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Enzyme immunoassay for measuring 25-hydroxyvitamin D₃ in serum

Center for Clinical & Basic Research, Ballerup Byvej 222, DK-2750 Ballerup, Denmark.
^a Author for correspondence. Fax +45 44 68 42 20; e-mail byrjal{at}biobase.dk

We developed a rapid, competitive enzyme immunoassay (EIA) for measuring 25-hydroxyvitamin D₃ [25(OH)D₃] in serum. The EIA was based upon 25(OH)D₃-3-hemisuccinate covalently coupled to secondary amino groups grafted onto the polystyrene surface of microtiter wells. Optimal coupling conditions were established, and we found that inclusion of 40 µmol/L chloramine T, an agent not previously described for use in coupling to these plates, resulted in both more reproducible coupling as well as more than a twofold increase in the coupling efficiency. Before EIA, 25(OH)D₃ was extracted from the serum samples by acetonitrile, and the redissolved extract was incubated with polyclonal rabbit antibody raised against 1,25-dihydroxyvitamin D₃-3-hemisuccinate conjugated to bovine serum albumin. Peroxidase-labeled antibody raised in goat against rabbit immunoglobulins was used for detection. The detection limit of the EIA was 4.4 µg/L; recovery 102%; on-plate CV 11%; within-run CV including extraction 12%, and between-run CV 15%. There was no clinically important cross-reactivity with other vitamin D metabolites, and results obtained by the EIA were compared with results obtained by a previously described RIA.

Key Words: indexing terms: vitamin D status • steroid hormones • cholecalciferol • EIA • covalent coupling

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