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.