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

Enzyme labeling of a steroid with glucose oxidase by the N-succinimidyl ester method was investigated. The activated ester of a testosterone derivative was treated with glucose oxidase to give a labeled antigen. Various molar ratios of steroid to enzyme, ranging from 2 to 100, were employed; the degrees of hapten substitution were found to be 0.7-13. Satisfactory immunoreactivities with an anti-testosterone antiserum in the enzyme immunoassay procedure were obtained with the labeled antigens prepared at molar ratios higher than 4. 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 sensitivity. A dose-response curve with a high sensitivity could be obtained by the use of the labeled antigen prepared at a molar ratio of 6. The active ester method proved to be useful for the preparation of glucose oxidase-labeled antigens as well as for alkaline phosphatase, β-galactosidase and horseradish peroxidase labelings, because of its simplicity and excellent reproducibility.