牛传染性鼻气管炎间接ELISA诊断方法的建立

以牛肾细胞系（MDBK）培养牛传染性鼻气管炎病毒（IBRV）Bartha Nu/67株,经超速离心纯化病毒,再经超声破碎处理后作为诊断抗原,建立了检测牛血清IBRV抗体的间接酶联免疫吸附试验.该ELISA的判定标准为：血清D490 nm值大于0.369的判为阳性,小于0.295的判为阴性,在0.295与0.369之间的为可疑.特异性和重复性试验结果表明,该方法特异性高、重复性好.与法国进口ELISA抗体诊断试剂盒比较,其符合率为96.3%;与中和试验比较,符合率为95.8%,且敏感性更高.应用该诊断方法调查了我国部分地区IBRV的感染情况,结果显示,这些地区的IBRV感染率为67.1%.     

Serodiagnosis of equine infectious anemia by agar gel immunodiffusion and ELISA using a recombinant p26 viral protein expressed in Escherichia coli as antigen

We used a p26 recombinant protein (p26r) from equine infectious-anemia virus (EIAV) expressed in Escherichia coli as antigen to standardize an agar-gel immunodiffusion (AGIDp26r) test and an indirect ELISA (ELISAp26r) for the detection of antibodies against EIAV in 720 equine sera from Brazil. We evaluated the tests's relative diagnostic sensitivities (relSe) and relative diagnostic specificities (relSp) against a commercial AGID kit (Idexx™, USA). We used three sera panels: panel A—196 AGID-negative sera from an AIE non-endemic controlled area; panel B—194 AGID-negative sera from an AIE endemic area and panel C—330 AGID-positive sera from an AIE endemic area. ELISAp26r cut-off value was defined with TG-ROC using sera from panels A and C. AGIDp26r showed an agreement of 100% with the commercial kit. When applied to sera from panels A and C, ELISAp26r showed an agreement of 100% with the kit, but, although relSe was 100% for panel C, the ELISAp26r had relSp of 93.3%.     

猫传染性腹膜炎病毒核衣壳蛋白的表达及间接ELISA法的建立

本研究对猫传染性腹膜炎病毒（FIPV）N蛋白的编码基因进行克隆和原核表达,并在纯化重组N蛋白的基础上,建立了FIPV抗体间接ELISA检测方法。研究结果显示,该重组纯化的N蛋白具有良好的抗原反应性,可用于FIPV阳性血清的筛查,为我国出入境检疫部门监控FIPV疫情提供技术支撑。     

马传贫驴白细胞弱毒疫苗株跨膜蛋白主要免疫决定区基因的克隆与表达

从感染驴白细胞的马传贫驴白细胞弱毒疫苗株前病毒DNA中克隆了编码跨膜蛋白主要免疫决定区(TMIR)的基因，并在大肠杆菌中进行了表达。所表达的融合蛋白有一部分是可溶的，其氨基端带有6个组氨酸的标签，因此可以用固定化金属离子亲和层析法在非变性条件下进行纯化。在间接酶联免疫吸附试验(ELISA)和免疫印迹试验中，重组的TMIR蛋白可与马传贫阳性血清样品发生反应，而与健康马血清无任何反应。这表明该重组蛋白具有良好的抗原性和特异性，可用于马传贫弱毒疫苗株在体内外复制、接种马体内免疫应答及马传贫诊断的研究。     

The efficacy of a commercial ELISA as an alternative to virus neutralisation test for the detection of antibodies to EAV

Infection with equine arteritis virus is a notifiable disease with sporadic occurrence in the UK. As stallions may harbour the virus after infection, horses are screened for exposure by serological testing prior to breeding. The virus neutralisation test is considered the 'gold standard' serological screening test, but it is time-consuming and labour intensive; consequently there is a move towards more rapid screening methodology. In this study, a commercially available EVA antibody ELISA is assessed. The ELISA performed poorly with a specificity [corrected] of 26% and a sensitivity [corrected] of 96% in the samples analysed. It was concluded that this ELISA would be of little value for reducing sample turnaround time. The study emphasises the need for in-house validation of commercially available kits.     

猪瘟抗体间接ELISA检测方法的建立和优化

采用真核系统表达纯化的猪瘟病毒E2蛋白作为包被物,通过对各个反应条件的摸索和优化,建立了检测猪瘟病毒抗体的猪瘟抗体间接ELISA检测方法。研究结果表明,E2蛋白的最佳包被浓度为0.25μg/mL,最佳包被条件为4℃16h;待检血清的最佳稀释倍数为100倍;特异性检测试验证明,该方法与猪常见病原的阳性血清没有交叉反应。通过对大量猪瘟抗体阴、阳性血清的检测,最终确定了本检测方法的判定标准。     

Validation and standardization of virus neutralizing test using indirect immunoperoxidase technique for the quantification of antibodies to rabies virus

A virus neutralizing test using an indirect immunoperoxidase technique (VNT-IIP) for rabies has been developed for the titration of dog and cat serum samples in Japan. The VNT-IIP has the advantage that results obtained can be viewed by the naked eye. The purpose of this study was to validate the VNT-IIP and compare it with one of the international standard methods, the fluorescent antibody virus neutralization test (FAVNT). The VNT-IIP showed satisfactory repeatability, high analytical specificity and good accuracy. Regarding the comparison between the VNT-IIP and the FAVNT, the VNT-IIP showed good agreement (91.9%), high sensitivity (92.8%) as well as specificity (87.0%) and good correlation (r = 0.92). As described above, the validation of the VNT-IIP was satisfactory and the performances of the test proved to be equivalent to those of an international standard method.     

流行性乙型脑炎病毒E蛋白的截短表达与间接ELISA诊断方法的建立

为建立一种快速检测流行性乙型脑炎病毒抗体的方法,本研究参照已发表的JEV基因组序列,应用RT-PCR扩增了长约1000bp的E基因片段,连接pET30a表达载体中,经诱导后获得了以包涵体形式表达的重组E蛋白。重组蛋白纯化后,经免疫印迹检测证明其具有良好的抗原性和特异性。以该蛋白作为诊断抗原,建立了检测流行性乙型脑炎病毒抗体的E-ELISA诊断方法。该诊断方法具有良好的敏感性、特异性和重复性,为JEV的快速诊断、免疫猪群抗体监测和JEV流行病学调查提供了一种快速、简便的血清学诊断方法。     

猪繁殖与呼吸综合征病毒重组N蛋白间接ELISA方法的建立

用纯化的猪繁殖与呼吸综合征病毒(PRRSV)重组核衣壳(N)蛋白作为包被抗原,建立了PRRSV抗体检测的间接ELISA方法,并优化确定了ELISA最佳工作条件:重组抗原包被浓度为13.3 μg/mL,37℃1h或4℃过夜;5％脱脂乳37℃封闭2h;血清1:40稀释,37℃作用90 min;酶标二抗1∶4000稀释,37...