This chapter describes the methodology associated with the application of the monoclonal antibodies toward the detection and characterization of the 1,25-(OH)2D3 receptor. The methodology involves the preparation of monoclonal antibodies, preparation and hormone labeling of l,25-(OH)2D3 receptors, sedimentation displacement analysis (SDA), immunoprecipitation analysis, radioligand immunoassay (RLIA), and immunoblot assay. Formation of 1,25-(OH)2D3 receptor complexes from both tissue as well as cultured cell extracts is achieved by incubating the cytosol with concentrations of 1,25-(OH)2[3H]D3 (120–170 Ci/mmol) ranging from 1 to 4 nM in a final volume containing 10% absolute ethanol. SDA is useful in determining the relative immunochemical similarities of various 1,25-(OH)2D3 receptor species. Preformed 1,25-(OH)2[3H]D3-receptor complexes (0.05–0.1 pmol) derived from chick intestinal cytosol or cytosols obtained from other tissues or cultured cell lines are incubated with preimmune or immune serum, hybridoma medium, or purified monoclonal antibody for periods of 1–4 hr at 4° in KETD-0.3 buffer. Immunoprecipitation represents an extremely valuable technique whereby immunoglobulin-bound 1,25- (OH)2[3H]D3 receptors can be quantitated and interactions between 1,25- (OH)2[3H]D3 receptors precisely assessed. Immunoprecipitation of 1,25-(OH)2[3H]D3-receptor complexes bound to monoclonal antibodies such as 9A7γ, 4A5γ, 8D3μ, or antiserum is achieved by the addition of carrier rat serum and anti-rat Ig antiserum, followed by overnight incubation at 4°. The RLIA for receptors is a competition assay in which the standard curve is created by competing chick intestinal cytosolic 1,25-(OH)2[3H]D3 receptor (16 fmol) with increasing concentrations of radioinert 1,25-(OH)2D3 receptor complex (0–240 fmol) for the 9A7γ monoclonal antibody. Immunoblot assay is essential in confirming that a particular protein species purified to homogeneity is in fact the 1,25-(OH)2D3 receptor.
ASJC Scopus subject areas
- Molecular Biology