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目的用免疫分析法检测水产品中无色孔雀石绿(LMG)的残留,研究LMG人工抗原及其多克隆抗体的制备和鉴定。方法通过混酸硝化还原法对LMG进行改造,获得氨基无色孔雀石绿(NH2-LMG),然后采用改良的碳二亚胺法制备LMG完全抗原。用该人工抗原免疫动物,获得多抗血清。采用酶联免疫法测定抗体效价。选择与LMG结构相似的5种药物进行交叉反应,计算交叉反应率。结果经紫外光谱及电泳法检测,LMG与载体蛋白偶联成功,偶联比为1∶17。利用该抗原免疫小鼠后获得了效价高于104的抗LMG抗体,交叉反应结果表明该抗体有较好的特异性。结论成功合成了LMG人工抗原,为进一步制备LMG单克隆抗体及研究LMG的新型、快速检测方法奠定了基础。
Objective To detect the residue of colorless malachite green (LMG) in aquatic products by immunoassay and to study the preparation and identification of LMG artificial antigen and its polyclonal antibody. Methods LMG was modified by mixed acid nitrification and reduction method to obtain amino-colorless malachite green (NH2-LMG), and then LMW complete antigen was prepared by modified carbodiimide method. Animals were immunized with this artificial antigen to obtain multiple antisera. Antibody titers were determined by enzyme-linked immunosorbent assay. Five drugs with similar structure to LMG were selected for cross-reaction and the cross-reactivity rate was calculated. The results of UV spectrophotometry and electrophoresis detection, LMG and carrier protein coupling success, the coupling ratio of 1:17. The anti-LMG antibody titer higher than 104 was obtained after the mouse was immunized with the antigen, and the cross-reaction result showed that the antibody has good specificity. Conclusion The LMG artificial antigen was successfully synthesized, which laid the foundation for the further preparation of the monoclonal antibody against LMG and the new and rapid detection of LMG.