A Selective Immunoaffinity Chromatography for Determination of Plasma 1α,25-Dihydroxyvitamin D3: Application of Specific Antibodies Raised against a 1α,25-Dihydroxyvitamin D3–Bovine Serum Albumin Conjugate Linked through the 11α-Position

Autor: Kazutake Shimada, Hidetoshi Mano, Junichi Kitahori, Norihiro Kobayashi, Tetsuya Imazu
Rok vydání: 1997
Předmět:
Zdroj: Analytical Biochemistry. 244:374-383
ISSN: 0003-2697
Popis: A selective and simple immunoaffinity chromatography of 1 alpha, 25-dihydroxyvitamin D3 has been developed and found to be a useful pretreatment tool for determining the metabolite in human plasma. A reasonably designed, haptenic derivative, 11 alpha-hemiglutaryloxy-1 alpha, 25-dihydroxyvitamin D3, was linked to bovine serum albumin, and rabbits were immunized repeatedly with the conjugate. The resulting polyclonal antibodies were specific to 1 alpha, 25-dihydroxyvitamin D3, recognizing both the A-ring and the side-chain structures. The antibodies were then immobilized on agarose gel to produce an immunosorbent which was stable and repeatedly usable. A plasma extract prepared with a Chem Elut column was applied to an affinity column containing the immunosorbent. After adequate washing, the adsorbed 1 alpha, 25-dihydroxyvitamin D3 was eluted selectively with a satisfactory recovery rate. This immunoaffinity chromatography enabled a simple radioreceptor assay for human plasma 1 alpha, 25-dihydroxyvitamin D3 which does not require any preparative high-performance liquid chromatography. The mean (+/-SD) values for 30 normal subjects and 8 patients with chronic renal failure were 36.0 (10.2) and 13.1 (2.1) pg/ml, respectively. The present method also gave reliable assay values for the plasma specimens from 1 alpha-hydroxyvitamin D3-administered volunteers, which have conventionally been difficult to measure unless complicated pretreatment is used.
Databáze: OpenAIRE
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