A heterologous enzyme immunoassay of prostaglandin E 2 using a stable enzyme-labeled hapten mimic

Abstract

A sensitive heterologous enzyme immunoassay for prostaglandin E 2 was developed using 9-deoxy-9-methylene-prostaglandin F 2α as a stable prostaglandin E 2 mimic. β-Galactosidase was conjugated to the hapten mimic. Anti-prostaglandin E 2 IgG was bound to a polystyrene tube. The enzyme-labeled hapten mimic mixed with unlabeled prostaglandin E 2 was allowed to react in a competitive manner with the immobilized antibody. Then, the β-galactosidase specifically bound to the antibody was assayed fluorometrically, and the enzyme activity was correlated with the amount of unlabeled prostaglandin E 2. According to the calibration curve thus obtained, prostaglandin E 2 could be determined in a range of 1.2–430 fmol. Prostaglandin E 2 was extracted from human urine by the use of an octadecylsilyl silica column. The crude extract contained a subsance(s) which disturbed the enzyme immunoassay and gave an apparently high content of prostaglandin E 2. The interfering substance was separated from prostaglandin E 2 by reverse-phase high-performance liquid chromatography. The purified urinary extract was examined by the enzyme immunoassay for prostaglandin E 2, and the validity of the results was confirmed by gas chromatography-selected ion monitoring.