A method for measuring testosterone-estradiol-binding globulin--new analysis of experimental data and comparison with...

Some binding assays have been suggested to measure serum testosterone--estradiol-binding globulin (TeBG) concentration. They have led to discrepant results. This study brought to surface the lack of these methods and the interest of a method using an accurate analysis of the binding isotherm. These observations led us to propose an absolute reference method especially suitable for the radioimmunological assays standardization. Our method used Con A Sepharose as a solid phase and DHT as a radioligand. We succeeded in solving the difficulties related to binding methods by evaluating the non-specific binding of the ligand and ligand--albumin binding. A careful analysis of experimental data, by setting equations specific to this DHT-TeBG system permitted the systematic errors to be corrected and allowed a precise determination of the serum TeBG concentration (27.3 +/- 6.0 nmol/l for 30 healthy men; 40 +/- 13 nmol/l for 15 healthy women). Moreover, the affinity constant of the ligand for the protein was accurately evaluated KDHT = (0.7 +/- 0.1)10(9) (mol/l-1) at 20 degrees C, which was not permitted by other methods. Two lines of evidence supported our assumptions and results. On the one hand, using estradiol as a radioligand instead of dihydrotestosterone, we found the same value for serum TeBG concentration; on the other hand, our assay favourably compared with two different kits commercially available. The correlation (r = 0.89) with 3H-SBP kit Merieux was good though this kit was not suitable for correctly assaying serum TeBG concentrations between 30 nmol/l and 60 nmol/l. An excellent correlation (r = 0.97) with SHBG IRMA Kit Farmos was found. This antibody-based assay and our binding assay yielded quite similar values that mutually validated both methods.