Abstract
The use of 3, 3', 5, 5'-tetramethylbenzidine (TMB) as a chromogen in a testosterone enzyme immunoassay system with a glucose oxidase label is described. Glucose oxidase activity was measured with glucose, peroxidase and TMB in acetate-citric acid buffers ; the optimum pH value was found to be 4.2. The glucose oxidase-labeled antigen was prepared by the N-succinimidyl ester method. In the competitive enzyme immunoassay, the bound and free enzyme-labeled antigens were separated by a double antibody method. A dose-response curve with a high sensitivity was obtained by the use of a minimum amount of the label at an appropriate dilution of anti-testosterone antiserum (Ka=2×1010M-1). The amount of testosterone needed to displace 50% of the bound lab el was 24 pg. The sensitivity was higher than that of the assay system using 2, 2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid), ABTS.
