Monoclonal antibody-based enzyme immunoassay for simultaneous quantification of 2- and 16α-hydroxyesterone in urine

Abstract

Alterations in the metabolism of estrogen have been implicated as an important factor in the etiology of diseases such as gynecological cancers and lupus erythematosus. The major metabolites of estradiol are hydroxylated at the C-2 or C-16α position yielding products with estrogen antagonist and agonist activities, respectively. A sensitive and specific immunodiagnostic assay to determine the balance between these competing pathways might serve as a routine biomarker for management of estrogen-related diseases. We describe here the generation of high affinity, specific murine monoclonal antibodies to 2-hydroxyesterone and 16α-hydroxyesterone by high efficiency fusion protocols. With these antibodies, we have developed a rapid and simple anzyme immunoassay (EIA) kit for the simultaneous quantitation of 2- and 16α-hydroxyestrone in unextracted urine. Initial validation studies established that urinary metabolite 2- and 16α-hydroxyestrone concentrations found by the EIA correlate well with values found by gas chromatography-mass spectroscopy. Preliminary studies with the EIA kit found total recovery of metabolites from spiked urine samples. The EIA inter- and intra-assay coefficients of variation for 2-hydroxyestrone and 16α-hydroxyestrone and the ratio of 2-hydroxyestrone to 16α-hydroxyestrone with the current EIA kit were consistently less than 9%. This kit, designated ESTRAMET ™ 2/16 may provide an important new tool for research in estrogen-related diseases.