Enzyme Labeling of Steroids by the N-Succinimidyl Ester Method. Preparation of Alkaline Phosphatase-Labeled Antigen for Use in Enzyme Immunoassay

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Enzyme labeling of a steroid with alkaline phosphatase by the N-succinimidyl ester method was investigated. The activated ester of 4-hydroxytestosterone 4-hemiglutarate was treated with alkaline phosphatase to give a labeled antigen. Various molar ratios of steroid to enzyme, ranging from 5 to 200, were employed. The loss of enzymic activity was less than 20% under the coupling conditions used. Satisfactory immunoreactivities with an anti-testosterone antiserum in the enzyme immunoassay procedure were obtained with the labeled antigens prepared at molar ratios higher than 20. The effect of steroid/enzyme molar ratio in the labeling on the sensitivity of the testosterone assay was then examined. It was found that the sensitivity of the assay is significantly influenced by the molar ratio, and a higher ratio results in a decrease in assay is significantly influenced by the molar ratio, and a higher ratio results in a decrease in assay sensitivity. A doserespared at a molar ratio of 30. The active ester method proved to be useful for the preparation of alkaline phosphatase-labeled antigens as well as for β-galactosidase and horseradish peroxidase labelings, because of its simplicity and excellent reproducibility.