Validation of radioimmunoassay for estradiol-17/3 by isotope dilution-mass spectrometry and by a test of radiochemical purity

Abstract

Isotope dilution-mass spectrometry (ID-MS) was used as a reference method to determine the concentration of estradiol-17β (E 2) in five different plasma pools (concentrations ranging from 0.040 to 65 nmol/1). The same plasma pools were also subjected to radioimmunoassay (RIA) using five different antisera of largely varying specificity. With the best antiserum (E), a direct RIA apparently gave accurate results (i.e. results statistically indistinguishable from those obtained by ID-MS) at all levels except the lowest one (0.040 nmol/1). It was shown, however, that the apparent accuracy of this RIA to some extent could be due to a lowering effect of lipids in the serum masking a lack of specificity of the antibodies. With the least specific antiserum (A), accurate results were obtained only after chromatography. However, in the assay of the lowest concentration of E 2 with this antiserum there was a significant overestimation, even after chromatography. The other three antisera (B, C, D) of average quality gave accurate results in assays of plasma diethyl ether extracts in various numbers of the plasma pools tested, depending on their intrinsic specificity. This specificity was not correlated with the cross-reaction reported for individual antisera. ID-MS is difficult to use in most laboratories. We demonstrate here that the validity of a RIA may in this case be assessed by a relatively simple method, the test of radiochemical purity (RP-test). This test is based on the measurements of specific activity (e.g. dpm/pg) in small consecutive fractions of the Chromatographie zone which is usually employed for the RIA.