitive assays for progesterone. The use of 17α-hydroxy progesterone-3-O-carboxymethyl oxime-HRP with the same antibodies against P-3-CMO-BSA and P-11-HS-BSA also proved to be better than P-3-CMO-HRP. These findings implied that the sensitivity and specificity of ELISA to a great extent depended on the nature of the antibody produced, while the choice of enzyme labels could be manipulated.