Abstract

A nonisotopic assay of vitamin B-12 in human serum or plasma is described, performed with the Abbott IMx analyzer. The sample is first treated at pH > 12.5 to release bound vitamin B-12 and to convert all forms to cyanocobalamin. Next, the analyte is bound, at lower pH, by vitamin B-12-specific binding protein, immobilized to a solid phase of polymeric microspheres. Detection involves monitoring the activity of the tracer enzyme (alkaline phosphatase) coupled to a derivative of cyanocobalamin. Total assay precision is 7.9% for vitamin B-12 at 200 ng/L, 6.6% at 400 ng/L, and 6.7% at 800 ng/L. Assay sensitivity, calculated as 2 SD from the zero calibrator, is 37 (+/- 9) ng/L. The dynamic range extends to 2000 ng/L. Analytical recovery of 300 and 600 ng/L additions of vitamin B-12 to sera with basal concentrations of 30-400 ng/L was 102.5%. Results of the assay correlated well with those of commercially available radioisotope assays. No interference was observed in specimens from patients with pernicious anemia, chronic or acute myelogenous leukemia, or renal failure. Cross-reactivity with cobinamide (1 g/L) was < 0.00003%. Vitamin B-12 measurements for blood specimens drawn into serum, EDTA, or heparinized plasma-collection tubes agreed within 3%.