Radioimmunological assay of prostacyclin synthetase activity

A radioimmunoassay for 6-oxoprostaglandin F1 alpha has been developed. 6-Oxoprostaglandin F1 alpha antiplasma was produced in rabbits by repeated immunization with 6-oxoprostaglandin F1 alpha coupled to bovine serum albumin. [125I] -labelled hapten with high specific radioactivity was prepared by radioiodination of 6-oxoprostaglandin F1 alpha-tyrosine methyl ester conjugate followed by purification with thin layer chromatography. The antibodies showed good specificity toward 6-oxoprostaglandin F1 alpha and crossreacted only significantly with prostaglandin F1 alpha. The radioimmunoassay was applied to the determination of prostacyclin synthetase activity in swine aorta microsomes using arachidonic acid and prostaglandin H2 as indirect and direct substrates. The enzyme assay was validated by the criteria that the formation of 6-oxoprostaglandin F1 alpha, immunoreactivity from either substrates was both time and enzyme protein dependent, and was inhibitable by specific inhibitors of the enzyme system.