ENZYME-IMMUNOASSAY FOR MEASURING 25-HYDROXYVITAMIN D-3 IN SERUM

We developed a rapid, competitive enzyme immunoassay (EIA) for measuri ng 25-hydroxyvitamin D-3 [25(OH)D-3] in serum. The EIA was based upon 25(OH)D-3-3-hemisuccinate covalently coupled to secondary amino groups grafted onto the polystyrene surface of microtiter wells. Optimal cou pling conditions were established, and we found that inclusion of 40 m u mol/L chloramine T, an agent not previously described for use in cou pling to these plates, resulted in both more reproducible coupling as well as more than a twofold increase in the coupling efficiency. Befor e EIA, 25(OH)D, was extracted from the serum samples by acetonitrile, and the redissolved extract was incubated with polyclonal rabbit antib ody raised against 1,25-dihydroxyvitamin D-3-3-hemisuccinate conjugate d to bovine serum albumin. Peroxidase-labeled antibody raised in goat against rabbit immunoglobulins was used for detection. The detection l imit of the EIA was 4.4 mu g/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 metabolite s, and results obtained by the EIA were compared with results obtained by a previously described RIA.