High performance liquid chromatography of the 1, 25-dihydroxyvitamin D₃ receptor: Its application toward resolution of receptor-monoclonal antibody complexes

The present report describes the application of high performance liquid chromatography (HPLC) towards the analysis of chick intestinal 1, 25-dihydroxyvitamin D₃ (1, 25(OH)₂D₃) receptors and their interaction with anti-receptor monoclonal antibodies. Hormone-labeled vitamin D₃ receptors were chromatographed under high pressure on an Altec Spherogel TSK 3000 sw column. Both radioactivity and DEAE-filtration measurements of the eluted fractions indicated a high yield (91%) of 1, 25(OH)₂D₃ receptor. Specific binding was both displaceable with 100-fold excess radioinert hormone and dissociable with 2 mM p-chloromercuribenzene sulfonate. In contrast, when 1, 25(OH)₂D₃ receptors were preincubated with anti-receptor monoclonal antibodies, the receptor's elution time was significantly accelerated to that expected for a larger macromolecule. This position corresponded to that observed for either pure or internally [³⁵S]methionine-labeled monoclonal antibody. These results suggest that HPLC analysis and resolution of both receptor and receptor-antibody complex may be of future practical value.