单核细胞增生性李斯特菌化学发光免疫检测法的建立及验证  

Development and verification of chemiluminescence immunoassay for Listeria monocytogenes

作　　者：栗克文 鲁倩茹 黄凤玲 邢珂慧 邵佩兰[1] LI Ke-wen;LU Qian-ru;HUANG Feng-ling;XING Ke-hui;SHAO Pei-lan(College of A griculture,Ningxia Uniwversity,Yinchuan 750021,Ningxia Hui A utonomous Region,China)

机构地区：[1]宁夏大学农学院,宁夏银川750021 [2]河南美凯生物科技有限公司,河南漯河462001

出　　处：《中国生物制品学杂志》2021年第8期989-993,1001,共6页Chinese Journal of Biologicals

基　　金：石家庄市科学技术研究与发展计划项目(177460166A)。

摘　　要：目的建立检测单核细胞增生性李斯特菌(简称单增李斯特菌)化学发光法并进行验证。方法以单增李斯特菌免疫原分别免疫BALB/c小鼠及新西兰大白兔,制备鼠源单抗和兔血清多抗,建立检测单增李斯特菌化学发光免疫法,优化兔血清多抗包被浓度及单增李斯特菌反应时间,验证方法的检测范围、灵敏度、特异性、准确性及稳定性。结果制备的单增李斯特菌兔血清多抗及鼠源单抗效价分别为1∶256 000和1∶128 000。兔血清多抗最适包被浓度为0.05 mg/mL,与单增李斯特菌最适反应时间为40 min。该方法检测单增李斯特菌浓度范围为1×10^(1)~1×10^(7)C^FU/m L,灵敏度小于10 CFU/mL;除单增李斯特菌ATCC19115外,单增李斯特菌CMCC54003、CMCC54004、英诺克李斯特菌也可检测到,金黄色葡萄球菌、大肠埃希菌,沙门菌未检出;添加1×10^(4)CFU/mL单增李斯特菌的火腿肠、牛奶及冰激凌溶液中回收率分别为90.3%、101.6%和969.3%;包被好的化学发光板密闭封装37℃放置28 d后,单增李斯特菌发光值为初始包被值的75.3%。结论成功建立了检测单增李斯特菌化学发光法,该方法具有检测时间短、范围宽,特异性强等特性,为促进食品安全检测提供了参考依据。Objective To develop and verify a chemiluminescence immunoassay for Listeria monocytogenes.Methods BALB/c mice and New Zealand rabbits were immunized with the L.monocytogenes as an immunogen to prepare mouse monoclonal antibody and rabbit polyclonal antibody respectively,based on which a chemiluminescence immunoassay for L.monocytogenes was developed.The polyclonal antibody concentration for coating and the reaction time with L.monocytogenes were optimized,and the developed method was verified for detection range,sensitivity,specificity,accuracy and stability.Results The titers of prepared polyclonal and monoclonal antibodies were 1∶256000 and 1∶128000 respectively.The optimal concentration of polyclonal antibody for coating was 0.05 mg/mL,while the optimal reaction time with L.monocytogenes was 40 min.The detection range of the developed method for L.monocytogenes was 1×10^(1)~1×10^(7) CFU/mL,while the sensitivity was less than 10 CFU/mL.Besides L.monocytogenes ATCC19115,L.monocytogenes CMCC54003,CMCC54004 and L.innocua were also detected by the developed method,while Staphylococcus aureus,E.coli and salmonella were undetected.The recovery rates of L.monocytogenes in ham sausage,milk and ice cream,added with 1×10^(4) CFU/mL L.monocytogenes,were 90.3%,101.6%and 969.3%respectively.After the coated chemiluminescence plate was closed and incubated at 37℃for 28 d,the chemiluminescence value of L.monocytogenes was 75.3%of the original value.Conclusion The chemiluminescence immunoassay for L.monocytogenes was successfully prepared,which was time-saving,abroad spectrum,specific,and provided a reference for promoting the food safety inspection.

关 键 词：单核细胞增生性李斯特菌 化学发光免疫法 双抗体夹心ELISA法 食源性病原菌 

分 类 号：R155[医药卫生—营养与食品卫生学]