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目的以单增李斯特菌(LM)内化素A蛋白(InlA)单克隆抗体为基础,研制其胶体金免疫层析检测试纸条。方法采用DNAStar软件对LM inlA全长基因编码蛋白进行抗原表位分析,截取部分inlA基因片段构建原核表达质粒,诱导表达和纯化获得重组蛋白。以该蛋白免疫BALB/c小鼠,筛选高效分泌抗InlA单克隆抗体的杂交瘤细胞株,制备单克隆抗体;以双抗体夹心的原理研制胶体金免疫层析检测试纸条,并对其特异性、敏感性、稳定性进行评价。结果筛选到2株高效分泌抗InlA单克隆抗体的杂交瘤细胞株,抗体属于IgG1亚类,小鼠腹水抗体效价为1∶64 000;研制的试纸条可与LM发生阳性反应,而与非LM李斯特菌、链球菌、鼠伤寒沙门菌、大肠杆菌O157∶H7等食源性致病菌均不发生阳性反应;LM纯培养物敏感性为2.4×105cfu/ml,模拟猪肉样品敏感性为4.0×106cfu/ml;4℃保存期可达16周以上。结论研制的胶体金免疫层析试纸条具有快速、特异、敏感等优点,可以用于样品中LM的快速检测。
OBJECTIVE To develop a colloidal gold immunochromatographic test strip based on the monoclonal antibody against Listeria monocytogenes (LM) endonuclease A protein (InlA). Methods DNAStar software was used to analyze the epitope of the full-length LM inlA gene. The prokaryotic expression plasmid was constructed by intercepting some of the inlA gene fragments, and the recombinant protein was induced and expressed. BALB / c mice were immunized with this protein, and the hybridoma cell line secreting anti-InlA monoclonal antibody was screened to prepare monoclonal antibody. The colloidal gold immunochromatographic test strip was developed based on the principle of double antibody sandwich, and its specificity Sex, sensitivity, stability evaluation. Results Two hybridoma cell lines secreting anti-InlA monoclonal antibodies were screened. The antibodies belonged to IgG1 subclass. The titer of mouse ascites antibody was 1:64 000. The test strip developed positive reaction with LM, Non-LM listeria, Streptococcus, Salmonella typhimurium, Escherichia coli O157: H7 and other food-borne pathogens were not positive reaction; LM pure culture sensitivity of 2.4 × 105cfu / ml, to simulate the sensitivity of pork samples 4.0 × 106cfu / ml; 4 ℃ preservation period of up to 16 weeks or more. Conclusion The colloidal gold immunochromatographic strip has the advantages of rapid, specific and sensitive, which can be used for the rapid detection of LM in the sample.