Antibody method for measurement of dihydrotestosterone receptors in cultured human skin fibroblasts.
We have adapted the method of Castañeda and Liao to the assay of DHT receptors in cultured fibroblasts arising from human skin. An antitestosterone antibody (100% crossreactivity for DHT) was coupled to CNBr-activated Sepharose. Confluent monolayers of fibroblasts were incubated with 3H-DHT (2 nM) at 37 degrees C for 30 min. Fibroblasts were then collected, sonicated, and centrifuged at 1200 x g for 15 min. The receptor assay was carried out on the supernatant; the antibody-sepharose was used to remove both unbound and nonspecifically bound DHT. Experience showed that the antibody did not entirely remove the nonspecifically bound and free DHT. A blank (sample heated at 60 degrees C for 3 min) was therefore subtracted to obtain an accurate value of specifically bound DHT. In spite of this, the antibody method, when compared to the gel filtration method, was more rapid and more convenient. Its reproducibility was similar to that of the gel filtration method, and its sensitivity was somewhat greater in patients with low levels of DHT-receptor complex. Improved sensitivity could be particularly useful when dealing with partial AIS.
D002478:Cells, Cultured; D002850:Chromatography, Gel; D013196:Dihydrotestosterone / Q000378:metabolism*; D005347:Fibroblasts / Q000378:metabolism*; D006801:Humans; D011863:Radioimmunoassay / Q000379:methods; D011944:Receptors, Androgen / Q000032:analysis*; D011987:Receptors, Steroid / Q000032:analysis*
D011944:Receptors, Androgen; D011987:Receptors, Steroid; D013196:Dihydrotestosterone