寨卡病毒感染早期快速抗原检测方法的建立
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摘要
目的建立检测非结构蛋白1(NS1)抗原的免疫层析胶体金试纸方法,用于寨卡病毒(ZIKV)感染的早期诊断。方法对已获得的5株人源抗ZIKV NS1的单克隆抗体(ZKns2E11、ZKns3G2、ZKns4B8、ZKns4F10、ZKns14G5)采用生物膜层光学干涉法进行竞争抑制实验,以得到它们在抗原结合位点的相互关系并根据抗体结合抗原表位的不同将其进行分类,又通过两两随机组合配对方法筛选出最佳捕获抗体及包被抗体,制备成胶体金试纸。分别用重组NS1蛋白、感染ZIKV细胞培养上清液以及感染登革病毒(DENV)病人血清/血浆多种检测样品以获得准确的特异性和敏感性。其中病毒定量采用FFA(focus-forming assay)方法。结果在竞争抑制实验结果中,根据抗体与抗原结合的剩余结合率(<20%被认为强竞争关系即结合位点相似甚至相同,>60%被认为无竞争关系即位点不同),抗体大致分为两类,并通过两两配对筛选出2株结合位点不一致的最佳配对抗体(ZKns2E11、ZKns3G2),最终建立检测ZIKV NS1抗原胶体金试纸的快速诊断方法。在试纸敏感性分析中,ZIKV重组NS1蛋白为31.25 ng/mL时肉眼仍可观测到阳性条带的出现,以2×10~4FFU/mL的ZIKV病毒量感染细胞所释放的NS1蛋白仍可被检测到。试纸在其特异性上,无论是重组蛋白、感染病毒细胞培养上清液还是临床样本,与DENV均不交叉,表现出良好的特异性。该检测方法的成功建立为ZIKV感染早期诊断带来了一定的补充作用。结论建立检测ZIKV NS1抗原胶体金试纸的方法可成功区分ZIKV和DENV,表现出良好的敏感性及特异性,为ZIKV与DENV共感染地区提供了技术储备。
Objective To establish a colloid gold-immunochromatography assay(GICA) for detecting of ZIKV NS1 antigen for early diagnosis of ZIKV infection. Methods Five monoclonal antibodies(ZKns2E11, ZKns3G2, ZKns4B8, ZKns4F10,ZKns14G5) against the human anti-ZIKV NS1 obtained were subjected to competitive inhibition experiments using biofilm layer optical interferometry to obtain their mutual relationship at the antigen binding site. The best capture antibody and coated antibody were screened by pairwise random combination pairing method to prepare colloidal gold test paper.Recombinant NS1 protein, infected ZIKV cell culture supernatant, and multiple samples of serum/plasma of dengue virus(DENV) patients were obtained to obtain accurate specificity and sensitivity, respectively. Among them, the virus was quantified by the focus-forming assay(FFA) method. Results Based on the residual of binding of antibodies to antigens(<20%is considered to be a strong competition, ie, the binding sites are similar or even the same, >60% are considered to be noncompetitive,ie, the sites are different),the antibodies were roughly divided into two types. A pair of optimal paired antibodies with different binding sites were screened by pairwise pairing,and a rapid diagnostic method for detecting ZIKV NS1 antigen colloidal gold test paper was finally established. In the sensitivity analysis of the test paper, the positive band was observed by naked eye when the ZIKV recombinant NS1 protein was 31.25 ng/mL, and the NS1 protein released by the infection of the ZIKV virus at 2×10~4 FFU/mL could still be detected. The test paper showed good specificity whether it was recombinant protein, infected virus cell culture supernatant or clinical sample, and did not cross with DENV. The successful establishment of this test method could provide early diagnosis of ZIKV infection. Conclusion The method of detecting ZIKV NS1 antigen colloidal gold test paper could successfully distinguish ZIKV and DEN V,showing good sensitivity and specificity, and providing technical support for diagnosis of ZIKV in the ZIKV and DENV co-infected areas.
Objective To establish a colloid gold-immunochromatography assay(GICA) for detecting of ZIKV NS1 antigen for early diagnosis of ZIKV infection. Methods Five monoclonal antibodies(ZKns2E11, ZKns3G2, ZKns4B8, ZKns4F10,ZKns14G5) against the human anti-ZIKV NS1 obtained were subjected to competitive inhibition experiments using biofilm layer optical interferometry to obtain their mutual relationship at the antigen binding site. The best capture antibody and coated antibody were screened by pairwise random combination pairing method to prepare colloidal gold test paper.Recombinant NS1 protein, infected ZIKV cell culture supernatant, and multiple samples of serum/plasma of dengue virus(DENV) patients were obtained to obtain accurate specificity and sensitivity, respectively. Among them, the virus was quantified by the focus-forming assay(FFA) method. Results Based on the residual of binding of antibodies to antigens(<20%is considered to be a strong competition, ie, the binding sites are similar or even the same, >60% are considered to be noncompetitive,ie, the sites are different),the antibodies were roughly divided into two types. A pair of optimal paired antibodies with different binding sites were screened by pairwise pairing,and a rapid diagnostic method for detecting ZIKV NS1 antigen colloidal gold test paper was finally established. In the sensitivity analysis of the test paper, the positive band was observed by naked eye when the ZIKV recombinant NS1 protein was 31.25 ng/mL, and the NS1 protein released by the infection of the ZIKV virus at 2×10~4 FFU/mL could still be detected. The test paper showed good specificity whether it was recombinant protein, infected virus cell culture supernatant or clinical sample, and did not cross with DENV. The successful establishment of this test method could provide early diagnosis of ZIKV infection. Conclusion The method of detecting ZIKV NS1 antigen colloidal gold test paper could successfully distinguish ZIKV and DEN V,showing good sensitivity and specificity, and providing technical support for diagnosis of ZIKV in the ZIKV and DENV co-infected areas.
引文
[1]Dick GW,Kitchen SF,AJ Haddow.Zika virus.I.Isolations and serological specificity[J].Trans R Soc Trop Med Hyg,1952,46(5):509-520.
[2]World Health Organization.The History of Zika Virus[EB/OL].https://www.who.int/emergencies/zika-virus/timeline/en/(Last accessed 10 December 2018).
[3]Duffy MR,Chen TH,Hancock WT,et al.Zika virus outbreak on Yap Island,Federated States of Micronesia[J].N Engl JMed,2009,360(24):2536-2543.
[4]Roth A,Mercier A,Lepers C,et al.Concurrent outbreaks of dengue,chikungunya and Zika virus infections-an unprecedented epidemic wave of mosquito-borne viruses in the Pacific 2012-2014[J].Euro Surveill,2014,19(41).pii:20929.
[5]Teixeira MG,Costa Mda C,de Oliveira WK,et al.The Epidemic of Zika Virus-Related Microcephaly in Brazil:Detection,Control,Etiology,and Future Scenarios[J].Am J Public Health,2016,106(4):601-605.
[6]World Health Organization.Zika situation report[EB/OL].http://www.who.int/emergencies/zika-virus/en/(Last accessed 11December 2018).
[7]Wu JY,Lun ZR,James AA,et al.Dengue Fever in mainland China[J].Am J Trop Med Hyg,2010,83(3):664-671.
[8]Xiong Y,Chen Q.Epidemiology of dengue fever in China since1978[J].Nan Fang Yi Ke Da Xue Xue Bao,2014,34(12):1822-1825.
[9]Zhao H,Zhang FC,Zhu Q,et al.Epidemiological and Virological Characterizations of the 2014 Dengue Outbreak in Guangzhou,China[J].PLoS One,2016,11(6):e0156548.
[10]Priyamvada L,Quicke KM,Hudson WH,et al.Human antibody responses after dengue virus infection are highly crossreactive to Zika virus[J].Proc Natl Acad Sci U S A,2016,113(28):7852-7857.
[11]Gao X,Wen Y,Wang J,et al.Delayed and highly specific antibody response to nonstructural protein 1(NS1)revealed during natural human ZIKV infection by NS1-based capture ELISA[J].BMC Infect Dis,2018,18(1):275.
[12]国家卫生和计划生育委员会.登革热诊疗指南(2014年第2版)[J].中国医药科学,2014,4(21):221-224.
[13]Brien JD,Lazear HM,Diamond MS.Propagation,quantification,detection,and storage of West Nile virus[J].Curr Protoc Microbiol,2013,31:15D.3.1-15D.3.18.
[14]Santiago GA,Vázquez J,Courtney S,et al.Performance of the Trioplex real-time RT-PCR assay for detection of Zika,dengue,and chikungunya viruses[J].Nat Commun,2018,9(1):1391.
[15]Kim YH,Lee J,Kim YE,et al.Development of a Rapid Diagnostic Test Kit to Detect IgG/IgM Antibody against Zika Virus Using Monoclonal Antibodies to the Envelope and Nonstructural Protein 1 of the Virus[J].Korean J Parasitol,2018,56(1):61-70.
[16]Muller DA,Depelsenaire AC,Young PR,et al.Clinical and Laboratory Diagnosis of Dengue Virus Infection[J].J Infect Dis,2017,215(suppl_2):S89-S95.
[17]Bosch I,de Puig H,Hiley M,et al.Rapid antigen tests for dengue virus serotypes and Zika virus in patient serum[J].Sci Transl Med,2017,9(409).pii:eaan1589.
[18]Stettler K,Beltramello M,Espinosa DA,et al.Specificity,cross-reactivity,and function of antibodies elicited by Zika virus infection[J].Science,2016,353(6301):823-826.
[2]World Health Organization.The History of Zika Virus[EB/OL].https://www.who.int/emergencies/zika-virus/timeline/en/(Last accessed 10 December 2018).
[3]Duffy MR,Chen TH,Hancock WT,et al.Zika virus outbreak on Yap Island,Federated States of Micronesia[J].N Engl JMed,2009,360(24):2536-2543.
[4]Roth A,Mercier A,Lepers C,et al.Concurrent outbreaks of dengue,chikungunya and Zika virus infections-an unprecedented epidemic wave of mosquito-borne viruses in the Pacific 2012-2014[J].Euro Surveill,2014,19(41).pii:20929.
[5]Teixeira MG,Costa Mda C,de Oliveira WK,et al.The Epidemic of Zika Virus-Related Microcephaly in Brazil:Detection,Control,Etiology,and Future Scenarios[J].Am J Public Health,2016,106(4):601-605.
[6]World Health Organization.Zika situation report[EB/OL].http://www.who.int/emergencies/zika-virus/en/(Last accessed 11December 2018).
[7]Wu JY,Lun ZR,James AA,et al.Dengue Fever in mainland China[J].Am J Trop Med Hyg,2010,83(3):664-671.
[8]Xiong Y,Chen Q.Epidemiology of dengue fever in China since1978[J].Nan Fang Yi Ke Da Xue Xue Bao,2014,34(12):1822-1825.
[9]Zhao H,Zhang FC,Zhu Q,et al.Epidemiological and Virological Characterizations of the 2014 Dengue Outbreak in Guangzhou,China[J].PLoS One,2016,11(6):e0156548.
[10]Priyamvada L,Quicke KM,Hudson WH,et al.Human antibody responses after dengue virus infection are highly crossreactive to Zika virus[J].Proc Natl Acad Sci U S A,2016,113(28):7852-7857.
[11]Gao X,Wen Y,Wang J,et al.Delayed and highly specific antibody response to nonstructural protein 1(NS1)revealed during natural human ZIKV infection by NS1-based capture ELISA[J].BMC Infect Dis,2018,18(1):275.
[12]国家卫生和计划生育委员会.登革热诊疗指南(2014年第2版)[J].中国医药科学,2014,4(21):221-224.
[13]Brien JD,Lazear HM,Diamond MS.Propagation,quantification,detection,and storage of West Nile virus[J].Curr Protoc Microbiol,2013,31:15D.3.1-15D.3.18.
[14]Santiago GA,Vázquez J,Courtney S,et al.Performance of the Trioplex real-time RT-PCR assay for detection of Zika,dengue,and chikungunya viruses[J].Nat Commun,2018,9(1):1391.
[15]Kim YH,Lee J,Kim YE,et al.Development of a Rapid Diagnostic Test Kit to Detect IgG/IgM Antibody against Zika Virus Using Monoclonal Antibodies to the Envelope and Nonstructural Protein 1 of the Virus[J].Korean J Parasitol,2018,56(1):61-70.
[16]Muller DA,Depelsenaire AC,Young PR,et al.Clinical and Laboratory Diagnosis of Dengue Virus Infection[J].J Infect Dis,2017,215(suppl_2):S89-S95.
[17]Bosch I,de Puig H,Hiley M,et al.Rapid antigen tests for dengue virus serotypes and Zika virus in patient serum[J].Sci Transl Med,2017,9(409).pii:eaan1589.
[18]Stettler K,Beltramello M,Espinosa DA,et al.Specificity,cross-reactivity,and function of antibodies elicited by Zika virus infection[J].Science,2016,353(6301):823-826.