间接竞争酶联免疫吸附测定水产品中的组胺残留

Determination of Histamine Residues in Aquatic Products by Indirect Competitive Enzyme-linked Immunosorbent Assay

摘要:

本文通过制备特异性识别组胺衍生物的多克隆抗体，建立了针对水产品中组胺的间接竞争酶联免疫吸附分析方法（ic-ELISA）。以组胺等为反应原料经三步化学反应合成组胺半抗原，采用活泼酯法将组胺半抗原分别与牛血清蛋白（BSA）和卵清蛋白（OVA）偶联作为包被原和免疫原，用紫外扫描法进行鉴定，结果显示组胺半抗原与载体蛋白偶联成功。通过免疫新西兰大白兔制备组胺抗血清，采用辛酸-硫酸铵方法纯化血清获得了相应的特异性多克隆抗体。通过对icELISA系列反应条件的摸索，确定了各组分的最适工作条件。试验结果表明：该方法对于组胺的IC50为0.91 ng/mL，与组氨酸、N-酰基组氨酸、3-甲基组胺等多种类似物及其衍生物均无交叉反应，方法特异性良好；线性检测范围为0.1~8.1 ng/mL，检出限为0.10 ng/mL；按照10、20、30 ng/g的添加量添加组胺至鱼、虾和贝类样品中，测得其回收率为98.9%~130.1%。方法检测的准确性好灵敏度高，适用于实际水产样品中组胺的检测。

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.