Enzyme immunoassay for measuring 25-hydroxyvitamin D3 in serum.
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 mumol/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 micrograms/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.
Lind
C
C
Center for Clinical & Basic Research, Ballerup, Denmark.
Chen
J
J
Byrjalsen
I
I
D000328:Adult; D000368:Aged; D000818:Animals; D002112:Calcifediol / Q000097:blood*; D002138:Calibration; D002417:Cattle; D005022:Ethyldimethylaminopropyl Carbodiimide / Q000737:chemistry; D006736:Horses; D006801:Humans; D007124:Immunoenzyme Techniques; D008832:Microchemistry / Q000379:methods; D008875:Middle Aged; D011817:Rabbits; D011863:Radioimmunoassay; D012680:Sensitivity and Specificity; D013386:Succinates / Q000737:chemistry; D013388:Succinimides / Q000737:chemistry
D013386:Succinates; D013388:Succinimides; C001426:N-hydroxysuccinimide; D002112:Calcifediol; D005022:Ethyldimethylaminopropyl Carbodiimide