Date of Award

Summer 1990

Document Type


Degree Name

Master of Science (MS)


Chemistry & Biochemistry



Committee Director

James H. Yuan

Committee Member

Mung H. Kim

Committee Member

Mark S. Elliot

Call Number for Print

Special Collections LD4331.C45 L5


An enzyme-labeled immunoassay of sufficient range and sensitivity was developed for serum testosterone determination. In this method, the testosterone in the serum sample and testosterone-HRPO conjugate compete with each other for the limited antibody binding sites on the anti-testosterone gamma globulin coated microwell. The assay can be performed in only 2.5 hours.

The gamma globulin fraction was isolated from the anti-testosterone whole serum by DEAE-cellulose column chromatography. Testosterone-peroxidase conjugate was prepared by a carbodiimide coupling method. The interference which arises from the binding of testosterone to steroid hormone binding protein was eliminated by the addition of 17 beta-estradiol. All assay conditions including incubation temperature, incubation time, incubation solution, and washing solution were analyzed and optimized.

The detection limit of the competitive enzyme immunoassay was determined to be 0.119 ng/mL. The coefficients of variation (CV) for intra-assay were found to be 8.48%, 11.6% and 13.4% at testosterone concentrations of 9.30 ng/mL, 3.00 ng/mL, and 0.460 ng/mL. The CVs for inter-assay were determined to be 9.16%, 12.1% and 14.6% at testosterone concentrations of 9.27 ng/mL, 2.88 ng/mL, and 0.493 ng/mL. The cross reactivities with 5 alpha-dihydrotestosterone, 17 beta-estradiol, 4- androstene-3,17-dione, and 5-androstene-3 beta,17 beta-diol were found to be 72%, 0.028%, 0.76%, and 0.30% respectively. Excellent agreement was obtained in the comparison study of serum samples with both EIA and RIA.


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