AN ENZYME-LINKED IMMUNOASSAY FOR THE 1,25-DIHYDROXYVITAMIN D-3 RECEPTOR PROTEIN

In this paper, we detail an enzyme-linked immunoassay for the 1,25-dih ydroxyvitamin D-3 receptor protein. The receptor protein of cell and t issue homogenates is bound between two monoclonal antibodies specific for different epitopes on the receptor protein, The first antibody is bound to the web of an ELISA plate and the second is biotinylated, The receptor-antibody complex is detected with avidin-alkaline phosphatas e and rho-nitrophenyl phosphate, The amount of receptor in each sample is determined by comparison with a standard curve made from purified receptor protein, This assay is highly sensitive, measuring as little as 2 fmol of receptor, and has an intra-assay coefficient of variation of 6.6% and an interassay coefficient of variation of 13.8%, The assa y can be used to measure the receptor from mammalian and avian species and is independent of the presence of hormone, By eliminating the nee d for a radio-iodinated monoclonal antibody and incorporating the ease of a plate assay, we have a significantly improved method for measuri ng the vitamin D receptor protein. This paper also presents Western an alysis of the antibodies used to demonstrate that they do not recogniz e other steroid hormone receptors.