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

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The sensitivities of colorimetric testosterone enzyme immunoassays using alkaline phosphatase (AP), β-gaIactosidase (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, ο-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 (K_a=2×1O^<10>_<M-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 fluorimetrie assay.
公益社団法人日本薬学会の論文
1987-08-25