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Competitive immunoassay for analysis of vitamin B12.

Sagaya Selvakumar, L. and Thakur, M. S. (2011) Competitive immunoassay for analysis of vitamin B12. Analytical Biochemistry, 418. pp. 238-246.

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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 (Ka) 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.

Item Type: Article
Uncontrolled Keywords: Vitamin B12, IgY antibody, ELISA, HPLC, Pharmaceutical and food analysis
Subjects: 600 Technology > 08 Food technology > 01 Analysis
600 Technology > 08 Food technology > 16 Nutritive value > 04 Vitamins
Divisions: Fermentation Technology and Bioengineering
Depositing User: Food Sci. & Technol. Information Services
Date Deposited: 20 Jan 2012 05:13
Last Modified: 16 Jan 2017 12:18
URI: http://ir.cftri.com/id/eprint/10533

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