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A RADIOIMMUNOASSAY FOR PLASMA TESTOSTERONE (1970)
Shunsuke Furuyama, Darrel M. Mayes, and Charles A. Nugent
ABSTRACT
A simple and reliable radioimmunoassay for plasma testosterone has been developed. Antiserum against testosterone was produced in rabbits immunized with testosterone-3-oxime-beef serum albumin. Plasma volumes of 0.1 ml from men and 0.5 ml from women were used for analysis. After a preliminary solvent wash, the plasma was extracted and the extracts were applied to AI203 microcolumns. Following elution, the dried purified extract was incubated with antiserum at room temperature for 2 hours. (NH4)2SO 4 was used to separate free from bound testosterone. 1,2-3H-testosterone was used for correction of losses and for determination of the % bound. The accuracy and precision of the method were satisfactory. The sensitivity was I0 pg per sample. The mean blank was 2 ~ 2 (SD) pg per sample. Analysis of plasma duplicates by an alternative method utilizing paper chromatography for purification of extracts did not give results that differed significantly from those obtained with the present method. The mean and range of plasma testosterone concentrations in normal men and women determined by this method were similar to those reported by other investigators using different methods.
INTRODUCTION
In recent years, numerous techniques have been developed for measuring plasma testosterone with double isotope derivative methods (I), gas-liquid chromatography (2), and competitive protein binding using testosterone binding globulin (3). Some of these methods are accurate, precise, sensitive, and specific; however, many are extremely tedious and all require at least one thin layer, paper, or gas-liquid chromatography for purification of the plasma extract in order to measure testosterone in plasma from men and women.
This report describes a reliable radioimmunoassay for testosterone in plasma from men and women which uses for purification only solvent partitioning and adsorption chromatography on a micro column of AI203.
Steroid-protein conjugates have been used as antigens (4) to develop antibodies which, in some cases, are more specific for certain steroids than are naturally occurring plasma binding proteins (5). Recently, radioimmunoassay methods have been reported for plasma estradiol (6), estrone (7), and aldosterone (8). The radioimmunoassay of testosterone was investigated by Niswender and Midgeley but details have not been published (9).
In Table III, the plasma testosterone concentrations determined by the present method (Column + antibody) are compared with the results of analyses of duplicate specimens determined by a modification of the method of Mayes and Nugent (10) using testosterone binding protein (Paper + TBP). In the original method (10), plasma extracts were purified by paper and thin layer chromatography before incubation with testosterone binding protein.
TABLE III. Specificity: Testosterone Concentrations Determined Using Different Methods* ng/100 ml of plasma
* Column + antibody refers to the method described in this report. Paper + TBP refers to the method using testosterone binding protein described earlier (10) with purification by paper chromatography but omitting thin layer chromatography.
For the present study, the thin layer chromatography was omitted since it has been shown that this omission did not result in a significant error (10). The means of the results (Table III) obtained with the present method were not significantly different from those obtained with the modified method of Mayes and Nugent in the analysis of plasma from men and women (p>0.5). In Table IV the testosterone concentrations in the plasma of normal men and women determined by the present method are compared with the results reported by others using different methods 1, 10, 13, 14).
TABLE. IV Plasma testosterone concentrations in ng/100 mL in normal subjects*
Discussion
The results of the assay of plasma specimens by the present method were not significantly different from those found on analysis of duplicate specimens by a modification of the method of Mayes and Nugent 10) utilizing paper chromatography for purification of extracts.- Also, the concentrations of plasma testosterone determined using this method in samples from normal men and women were similar to the values reported by other investigators using different methods.
One technician can complete analysis of 20 plasma samples plus 2 samples of ether-extracted plasma fortified with known amounts Of testosterone in addition to a set of direct standards in 1½ working days.
Shunsuke Furuyama, Darrel M. Mayes, and Charles A. Nugent
ABSTRACT
A simple and reliable radioimmunoassay for plasma testosterone has been developed. Antiserum against testosterone was produced in rabbits immunized with testosterone-3-oxime-beef serum albumin. Plasma volumes of 0.1 ml from men and 0.5 ml from women were used for analysis. After a preliminary solvent wash, the plasma was extracted and the extracts were applied to AI203 microcolumns. Following elution, the dried purified extract was incubated with antiserum at room temperature for 2 hours. (NH4)2SO 4 was used to separate free from bound testosterone. 1,2-3H-testosterone was used for correction of losses and for determination of the % bound. The accuracy and precision of the method were satisfactory. The sensitivity was I0 pg per sample. The mean blank was 2 ~ 2 (SD) pg per sample. Analysis of plasma duplicates by an alternative method utilizing paper chromatography for purification of extracts did not give results that differed significantly from those obtained with the present method. The mean and range of plasma testosterone concentrations in normal men and women determined by this method were similar to those reported by other investigators using different methods.
INTRODUCTION
In recent years, numerous techniques have been developed for measuring plasma testosterone with double isotope derivative methods (I), gas-liquid chromatography (2), and competitive protein binding using testosterone binding globulin (3). Some of these methods are accurate, precise, sensitive, and specific; however, many are extremely tedious and all require at least one thin layer, paper, or gas-liquid chromatography for purification of the plasma extract in order to measure testosterone in plasma from men and women.
This report describes a reliable radioimmunoassay for testosterone in plasma from men and women which uses for purification only solvent partitioning and adsorption chromatography on a micro column of AI203.
Steroid-protein conjugates have been used as antigens (4) to develop antibodies which, in some cases, are more specific for certain steroids than are naturally occurring plasma binding proteins (5). Recently, radioimmunoassay methods have been reported for plasma estradiol (6), estrone (7), and aldosterone (8). The radioimmunoassay of testosterone was investigated by Niswender and Midgeley but details have not been published (9).
In Table III, the plasma testosterone concentrations determined by the present method (Column + antibody) are compared with the results of analyses of duplicate specimens determined by a modification of the method of Mayes and Nugent (10) using testosterone binding protein (Paper + TBP). In the original method (10), plasma extracts were purified by paper and thin layer chromatography before incubation with testosterone binding protein.
TABLE III. Specificity: Testosterone Concentrations Determined Using Different Methods* ng/100 ml of plasma
* Column + antibody refers to the method described in this report. Paper + TBP refers to the method using testosterone binding protein described earlier (10) with purification by paper chromatography but omitting thin layer chromatography.
For the present study, the thin layer chromatography was omitted since it has been shown that this omission did not result in a significant error (10). The means of the results (Table III) obtained with the present method were not significantly different from those obtained with the modified method of Mayes and Nugent in the analysis of plasma from men and women (p>0.5). In Table IV the testosterone concentrations in the plasma of normal men and women determined by the present method are compared with the results reported by others using different methods 1, 10, 13, 14).
TABLE. IV Plasma testosterone concentrations in ng/100 mL in normal subjects*
Discussion
The results of the assay of plasma specimens by the present method were not significantly different from those found on analysis of duplicate specimens by a modification of the method of Mayes and Nugent 10) utilizing paper chromatography for purification of extracts.- Also, the concentrations of plasma testosterone determined using this method in samples from normal men and women were similar to the values reported by other investigators using different methods.
One technician can complete analysis of 20 plasma samples plus 2 samples of ether-extracted plasma fortified with known amounts Of testosterone in addition to a set of direct standards in 1½ working days.
