An immunoradiometric assay for 1,25-dihydroxyvitamin D3 receptor.
A ligand-independent, quantitative assay has been developed for the measurement of 1,25-dihydroxyvitamin D receptor utilizing purified receptor from pig intestine as a standard and two high affinity monoclonal antibodies directed to two different epitopes on the receptor. In this assay a fixed amount of 125I-labeled antibody is incubated with a fixed amount of a second antireceptor antibody linked to biotin and increasing amounts of purified receptor protein or sample. Antibody-receptor complexes can then be immunoprecipitated with avidin-Sepharose beads and counted. This method is highly reproducible and can detect 150 pg of 1,25-dihydroxyvitamin D3 receptor in crude extracts with intra- and interassay coefficients of variation of 8.6 and 18.2%. The monoclonal antibodies used recognize both native and denatured receptors from several different species, including human. This immunoradiometric assay should prove useful for studies of receptor regulation, occupancy, distribution, and turnover.
Sandgren
M E
ME
Department of Biochemistry, University of Wisconsin-Madison 53706.
Deluca
H F
HF
D000818:Animals; D000911:Antibodies, Monoclonal; D002117:Calcitriol / Q000494:pharmacology; D002417:Cattle; D000939:Epitopes / Q000276:immunology; D006801:Humans; D015592:Immunoradiometric Assay / Q000379:methods*; D051379:Mice; D051381:Rats; D018167:Receptors, Calcitriol; D011987:Receptors, Steroid / Q000032:analysis*; D015203:Reproducibility of Results; D013045:Species Specificity; D013256:Steroids / Q000378:metabolism; D013552:Swine
D000911:Antibodies, Monoclonal; D000939:Epitopes; D018167:Receptors, Calcitriol; D011987:Receptors, Steroid; D013256:Steroids; D002117:Calcitriol