INFLUENCE OF DIFFERENT HOMOLOGOUS AND HETEROLOGOUS COMBINATIONS OF ANTIBODIES AND ENZYME CONJUGATES OF DEHYDROEPIANDROSTOSTERONE ON THE SENSITIVITY AND SPECIFICITY OF DHEA ELISA

Abstract

Anti-sera were raised against three immunogen: dehydroepiandrostosterone-17-carboxymethyl-oxime-bovine serum albumin (DHEA-17-CMO-BSA), DHEA-7-CMO-BSA, and dehydroepiandrostosterone-3-hemisuccinate-bovine serum albumin (DHEA-3-HS-BSA). They were evaluated with horseradish peroxidase (HRP)-labeled DHEA-17-CMO, DHEA-7-CMO, DHEA-3-HS enzyme conjugates for their influence on the sensitivity and specificity of ELISA. Of the various combinations, DHEA-3-HS-BSA antiserum along with DHEA-7-CMO-horseradish peroxidase (DHEA-7-CMO-HRP) enzyme conjugate showed no cross-reaction with any of the closely related steroids. All the homologous combinations appeared to be less sensitive due to their low affinity for dehydroepiandrostosterone. Out of six heterologous systems tested, only three combinations, (1) anti-DHEA-17-CMO antiserum and DHEA-7-CMO-horseradish peroxidase, (2) anti-DHEA-7-CMO-antiserum and DHEA-3-HS-horseradish peroxidase, and (2) anti-DHEA-3-HS-antiserum and DHEA-7-CMO-horseradish peroxidase, showed displacement. The former two assays were less specific; the first one showed 15.38% and 16.66% cross-reaction with androstenediol and testosterone, respectively, whereas the second assay showed 30.3%, 22.72%, 111.1%, 62.5%, and 31.25% cross-reaction with DHEA-glucuronide, 16-dihydroxyprogesterone, androstenediol, etiocholon-3-β-ol-17-one, and aldosterone, respectively. The ability of DHEA to displace the DHEA-enzyme conjugate and the specificity of the assay appear to depend on the position of the enzyme label on the DHEA molecule as well as on the availability of antigenic sites in particular combinations of antibody and DHEA-enzyme conjugates.

Keywords:

• dehydroepiandrosterone
• heterologous
• homologous
• immunoassay
• site heterology

ACKNOWLEDGMENTS

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

Notes

The nine assay combinations were tested in the standard displacement assay as described in the Experimental section.

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

N = Number of times assays carried out for inter-assay variation.