Improved radioimmunoassay for vitamin D and its use in assessing vitamin D status.
We describe a faster, more-sensitive radioimmunoassay for vitamin D in plasma. Antibodies were generated in rabbits immunized with a new vitamin D analog, the 23,24,25,26,27-pentanor-C(22)-carboxylic acid of vitamin D, coupled directly with bovine serum albumin. After several months, Rivanol-treated sera from the rabbits contained high-titer antibodies, as determined by their abilities to bind 25-hydroxy-[3H]cholecalciferol. The antibody, used at a 1:15 000 final dilution, cross reacted equally with all cholecalciferol and ergocalciferol metabolites tested except 1,25-dihydroxycalciferol metabolites and the parent calciferols. 25-Hydroxycalciferol and similar forms were efficiently extracted from plasma or serum with acetonitrile. We separated bound from free 25-hydroxy-[3H]cholecalciferol by using a second antibody: goat antiserum to rabbit serum. The detection limit of the assay was 3.0 pg of 25-hydroxycholecalciferol and its equivalents per tube; thus only 1 microL of plasma is needed per assay tube. Results compared well with those from a liquid-chromatographic procedure involving specific ultraviolet detection of 25-hydroxycalciferol in plasma.
D000818:Animals; D000917:Antibody Formation; D002851:Chromatography, High Pressure Liquid; D003429:Cross Reactions; D006801:Humans; D007223:Infant; D011817:Rabbits; D011863:Radioimmunoassay; D012710:Serum Albumin, Bovine; D013056:Spectrophotometry, Ultraviolet; D014807:Vitamin D / Q000097:blood*; D014808:Vitamin D Deficiency / Q000175:diagnosis
D014807:Vitamin D; D012710:Serum Albumin, Bovine