Influence of antisera, purification procedures, and tracers on validity of a testosterone radioimmunoassay

The levels of testosterone in pooled plasma from men and from women were measured by radioimmunoassay. Testosterone was isolated from plasma either by ether extraction (direct procedure) or by chromatography of the ether extract (chromatographic procedure). Three different antisera (all raised against testosterone-3-oxime albumin) were employed. An additional variable was the use of two tracers, [3H] testosterone and [125I] histamine testosterone. The latter tracer was employed in both a freshly prepared form and two months after labelling. The old tracer was used both in a nonpurified and repurified form. The results were compared with those given by one of the antisera using the assay with chromatography. The validity of this assay was verified earlier using a test of radiochemical purity. In the statistical evaluation of the data, it was found that the assays of both plasma pools gave the same results as the validated assay, even with two other antisera, but only when the chromatographic purification was used. The direct assays gave significantly increased values for testosterone in the male plasma pool with one antiserum, and in the female plasma pool with all three antisera. It is concluded that those assays maybe considered valid which give results statistically indistinguishable from those obtained by the valid assay. These were all chromatographic assays and, in the man, also two direct assays. The comparison of [3H] testosterone and [125I] histamine testosterone as tracers did not indicate any major differences in the validity of the assays. When the 125I-tracer was employed, a moderate (1.5-2 fold) increase of sensitivity was seen with all three antisera, and the antisera could be used in a 10-40 times higher dilution. Furthermore, it was possible to use [125I] histamine testosterone and obtain practically the same results after two months, whether repurified or not.