孔雀石绿间接竞争ELISA检测方法的建立

采用无色孔雀石绿(Leucomalachite green,LMG)单克隆抗体建立了水产品中孔雀石绿(Malachite green,MG)残留的间接竞争ELISA(ciELISA)检测方法。结果表明,LMG-McAb最佳稀释倍数为1∶80 000,包被抗原最佳质量浓度为0.80μg/mL;竞争反应时的LMG理想稀释液为40%乙腈水溶液;标准曲线呈线性相关,相关系数R2=0.9823,最适检测范围1 ng/mL~256 ng/mL,最低检测限为1.29 ng/mL,批内和批间变异系数分别为4.307%和4.566%;鳗鱼肉样的平均添加回收率为90%~110%;该检测方法与隐性结晶紫、孔雀石绿、结晶紫的交叉反应(CR%)分别为40.67%、13.50%和5.89%,与其他抗生素无交叉反应。

Establishment of an indirect competitive enzyme-linked immunosorbent assay for detecting the residues of Malachite green

An indirect competitive enzyme-linked immunosorbent assay(ciELISA) based on monoclonal antibody was developed for the detection of Malachite green residues in fishery product.The results demonstrated that the optimal concentrations of the coating antigen and monoclonal antibody against Leucomalachite green(LMG) were 1∶80 000 and 0.80 μg/mL,respectively.Desirable dilution to LMG was Methyl Cyanides / demineralized water mixture(2∶3;V/V).Linear detection of ciELISA covered a range from 1 ng/mL to 256 ng/mL(R2=0.9823) and it had a sensitivity of 1.29 ng/mL.The intra-assay and inter-assay CVs were 4.307% and 4.566%,respectively.And the analytical recovery of the method was 90%~110%.The ELISA had 40.67%,13.50% and 5.89% of cross-reactivity with Leucocrystal violet,Malachite green and crystal violet,respectively.There was no cross-reactivity with other antibiotics.