胞内劳森菌夹心ELISA检测方法的建立与应用  

Establishment and Application of Sandwich ELISA Method for Detecting Lawsonia intracellularis

作　　者：洪润婧 周红[1] 蔺辉星[1] 范红结[1] HONG RunJing;ZHOU Hong;LIN HuiXing;FAN HongJie(College of Veterinary Medicine,Nanjing Agricultural University,Nanjing 210095)

机构地区：[1]南京农业大学动物医学院,南京210095

出　　处：《中国农业科学》2025年第5期1032-1042,共11页Scientia Agricultura Sinica

基　　金：国家重点研发计划课题(2021YFD1800404);江苏省农业科技自主创新资金项目(CX(23)1029);江苏高校优势学科建设工程专项资金项目(PAPD)。

摘　　要：【目的】通过建立检测胞内劳森菌(Lawsonia intracellularis,LI)的夹心ELISA方法,进行猪增生性肠炎(porcine proliferative enteropathy,PPE)灭活疫苗研制中抗原含量的测定。【方法】以LI为免疫原免疫新西兰大白兔制备抗LI多克隆抗体;同时免疫6周龄BALB/c小鼠,通过杂交瘤细胞技术制备抗LI单克隆抗体。用Western Blot、间接免疫荧光试验(IFA)和免疫过氧化物酶单层试验(IPMA)对制备的抗体进行鉴定。将单克隆抗体作为捕获抗体,多克隆抗体作为检测抗体,建立检测LI的夹心ELISA方法,优化反应条件,并对该方法进行特异性、敏感性和重复性评价。用优化后的检测方法对灭活后的LI进行定量检测。【结果】制备的多克隆抗体效价达1:409600。经三轮亚克隆后共筛选出3株阳性杂交瘤细胞,分别命名为1B7、3C7、4F10,腹水效价均为1:204800。3株单克隆抗体均与LI发生特异性反应,与肠致病性大肠杆菌(E.coli O157:H7)、猪霍乱沙门氏菌(Salmonella cholerasuis,S.Choleraesuis)、鼠伤寒沙门氏菌(Salmonella typhimurium,S.typhimurium)均无交叉反应。夹心ELISA方法经优化后条件为:捕获抗体为4F10;捕获抗体与检测抗体的工作浓度分别为1.25和0.625μg·mL^(-1);包被液为磷酸盐缓冲液,包被条件为4℃14 h;封闭液为1%明胶,封闭条件为37℃1.5 h;抗原孵育条件为37℃1 h;检测抗体孵育条件为37℃0.5 h;酶标抗体稀释度为1:16000,孵育条件为37℃1.5 h;显色条件为室温孵育10 min。该方法对其他常见的肠道病原菌检测均为阴性,对LI的最低检测限为1×10^(5)个/mL,且批内、批间变异系数均小于10%。LI浓度在1×10^(5)—1×10~8个/mL范围内与P/N值呈线性关系,标准方程:y=2.7349x-11.643(R^(2)=0.9966)。使用该方法对该实验室制备的PPE灭活疫苗进行抗原定量,结果显示灭活前后抗原含量基本不变,且两组疫苗样品定量结果批内重复性良好。120份猪临床粪便样品检测结果显示,【Objective】This study established a sandwich ELISA method for detecting Lawsonia intracellularis(L.intracellularis),which could be used to measure antigen content in the development of porcine proliferative enteropathy(PPE)inactivated vaccines.【Method】L.intracellularis was used as the immunogen,and polyclonal antibody against L.intracellularis was obtained by immunizing New Zealand rabbit.Additionally,6-week-old BALB/c mice were immunized to prepare monoclonal antibodies against L.intracellularis by hybridoma cell technique.The antibodies were identified by Western Blot,indirect immunofluorescence(IFA)and immunoperoxidase monolayer assay(IPMA).A sandwich ELISA method was established for detecting L.intracellularis,using monoclonal antibody as capture antibody and polyclonal antibody as detection antibody.the optimized reaction conditions and the specificity,sensitivity and reproducibility of the method were evaluated.The optimized sandwich ELISA was used to quantify inactivated L.intracellularis.【Result】The polyclonal antibody titer reached 1﹕409600.After three rounds of subcloning,three positive hybridoma cell lines were screened,named 1B7,3C7,and 4F10,respectively,and the titer of ascites were all 1﹕204800.All three monoclonal antibodies had specific reactions with L.intracellularis and showed no cross-reactivity with E.coli O157:H7,S.choleraesuis,and S.typhimurium.The optimal conditions for the sandwich ELISA were as follows:capture antibody was 4F10;working concentrations for capture and detection antibodies were 1.25 and 0.625μg·mL^(-1),respectively.The coating solution was phosphate buffer,and the coating condition was 4℃for 14 h.Blocking solution was 1%gelatin and blocking condition was 37℃for 1.5 h.The reaction condition of antigen was 37℃for 1 h,and the reaction condition for detection antibody was 37℃for 0.5 h.HRP-antibody dilution was 1﹕16000 and reaction condition of HRP-antibody was 37℃for 1.5 h.The developing time was 10 min at room temperature.This method was negative

关 键 词：胞内劳森菌 单克隆抗体 夹心ELISA 抗原定量 

分 类 号：R28[医药卫生—中药学]