Development of an Indirect Enzyme Immunoassay using Monoclonal Antibodies for the Measurement of 17α-Hydroxyprogesterone in Human Serum

Abstract

An indirect enzyme immunoassay for the measurement of total 17α-hydroxyprogesterone (17OHP) in serum using monoclonal antibodies generated in our laboratory was developed. Here, (a) instead of extraction with solvents, serum was heated to free protein-bound 17OHP and assay was performed at pH 9.6, (b) to ensure uniform assay conditions for both standards and samples, buffer for standards contained charcoal-stripped pre-heated pooled cord serum. Assays were done in 96-well EIA microplates pre-coated with 17α-hydroxyprogesterone-3-(o-carboxymethyl)oxime: bovine serum albumin. Secondary antibody was horseradish peroxidase-linked sheep anti-mouse IgG polyclonal antibody. The method was accurate and suitable for screening for congenital adrenal hyperplasia.

Keywords:

• 17α-Hydroxyprogesterone
• Indirect enzyme immunoassay
• Monoclonal antibody

ACKNOWLEDGMENTS

We wish to thank Dr. J. V. Gavilondo and Dr. M. F. de Cossio from the Center of Genetic Engineering and Biotechnology, Havana, Cuba, for their excellent technical assistants and technology transfer whose travel was supported by a grant from The International Atomic Energy Agency, Vienna. This work is supported by grants from the Malaysia Government Ministry of Science, Technology and Innovation (MOSTI) and the University of Malaya. Dr. Chong was a recipient of a National Science Foundation training grant from MOSTI during her PhD training. This work was conducted in partial fulfillment for Dr. Chong's PhD degree at the University of Malaya.

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Notes

∗Percentage indicates the cross-reactivity for each steroid at 50% displacement, calculated using criteria described by Abraham, 1969.