Competitive immunoassay for analysis of vitamin B(12)

In the current work, direct competitive enzyme-linked immunosorbent assay (ELISA) was developed for derivatized vitamin B₁₂ by generating chicken egg yolk immunoglobulins (IgY) against derivatized vitamin B₁₂ and purified using affinity chromatography. Checkerboard assay was performed with vitamin B₁₂ antibody and vitamin B₁₂–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 B₁₂ antibody was found to be 4.23 × 10⁸ L/mol. Cross-reactivity with other water-soluble vitamins was found to be less than 0.01% except for analogs of vitamin B₁₂ 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 B₁₂ 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 B₁₂ using Amberlite XAD, the developed ELISA method correlated well with the established HPLC method with a correlation coefficient of 0.90.