6种虫媒病毒蛋白芯片检测方法的建立  

Development of antibody-array for detection of six arboviruses

作　　者：林方[1,2] 康晓平[1] 李裕昌[1] 朱晓磊[1] 范丽[1] 魏婧靖[1] 杨银辉[1] 祝庆余[1] 

机构地区：[1]军事医学科学院微生物流行病研究所,北京100077 [2]第二炮兵总医院检测科,北京100088

出　　处：《中华微生物学和免疫学杂志》2011年第11期1035-1040,共6页Chinese Journal of Microbiology and Immunology

摘　　要：目的 建立针对6种虫媒病毒的蛋白芯片检测方法,用以检测流行性乙型脑炎病毒、蜱传脑炎病毒、登革病毒（1～4型）、西尼罗病毒、西部马脑炎病毒和东部马脑炎病毒的特异性抗体.方法 将病毒特异性抗原作为捕获抗原点样制备蛋白芯片,利用双抗夹心ELISA原理检测血清中的病毒特异性抗体.首先利用免疫兔血清进行特异性诊断抗原的筛选,并对抗体芯片检测条件进行优化,然后采用56份临床疑似的阳性血清标本及阴性对照标本对该方法进行验证,并与常规ELISA方法进行比对.结果 共筛选出11个特异性较好的重组诊断抗原.抗原点样浓度在0.125 ～0.900mg/ml时可获得良好的检测效果,血清检测范围为1：100～1：1000.对26份临床疑似的蜱传脑炎病毒血清标本,22份登革病毒血清标本及8份流行性乙型脑炎病毒临床血清标本的检测结果为：共检测出蜱传脑炎病毒IgG阳性血清标本20份,阳性检出率为76.9％,IgM阳性血清标本17份,阳性检出率65.3％,与ELISA检测符合率分别为96.1％和84.6％.乙型脑炎病毒IgG阳性血清4份,阳性检出率50.0％,IgM阳性血清5份,阳性检测率62.0％,与ELISA检测符合率分别为87.5％和100％.登革病毒IgG阳性血清标本13份,阳性检出率63.6％,IgM阳性血清标本14份,阳性检测率68.1％,与ELISA检测符合率分别为86.3％和90.1％,结果经一致性Kappa检验后,与ELISA检测结果一致性良好.阴性对照血清结果显示检测特异性为100％.结论 本研究建立的虫媒病毒抗体芯片检测方法具有较高的特异性和可靠性,可用于6种虫媒病毒抗体的临床检测.Objective To develop an antibody-array system for multiple detection of antibodies against Japanese B encephalitis virus （JEV）,Tick-borne encephalitis virus （TBV）,Dengue virus （ DENV ）,West Nile virus （WNV）,Western equine encephalitis virus （WEEV） and East Equine encephalitis virus （EEEV）.Methods Recombined antigens were spotted on array as capture antigens.Specific antibodies were detected by using a sandwich ELISA format.Rabbit antiserum was employed to select and confirm the specificity of antigens and to optimize the conditions of the assay.The detection efficiency of the system was validated by 40 clinical suspected serum samples and compared with the relative ELISA assays.Results Eleven recombined antigens were selected as diagnostic antigens with high specificity.Better detection could be achieved when scale of antigen concentrations were within 0.125-0.900 mg/ml and the serum dilutions were 1：100-1：1000.When detecting the 26 clinical suspected TBE serum samples,20 were IgG positive （76.9%）,and 17 were IgM positive （65.3%） which was 96.1% and 84.6% consistent with the relevant ELLSA tests,the 8 clinical suspected JEV serum samples,4 were IgG positive （50.0%）,and 5 were IgM positive （62.0%）,which was 86.3% and 90.1% consistent with the relevant ELLSA tests.As for the 22 DEN serum samples,13 were IgG positive （60%） and 15 were IgM positive （68%） which was 85% and 93% consistent with ELISA.The specificity of the assay was 100% and the sensitivity was higher than the relative ELISAs.Conclusion The developed antibody-array is highly specific and reliable,which could be used for the detection of antibodies against the 6 arboviruses.

关 键 词：虫媒病毒 抗体芯片 检测 

分 类 号：S[农业科学] R[医药卫生]