H1与H3亚型猪流感病毒IgG抗体双重荧光微球免疫学检测方法的建立
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摘要
有效防治猪流感需要借助于准确而快速的检测方法,为了建立一种高效、快速的多重检测方法,本研究应用H1和H3亚型猪流感病毒(SIV)的血凝素(HA)蛋白抗原偶联不同编码的荧光微球,应用间接法检测模式,建立了H1、H3亚型猪流感病毒IgG抗体荧光微球免疫学检测方法,并将该方法与血凝抑制试验(HI)、病毒中和试验(VN)和酶联免疫吸附试验(ELISA)进行比较分析。结果表明,该方法的抗体检测灵敏度比ELISA方法高100～1 000倍;与猪繁殖与呼吸综合征病毒、猪圆环病毒2型、猪瘟病毒、伪狂犬病病毒及日本乙型脑炎病毒的阳性血清均无交叉反应,且两种亚型血清之间也不存在交叉反应。重复性试验表明,批内和批间精密度测试结果分别为10.2%和12.8%。与HI、VN和ELISA方法比较,总符合率分别为97.82%、98.27%和97.83%。综上,本研究建立的检测方法具有双重检测特性,特异性好、灵敏性高、重复性强,可用于H1、H3亚型SIV的鉴别诊断。
Effective prevention and control of swine influenza requires the help of accurate and rapid detection methods.In order to establish a highly efficient and rapid multiple detection method,this study applied H1 and H3 subtypes swine influenza virus(SIV) hemagglutinin(HA) protein antigen coupled with different encoded fluorescent microspheres,and used the indirect method for detectingthe IgG antibodies against H1 and H3 subtypes SIV.The immunological microspheres of IgG antibodies against swine influenza virus were detected and compared with the methods of blood coagulation inhibition test(HI),virus neutralization test(VN) and enzyme linked immunosorbent assay(ELISA).The results showed that the detection sensitivity of the antibodies was 100─1 000 times higher than that of the ELISA method.There was no cross-reactivity with the positive serum with porcine reproductive and respiratory syndrome virus,type II porcine circovirus,swine fever virus,pseudorabies virus and Japanese encephalitis virus and there was no cross-reaction between the H1 and H3 subtype SIV sera.Repeatability experiments showed that the intra and inter batch precision test indexes were 10.2% and12.8% respectively.Compared with HI,VN and ELISA,the total coincidence rates were 97.82%,98.27%and 97.83% respectively.In conclusion,the detection method established in this study has dual detection characteristics,good specificity,high sensitivity and strong repeatability,which can be used for the differential diagnosis of the antibodies of H1 and H3 subtype SIV.
Effective prevention and control of swine influenza requires the help of accurate and rapid detection methods.In order to establish a highly efficient and rapid multiple detection method,this study applied H1 and H3 subtypes swine influenza virus(SIV) hemagglutinin(HA) protein antigen coupled with different encoded fluorescent microspheres,and used the indirect method for detectingthe IgG antibodies against H1 and H3 subtypes SIV.The immunological microspheres of IgG antibodies against swine influenza virus were detected and compared with the methods of blood coagulation inhibition test(HI),virus neutralization test(VN) and enzyme linked immunosorbent assay(ELISA).The results showed that the detection sensitivity of the antibodies was 100─1 000 times higher than that of the ELISA method.There was no cross-reactivity with the positive serum with porcine reproductive and respiratory syndrome virus,type II porcine circovirus,swine fever virus,pseudorabies virus and Japanese encephalitis virus and there was no cross-reaction between the H1 and H3 subtype SIV sera.Repeatability experiments showed that the intra and inter batch precision test indexes were 10.2% and12.8% respectively.Compared with HI,VN and ELISA,the total coincidence rates were 97.82%,98.27%and 97.83% respectively.In conclusion,the detection method established in this study has dual detection characteristics,good specificity,high sensitivity and strong repeatability,which can be used for the differential diagnosis of the antibodies of H1 and H3 subtype SIV.
引文
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[3] KARASIN A I,SCHUTTEN M M,COOPER L A,et al.Genetic characterization of H3N2 influenza viruses isolated from pigs in North America,1977-1999:Evidence for wholly human and reassortant virus genotypes[J].Virus Research,2000,68(1):71-85.
[4] WEBBY R J,WEBSTER R G.Emergence of influenza A viruses[J].Philosophical Transactions of the Royal Society Biological Sciences,2001,356(1416):1817-1828.
[5] CASTRUCCI M R,DONATELLI I,SIDOLI L,et al.Genetic reassortment between avian and human influenza A viruses in Italian pigs[J].Virology,1993,193(1):503-506.
[6] MA W,KAHN R E.RICHT J A.The pig as a mixing vessel for influenza viruses:Human and veterinary implications[J].Journal of Molecular and Genetic Medicine,2008,3(1):158-166.
[7] CHOMEL J J,REMILLEUX M F,MARCHAND P,et al.Rapid diagnosis of influenza A.Comparison with ELISA immunocapture and culture[J].Journal of Virological Methods,1992,37(3):337-343.
[8] CODICES V,MARTINS C,NOVO C,et al.Dynamics of cytokines and immunoglobulins serum profiles in primary and secondary Cryptosporidium parvum infection:usefulness of Luminex(R)xMAP technology[J].Experimental Parasitology,2013,133(1):106-113.
[9] RESLOVA N,MICHNA V,KASNY M,et al.xMAP technology:Applications in detection of pathogens[J].Front Microbiol,2017,8:55.
[10]张立营,刘国凤,李克峰.液相芯片技术在医学检验诊断中的应用[J].实用医药杂志,2016,33(2):179-180.ZHANG Li-ying,LIU Guo-feng,LI Ke-feng.Application of liquid chip technology in medical examination[J].Practical Journal of Medicine Pharmacy,2016,33(2):179-180.(in Chinese)
[11] BAY J T,GARRED P.Rapid bead-based immunoas say for measurement of mannose-binding lectin[J].Scandinavian Journal of Immunology,2009,69(6):570-575.
[12]曾梦,纪方晓,冀池海,等.猪繁殖与呼吸综合征病毒抗体的荧光微球免疫学检测方法的建立[J].中国兽医科学,2017,47(12):1497-1502.ZENG Meng,JI Fang-xiao,JI Chi-hai,et al.De velopment of a fluorescent microbead-based immunoassay for the detection of swine porcine reproductive and respiratory syndrome virus IgG[J].Chinese Veterinary Science,2017,47(12):1497-1502.(in Chinese)
[13]纪方晓.PRRSV、SIV、HEV抗体多重液相蛋白芯片检测方法的建立[D].广州:华南农业大学,2016.JI Fang-xiao.Development of a multiplex fluorescent microsphere immunoassay(FMIA)for the detection of antibodies against to PRRSV,SIV and HEV[D].Guangzhou:South China Agricultural University,2016.(in Chinese)
[14]纪方晓,陈济铛,梁焕斌,等.猪戊型肝炎病毒液相蛋白芯片检测方法的建立[J].中国兽医学报,2017,37(1):60-65.JI Fang-xiao,CHEN Ji-dang,LIANG Huan-bin,et al.Development of a fluorescent micros phere-based immunoassay for detection of an-tibodies a gainst swine hepatitis E virus[J].Chinese Journal of Veterinary Science,2017,37(1):60-65.(in Chinese)
[15]方开泰.正交与均匀设计试验[M].北京:科学出版社,2001:52-80.FANG Kai-dai.Orthogonal and Uniform Design Tests[M].Beijing:Science Press,2001:52-80.(in Chinese)
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