腈菌唑单克隆抗体制备与胶体金检测方法的建立

Preparation of monoclonal antibody against myclobutanil and establishment of a method for detection of colloidal gold

目的制备高灵敏度腈菌唑单克隆抗体,建立芹菜样本中腈菌唑胶体金快速检测方法。方法对腈菌唑化学结构进行改造,合成两种半抗原,与载体蛋白偶联,免疫小鼠,制备单克隆抗体。结合胶体金技术,优化抗体标记参数等,建立胶体金的检测方法,并对检测性能进行评价。结果由所制备的单克隆抗体开发的胶体金试纸条半数抑制浓度为2.10μg/L,线性范围为0.62~7.25μg/L,与其他三唑类杀菌剂和芹菜中常见杀虫剂的交叉反应率均小于5%;可定性检测芹菜样本中腈菌唑是否超过GB2763—2021《食品安全国家标准食品中农药最大残留限量》限量,检出限为50μg/kg;通过实际阳性样本测试与气相色谱-串联质谱法比较,符合性较好,结果可靠。结论基于两种半抗原免疫制备的抗体,可建立腈菌唑胶体金检测方法,检出限满足国家标准限量要求,可应用于现场的快速筛查。

Objective To prepare high-sensitivity monoclonal antibodies against myclobutanil and establish a rapid detection method for colloidal gold of myclobutanil in celery samples.Methods The chemical structure of myclobutanil was modified to synthesize 2 kinds of haptens,which were coupled with carrier protein to immunize mice and prepare monoclonal antibodies.Combined with colloidal gold technology,the parameters of antibody labeling were optimized,and the detection method of colloidal gold was established and the detection performance was evaluated.Results The half maximal inhibitory concentration and linear range of the colloidal gold strip developed from the prepared monoclonal antibody were 2.10μg/L and 0.62‒7.25μg/L,respectively,and the cross-reaction rates with other triazole fungicides and common insecticides in celery were less than 5%.The limit of detection was 50μg/kg,which could qualitatively detect whether myclobutanil in celery samples exceeds the GB 2763—2021 National food safety standard-Maximum residue limits for pesticides in food national standard limit.Compared with gas chromatography-tandem mass spectrometry,the actual positive sample test had good coincidence and reliable results.Conclusion Based on the antibodies prepared by 2 kinds of haptens immunizations,the detection method of myclobutanil colloidal gold can be established,and the limit of detection can meet the requirements of the national standard limit,which can be used in the field of rapid screening.