An Immunoaffinity Gel Column Assay for Visualizable Detection of Furaltadone Metabolites
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Abstract:
To establish a visualizable immunoaffinity gel column detection method for analysis of the furoxone metabolites (AMOZ) in animal-derived foods, derivatization of AMOZ with aldehyde benzoic acid (CPA) was conducted to yield the derivative CPAMOZ for the preparation of AMOZ complete antigen. The CPAMOZ polyclonal antibody was obtained via immunization of the mouse, and the titer and specificity of the antibody were determined using an indirect ELISA. CPAMOZ antibody gel and HRP antibody gel were prepared by coupling the cyanogen bromide-activated agarose gel with CPAMOZ antibody and HRP antibody, respectively, as a detection layer and a quality control layer of the affinity gel column. The AMOZ visualized immunoaffinity gel column analysis method was established with the optimal working conditions as: dilution factor 5000, HRP gel dilution 20 times, and CPAMOZ antibody gel dilution 15 times. The experimental results showed that the CPAMOZ polyclonal antibody had a titer of 1:64000 and an IC50 value of 2.19 μg/L, with good sensitivity and specificity. The detection limit of the established AMOZ visualizable immunogel column method was 4 μg/L, and the detection limit of AMOZ in the actual sample of animal origin was 2 μg/kg. The method is fast and convenient and meets the requirements for high-throughput screening of AMOZ in foods in China.
