Tandem immunoaffinity chromatography for plasma 1α,25-dihydroxyvitamin D3 utilizing two antibodies having different specificities: A novel and powerful pretreatment tool for 1α,25-dihydroxyvitamin D3 radioreceptor assays

We report here a novel and powerful pretreatment method for radioreceptor assays (RRAs) for human plasma 1α,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) based on “Tandem” immunoaffinity chromatography (Tandem IAC). Two antibodies having different specificities were each immobilized on agarose gel with cyanogen bromide to produce immunosorbents which were stable and repeatedly usable. An ethyl ether extract of plasma was applied to the first affinity column, from which 1,25(OH)₂D₃ could be preferentially eluted and separated from 1α-deoxy type metabolites. The effluent was then submitted to the second column, and the 1,25(OH)₂D₃ retained was eluted after non- or weakly-adsorbed interfering substances were washed out. This procedure allowed efficient purification without careful handling or strict time-management in the entire operation and enabled avoiding preparative high-performance liquid chromatography (HPLC) from RRA even with a conventional chick intestinal vitamin D receptor. Mean (± SD) plasma 1,25(OH)₂D₃ values of 56 normal subjects and 10 patients with chronic renal failure, obtained with this Tandem IAC/RRA system, were 36.4 (8.7) and 11.2 (4.0) pg/ml, respectively. The Tandem IAC will also be useful for developing immunoassays or gas chromatography-mass spectrometry of 1,25(OH)₂D₃.