An improved competitive protein-binding assay for 25-hydroxy vitamin D

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Abstract
A competitive protein binding assay for 25-hydroxy vitamin D in serum is described using serum from a pregnant woman as source of binding protein. Introduction of a polyvinyl alcohol to the reaction buffer enhanced the solubility of 25-hydroxy vitamin D in aqueous solutions and nearly eliminated loss of material to the walls of reaction tubes. Addition of a protein fraction obtained by gel filtration of human serum, shown to bind only vitamin D, enhanced the specificity and reproducibility of the assay without interfering with the binding properties of 25-hydroxy vitamin D. Serum samples were extracted by chloro-form-methanol. Comparison of column chromatography on silicic acid and Sephadex LH-20 showed that both systems gave good separation of vitamin D and 25-hydroxy vitamin D and with a similar recovery of about 80%. The silicic acid columns were less time consuming, easier to handle and cheaper in use, and was therefore preferred as the routine separation method. The assay has a detection limit of 0.4 nmol/l in serum, and an intra- and inter-assay coefficient of variation of 6.8 and 8.1%, respectively.

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