Abstract
Radioimmunoassay (RIA) is a sensitive, specific and handy method, which has been widely utilized in the determination of prostaglandin (PG) and thromboxane (TX). In consideration of the disadvantage of RIA depending on the use of radioisotope we made attempts to apply enzyme immunoassay (EIA) to the assay of PG and TX, in which enzyme-labeled hapten molecule is used instead of radioactive compound.
The mixed anhydride reaction was performed to conjugate the amino group of β-galactosidase to the carboxyl group of PGF2α, TXB2 and 6-keto-PGF1α, respectively. The enzyme-labeled antigen could be stored at 4° without an appreciable loss of enzyme activity and antigenicity for more than a year.
After competitive immunoreaction with antibody between enzyme-labeled antigen and unlabeled antigen the immunoprecipitate formed was separated by the double antibody method. The enzyme activity of the precipitate was assayed fluorometrically with 4-methylumbelliferyl-β-Dgalactoside as substrate. PGF2α, TXB2 and 6-keto PGF1α could be measured quantitatively in the range of 0.06-30pmol, 0.1-30pmol, 0.14-30pmol, respectively. The sensitivity was comparable to that of RIA. The cross-reactivities with other PGs and their metabolites were less than 1% in most cases.
A column of octadecyl silica was used to extract TXB2 which was added to human plasma, and the extract was subjected to RIA and EIA. A good correlation was obtained between the amount of added TXB2 and that of measured TXB2 (y=1.09x+11.07pmol/ml, r=0.99) A satisfactory interrelation was also observed between RIA and EIA (y=0.92x+4.64pmol/ml, r=0.96).
