A selective immunoaffinity chromatography for determination of plasma 1 alpha, 25-dihydroxyvitamin D3: application of specific antibodies raised against a 1 alpha, 25-dihydroxyvitamin D3-bovine serum albumin conjugate linked through the 11 alpha-position.
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.
Kobayashi
N
N
Faculty of Pharmaceutical Sciences, Kanazawa University, Japan.
Imazu
T
T
Kitahori
J
J
Mano
H
H
Shimada
K
K
D000818:Animals; D000906:Antibodies / Q000276:immunology; D002117:Calcitriol / Q000031:analogs & derivatives / Q000097:blood* / Q000276:immunology; D002417:Cattle; D002846:Chromatography, Affinity / Q000379:methods*; D005260:Female; D005977:Glutarates / Q000276:immunology; D006241:Haptens / Q000276:immunology; D006801:Humans; D011817:Rabbits; D012680:Sensitivity and Specificity; D012710:Serum Albumin, Bovine
C104974:11-hemiglutaryloxy-1,15-dihydroxyvitamin D3; D000906:Antibodies; D005977:Glutarates; D006241:Haptens; D012710:Serum Albumin, Bovine; D002117:Calcitriol