Competitive immunoassay for analysis of vitamin B(12).

In the current work, direct competitive enzyme-linked immunosorbent assay (ELISA) was developed for derivatized vitamin B(12) by generating chicken egg yolk immunoglobulins (IgY) against derivatized vitamin B(12) and purified using affinity chromatography. Checkerboard assay was performed with vitamin B(12) antibody and vitamin B(12)-alkaline phosphatase conjugate followed by its conjugate characterization using ultraviolet (UV) spectroscopy and high-performance liquid chromatography (HPLC). The limit of detection was 10 ng/ml with a linear working range of 10 to 10,000 ng/ml. The affinity constant (K(a)) of the vitamin B(12) antibody was found to be 4.23×10(8) L/mol. Cross-reactivity with other water-soluble vitamins was found to be less than 0.01% except for analogs of vitamin B(12) that showed 12% to 35%. The intra- and interassay coefficients of variation were found to be in the ranges from 0.0005% to 1.2% and 0.009% to 1.03%, respectively. The assay was validated with the HPLC method in terms of sensitivity, specificity, precision, and recovery of vitamin B(12) with spiked multivitamin injections, tablets, capsules, and chocolates. The HPLC method had a detection limit of 500 ng/ml with a linear working range of 1000 to 10,000 ng/ml. After extraction of vitamin B(12) using Amberlite XAD, the developed ELISA method correlated well with the established HPLC method with a correlation coefficient of 0.90.