Fluorescence Polarization Immunoassay of Progesterone

• Published in: Biological & pharmaceutical bulletin Vol. 20; no. 4; pp. 309 - 314
• Main Authors: CHOI, Myung Ja, CHOI, Jeongeun, YOON, Do Young, PARK, Jongsei, EREMIN, Sergei A.
• Format: Journal Article
• Language: English
• Published: Tokyo The Pharmaceutical Society of Japan 01.04.1997; Maruzen; Japan Science and Technology Agency
• Subjects: Analysis; Analytical, structural and metabolic biochemistry; Biological and medical sciences; fluorescence; fluorescence polarization immunoassay; Fluorescence Polarization Immunoassay - methods; Fundamental and applied biological sciences. Psychology; General pharmacology; immunoassay; Immunoenzyme Techniques; immunogen; Medical sciences; Other biological molecules; Pharmacology. Drug treatments; progesterone; Progesterone - urine; Sensitivity and Specificity; Terpenes, steroids. Hormones; tracer; Urine; Biological fluid; Fluorescence polarization immunoassay; Progesterone; Sex steroid hormone; Quantitative analysis; Immunological method
• ISSN: 0918-6158; 1347-5215
• DOI: 10.1248/bpb.20.309

A homogeneous fluorescence polarization immunoassay (FPIA) was developed to measure levels of progesterone in urine using a TDx analyzer in photocheck mode (Abbott Labs). Two tracers of ethylenediamine fluorescein thiocarbamyl (EDF) were employed; one was synthesized from 11α-hydroxyhemisuccinate progesterone (Prog-11OH-HS) and the other was synthesized from 3-(o-carboxymethyl)oxime progesterone (Prog-3CMO). Each derivative of progesterone was conjugated with bovine serum albumin and used as an immunogen which produced monoclonal antibody clone 15A (MAb 15A, anti-Prog-11OH-HS) and clone 2B7 (MAb 2B7, anti-Prog-3CMO), respectively. Different combinations of tracers and antibodies were investigated in the FPIA system. Similar sensitivity was observed when using the pair, MAb 2B7 and its homologous tracer, Prog-3CMO-EDF, or MAb 15A and its homologous tracer, Prog-11OH-HS-EDF. In this immunoassay, no separation step was required and the total time for an assay of 10 samples was approximaterly 7 min. The progesterone detection limit in a 10 μl sample was 3 ng/ml. The cross-reactivity results indicate that the A-, B- and D-ring of a steroid are buried in the binding pocket of MAb 15A, while the C-ring faced outward, resulting in cross-reactivity with 11-αhydroxy progesterone. The A-, B- and C-ring of a steroid of MAb 2B7, in contrast, are buried deep in the pocket leaving the D-ring facing outward, resulting in some different degrees of cross-reactivity with C17 position substituted steroids.