Enzyme-linked immunosorbent assay for plasma 24,25-dihydroxyvitamin D3

A highly sensitive enzyme-linked immunosorbent assay (ELISA) of (24R)-24,25-dihydroxyvitamin D-3 [24,25(OH)(2)D-3] has been developed for measuring its plasma levels. An antibody elicited against 24,25(OH)(2)D-3 3-hemiglutarate conjugated with bovine serum albumin was used for this purpose. Two enzyme (beta-galactosidase)-labeled antigens linked with the above hapten or its homologue (succinate) were prepared by the N-succinimidyl ester method. Both homologous and bridge-heterologous ELISA systems employing the sequential saturation method exhibited a high sensitivity with a midpoint of 25 pg, which was about one-tenth of the conventional competitive protein binding assay. A lipid ex:tract from plasma was treated with alkaline buffer and purified with an Isolute C18 (EC) cartridge, and 24,25(OH)(2)D-3 levels were determined with the homologous ELISA. This ELISA afforded reliable plasma 24,25(OH)(2)D-3 levels which were confirmed by a serial dilution study and recovery test. The overall recovery rate through the pretreatment was satisfactory and constant (84.7 +/- 4.3%, n=52, mean+/-SD), and recovery revision by the addition of H-3-labeled compound to each plasma sample was omitted. Intra- and inter-assay coefficients of variation were less than 13%. (C) 1998 Elsevier Science B.V.