Estimation of true values in radioimmunoassays

We tested the accuracy of radioimmunoassays for two hormones, progesterone and estradiol, in relation to the use of two different antisera in each assay and to the degree of purification from plasma before the assay was done. Each analyte was assayed either in a diethyl ether extract, in a zone eluted from a Sephadex LH-20 chromatographic column, or in fractions of a chromatographic zone (from Sephadex LH-20 or Celite) that passed a test of "radiochemical purity." Statistically indistinguishable results were obtained in the assay of radiochemically pure fractions of both analytes, irrespective of the antiserum used. In addition, one of the antisera from each hormone gave equivalent results in the radioimmunoassay of ether extracts, even with no preceding chromatography. We demonstrated in this way that results obtained with use of highly specific antisera may, after a single chromatography, but even without any chromatographic purification, be as nearly accurate and as closely reflect the true value as those obtained in the assay of radiochemically pure hormones, an assay that has a character of a reference method (as defined by the IFCC).