Determination of Histamine Residues in Aquatic Products by Indirect Competitive Enzyme-linked Immunosorbent Assay
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Abstract:
In this work, a novel monoclonal antibody against histamine derivative was obtained, and an indirect competitive enzyme-linked immunoassay (ic-ELISA) was developed for the analysis of histamine in aquatic products. The histamine hapten was prepared via three steps of chemical reactions, and then, the hapten was conjugated to carrier proteins of BSA and OVA by the active ester method to form immounogen and coating antigen, respectively. The results of UV scanning showed that histamine haptens were successfully conjugated with carrier protein. Histamine antiserum was prepared by immunizing New Zealand white rabbits, and then purified by caprylic acid-ammonium sulfate method. Under the optimized conditions, The IC50 of this developed ic-ELISA was 0.91 ng/mL, and negative cross reactions (CR) of antibody to Histidine, n-acyl histidine, 3-methyl histamine and other histamine analogues and their derivatives were observed, indicating the good specific method. The calibration curve of this ic-ELISA had a linear detection of 0.1~8.1 ng/mL, with the detect limit detection of 0.10 ng/mL. The average recoveries of histamine in fortified aquatic products were in the range of 98.9~130.1%. Therefore, the established ic-ELISA in this work is a practical method for determination of histamine residues in aquatic products.
