摘要
旨在建立猪轮状病毒(porcine rotavirus,PoRV)的抗体检测方法。本研究以PoRV截短的VP6*(aa149-332)蛋白作为检测抗原,建立PoRV抗体的间接ELISA检测方法。结果显示:截短的重组VP6*(aa149-332)以包涵体和可溶性两种形式存在,大小为22.5 ku。优化后的ELISA反应条件:VP6*(aa149-332)包被量为25 ng·孔^(-1),抗原4℃过夜包被,5%脱脂奶粉溶液37℃封闭2 h,待检血清1∶400稀释、37℃孵育30 min,酶标羊抗鼠IgG二抗1∶24000稀释、37℃孵育30 min,显色15 min。试验确定阴阳性临界值OD_(450 nm)=0.418,可检测到抗体的最大稀释度为1∶3200,批内和批间重复变异系数均小于10%,并具有较好的特异性。28份血清与Western blot结果符合率为96.42%。ELISA OD_(450 nm)值与中和抗体效价log2相关系数R^(2)=0.8785。综上所述,本研究建立的PoRV抗体间接ELISA具有较高的特异性和敏感性,可应用于PoRV的临床血清样本抗体检测。
This study was designed to establish an antibody detection method for porcine rotavirus(PoRV).We utilized the truncated VP6*(aa149-332)protein of PoRV as the detection antigen to develop an indirect ELISA detection method for PoRV antibodies.The truncated recombinant VP6*(aa149-332)exists in two forms:inclusion bodies and soluble,with a size of 22.5 ku.The ELISA reaction conditions were as follows:the antigen was coated at 25 ng/well and left overnight at 4℃,the wells were blocked with 5%skimmed milk at 37℃for 2 hours,the test serum was diluted 1∶400 and incubated at 37℃for 30 minutes,the secondary antibody was diluted 1∶24000 and incubated at 37℃for 30 minutes,The optimized reaction conditions of the ELISA were as follows:the optimal coating antigen concentration,coating time and temperature were found to be 25 ng per well,and left overnight at 4℃;the plates were blocked with 5%skimmed milk at 37℃for 2 hours;the optimal dilution of test serum and the secondary antibody were found to be 1∶400 and 1∶24000,respectively.The optimal incubation time and temperature for test serum and the secondary antibody were 30 minutes and 37℃,respectively,and the color development time was 15 minutes.Testing 30 negative sera determined the cutoff value at OD_(450 nm)=0.418,the maximum dilution of detectable antibodies is 1∶3200.The intra-batch and inter-batch coefficient of variation were both less than 10%,and it exhibited good specificity.The agreement rate with Western Blot results for 28 sera was 96.42%.The correlation coefficient(R^(2))between ELISA OD_(450 nm)values and neutralizing antibody log2 values was 0.8785.In summary,the indirect ELISA established in this study for detecting porcine rotavirus antibodies has high specificity and sensitivity,making it applicable for clinical sample testing of porcine rotavirus.
作者
高力国
申翰钦
陈诒全
陈胜
蔺文成
陈峰
GAO Liguo;SHEN Hanqin;CHEN Yiquan;CHEN Sheng;LIN Wencheng;CHEN Feng(College of Animal Science,South China Agricultural University,Guangzhou 510642,China;Guangdong Jingjie Inspection and Testing Co.,Ltd,Yunfu 527400,China)
出处
《畜牧兽医学报》
CAS
CSCD
北大核心
2024年第9期4021-4028,共8页
ACTA VETERINARIA ET ZOOTECHNICA SINICA
基金
广东省乡村振兴战略专项资金(“大专项+任务清单”)项目(220628175593089)。
作者简介
高力国(1998-),男,河南商丘人,硕士生,主要从事动物免疫与生物安全方向的研究,E-mail:gaoliguo@stu.scau.edu.cn;通信作者:陈峰,主要从事畜禽健康养殖研究,E-mail:fengch@scau.edu.cn。
