A chemiluminescence-based assessment of androgen-binding activity in a large pedigree affected with androgen insensitivity syndrome.
We report a novel chemiluminescence (CL)-based method for assaying the ligand-binding activity of the androgen receptor. The central parts of this method are the utilization of the steroid CL marker as the replacement of the radioactive label in the conventional ligand-binding assay and the determination of the binding activity by the light measurement of the bound CL-label under an H(2)O(2)-microperoxidase system. The properties and reliability of this assay were investigated and verified using genital skin fibroblasts (GSF) from seven normal males. The method is precise (CV < 7% for both B(max) and K(d)) with high correlation coefficients (r > 0.93) in each Scatchard linear regression analysis. This assay can determine the androgen binding properties using only a quarter of the cells (approximately 40 000 cells/data point) of that required by the radiolabelling approach. The utility of the method was illustrated by binding experiment on the GSFs of several patients from a large Chinese family affected with androgen insensitivity syndrome. The familial distinct feature is that all patients shared an identical Arg840Cys substitution in the androgen receptor but displayed high phenotypic variation in disorders of male sexual development. The patients selected for the present study represent a wide spectrum of this phenotypic variation. This study thus provides insights on the pleiotropic effects of the mutation. In conclusion, the CL-based method can serve as an effective, precise and reliable replacement for the radiolabelling approach and has the advantages of simplicity, cost-effectiveness and health and environmental safety over the counterpart.
Wang
Jiu-Cun
JC
China's State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, and Morgan-Tan International Center for Life Sciences (MTIC), Fudan University, Shanghai, People's Republic of China.
Wang
Minghua
M
Zhang
Rongmei
R
Zhao
Zhimin
Z
Chen
Jie
J
Luo
Zewei
Z
D019943:Amino Acid Substitution; D013734:Androgen-Insensitivity Syndrome / Q000209:etiology* / Q000235:genetics; D044466:Asian People; D002478:Cells, Cultured; D005347:Fibroblasts / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide; D008163:Luminescent Measurements / Q000379:methods*; D008297:Male; D008722:Methods; D010375:Pedigree; D011485:Protein Binding; D011944:Receptors, Androgen / Q000032:analysis / Q000235:genetics / Q000378:metabolism*
C410798:AR protein, human; D011944:Receptors, Androgen; D006861:Hydrogen Peroxide