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

Enzyme labeling of a steroid with horseradish peroxidase by the N-succinimidyl ester method has been investigated in comparison with that with β-galactosidase. The reaction of the activated ester of 4-hydroxytestosterone 4-hemiglutarate with horseradish peroxidase provided a labeled antigen showing high immunoreactivity with an anti-testosterone antiserum in the enzyme immunoassay procedure. The effect of steroid/enzyme molar ratio, ranging from 2 to 60, in the labeling on the assay sensitivity was then examined. It was found that, in contrast to the case of β-galactosidase, the sensitivity of the assay using horseradish peroxidase-labeled antigen is not significantly influenced by the molar ratio. Dose-response curves with high sensitivities could be obtained by the use of these labeled antigens at an appropriate dilution of the antiserum. The active ester method proved to be useful for the preparation of enzymelabeled antigens because of its simplicity and excellent reproducibility.