A solid-phase enzyme immunoassay of thromboxane B2.

Abstract

A solid-phase enzyme immunoassay for thromboxane B2 was developed using a conjugate of thromboxane B2 and beta-galactosidase. Anti-thromboxane B2 IgG was bound to a polystyrene tube, and the enzyme-labeled and unlabeled thromboxane B2 were allowed to react in a competitive manner with the immobilized antibody. Then, the specifically bound beta-galactosidase was assayed fluorimetrically, and the enzyme activity was correlated with the amount of unlabeled thromboxane B2. By using a calibration curve, thromboxane B2 was determined in the range of 20 fmol-14 pmol. 2,3-Dinor- and 2,3,4,5-tetranor-thromboxane B2 cross-reacted with thromboxane B2 to the extents of 18.6% and 0.4%, respectively. Most prostaglandins and their metabolites tested showed cross-reactivities of less than 1%. In application of the method to human blood and urine, an octadecylsilyl silica column was utilized for extraction and concentration of thromboxane B2. The crude extract contained a substance(s) which disturbed the enzyme immunoassay and gave an apparently high value of thromboxane B2, and the interfering substance was separated from thromboxane B2 by reverse-phase HPLC. Various amounts of authentic thromboxane B2 added to the purified material from human plasma could be determined by the enzyme immunoassay with a recovery of about 80% and the results correlated well with the values obtained by radioimmunoassay (r = 0.979). When the extract from human urine was analyzed by reverse-phase HPLC, the 2,3-dinor metabolite rather than thromboxane B2 was the predominant compound detected by the enzyme immunoassay.