HETEROLOGOUS ENZYME LINKED IMMUNOSORBENT ASSAY FOR MEASUREMENT OF TESTOSTERONE IN SERUM

Abstract

In steroid enzyme immunoassay (EIA), homologous and heterologous combinations of enzyme conjugate with immunogen influences labeled steroid recognition by antibodies that affect sensitivity of the assay. To develop testosterone enzyme linked immunosorbent assay (ELISA), antibodies were generated against testosterone-3-carboxymethyloxime-bovine serum albumin (T-3-CMO-BSA), testosterone-11-hemisuccinate-bovine serum albumin (T-11-HS-BSA), testosterone-17-hemisuccinate-bovine serum albumin (T-17-HS-BSA), testosterone-17-glucuronide-bovine serum albumin (T-17-G-BSA), and testosterone-19-carboxymethylether-bovine serum albumin (T-19-CME-BSA). Testosterone horseradish peroxidase (HRP) enzyme conjugate were prepared using carboxyl derivatives of 11-keto-testosterone (11-keto-T) and 1-dehydrotestosterone (1-Dehydro-T). Ten combinations of heterologous assays were evaluated. The data of the present study revealed that the use of the T-11-HS-BSA antibody in antigen plus site heterologous assay that employed 11-keto-testosterone-3-CMO-HRP as the label showed binding and displacement, and led to the development of sensitive and specific assay.

Keywords:

• antigen heterologous
• heterologous
• homologous
• immunoassay
• site heterologous
• testosterone

ACKNOWLEDGMENTS

The National Institute of Health and Family Welfare, New Delhi, India, supported this study. We are grateful to Profs. Jayanta K. Das and K. Kalaivani, for their keen interest and encouragement in the present study.

Notes

The recovery of the assay is in the range of 95.38–100%.

n = Number of times the same sample was analyzed for intra-assay variation.

N = Number of times the assays were carried out for interassay variation.

^a Mean ±SD.

n = Number of samples assayed.