Sensitivity of Steroid Enzyme Immunoassays. Comparison of Alkaline Phosphatase, β -Galactosidase and Horseradish Peroxidase as Labels in a Colorimetric Assay System

Abstract

The sensitivities of colorimetric testosterone enzyme immunoassays using alkaline phosphatase (AP), β-galactosidase (β-GAL) and horseradish peroxidase (HRP) as labels were compared. Enzyme labeling of testosterone was carried out by the N-succinimidyl ester method at an appropriate molar ratio of steroid to enzyme. In the competitive immunoassay, the bound and free enzyme-labeled antigens were separated by a double antibody method and the enzymic activity of the immune precipitate was determined by spectrophotometric methods. The AP activity was measured in four ways, using p-nitrophenyl phosphate, phenolphthalein monophosphate, phenyl phosphate, and nicotinamide adenine dinucleotide phosphate (NADP) as substrates. In the cases of β-GAL and HRP, o-nitrophenyl β-D-galactopyranoside and 3,3',5,5’-tetramethylbenzidine were used, respectively. A dose—response curve with a satisfactory sensitivity was obtained in each testosterone assay system by the use of a minimum amount of the enzyme-labeled antigen at an appropriate dilution of anti-testosterone antiserum (Ka = 2 x 10l0M-1). The amount of testosterone needed to displace 50% of the bound label ranged from 9 to 90 pg. It was found that the highest sensitivity was obtained by the use of HRP, with AP next and β -GAL third; the former two sensitivities were each comparable to that of the corresponding fluorimetric assay. © 1987, The Pharmaceutical Society of Japan. All rights reserved.