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A highly sensitive enzyme-linked immunosorbent assay (ELISA) of (24R)-24,25-dihydroxyvitamin D3 [24,25(OH)2D3] has been developed for measuring its plasma levels. An antibody elicited against 24,25(OH)2D3 3-hemiglutarate conjugated with bovine serum albumin was used for this purpose. Two enzyme (β-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 extract from plasma was treated with alkaline buffer and purified with an Isolute C18 (EC) cartridge, and 24,25(OH)2D3 levels were determined with the homologous ELISA. This ELISA afforded reliable plasma 24,25(OH)2D3 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 3 H -labeled compound to each plasma sample was omitted. Intra- and inter-assay coefficients of variation were less than 13%. |
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