A rapid radioimmunoassay for serum testosterone

This technique of R.I.A was developed for students in classroom laboratory situations where rat serum testosterone assays could be completed within two 3-hour laboratory periods. The assay utilizes commercially prepared testosterone R.I.A. Pak. Plasma samples and extraction controls are prepared for extraction with a 1:10 dilution in phosphate buffer of which 0.1 ml is then incubated with 40 units of the enzyme Subtilisin Carlsberg (for steroid extraction from the Sex Hormone Binding Protein and otherplasma proteins) for a period of two hours. After extraction, the enzyme is heat inactivated for two minutes at 100 degrees C. At this point, the assay can be stopped for 24-48 hours by storage of extraction samples at 2-3 degrees C. The assay is concluded with assembly of standard curve tubes and by addition of antibody, antigens system to all tubes for the final two hour incubation followed by the Dextran charcoal separation of unbound components and the decanting of bound complexes into scintillation counting vials. tthis assay technique has a range of 0.05 ng/ml to 5.0 ng/ml. The mean serum testosterone level of fifteen adult male Sprague-Dawley rats was determined to be 262 ng/100 ml.