O型口蹄疫病毒固相竞争ELISA抗体检测方法的建立

本研究建立了O型口蹄疫病毒固相竞争ELISA抗体检测方法,确立了O型口蹄疫病毒固相竞争ELISA抗体检测方法检测O型口蹄疫血清阴阳性判定标准：抗体效价大于或等于1：32时判为O型口蹄疫抗体阳性;1：16～1：32时判为O型口蹄疫抗体可疑。检测131份阴性血清、口蹄疫亚洲1型阳性血清和A型阳性血清,固相竞争ELISA方法和液相阻断ELISA方法的特异性分别是95．5％,84．7％。固相竞争ELISA方法检测口蹄疫亚洲1型、口蹄疫A型阳性血清时,无交叉反应,O型口蹄疫病毒感染牛、免疫牛和感染猪血清的阳性检出率均为100％,免疫猪检出率为86．7％。     

猪口蹄疫O型合成肽VP1结构蛋白ELISA抗体检测方法的建立

根据猪口蹄疫O型流行毒株VP1序列设计出5条合成肽,以固相法合成并以此合成肽作为包被抗原建立猪口蹄疫O型合成肽VP1结构蛋白ELISA抗体检测方法,并对该方法敏感性、特异性、重复性等进行验证。结果表明:该检测方法敏感性为96.0%,特异性为99.1%,批内与批间重复试验变异系数小于10.0%。比对试验结果显示,该检测方法与UBI猪口蹄疫病毒VP1结构蛋白抗体酶联免疫吸附试验诊断试剂盒符合率为93.2%,与中国农业科学院兰州兽医研究所口蹄疫O型液相阻断ELISA抗体检测试剂盒符合率为85.7%。该猪口蹄疫O型合成肽VP1结构蛋白ELISA抗体检测方法敏感性好、特异性强、稳定性高、操作简便,可用于检测O型口蹄疫抗体水平。     

以合成肽为抗原检测O型口蹄疫病毒抗体 ELISA方法的建立和评价

作者以固相法合成特异性FMDV主要保护性抗原VP1上的表位肽,将其与载体蛋白BSA偶联,作为包被抗原,制备检测抗O型口蹄疫病毒(FMDV)抗体的ELISA试剂盒,并对该试剂盒进行方法考核.结果表明该方法的敏感性为95.12%,特异性为100%.检测199份血清标本,与UBI FMD VP1试剂盒的符合率达到98.49%,与液相阻断ELISA试剂盒的符合率达到96.98%.该多肽ELISA试剂盒特异、敏感、稳定、操作简便,可用来监控口蹄疫抗体水平.     

一致性检验比较牛口蹄疫病毒抗体LBE检测试剂盒

用660份牛血清比较牛口蹄疫病毒O型、A型抗体液相阻断ELISA检测试剂盒检测结果的一致性,并用u检验排除抽样误差的影响。结果表明:待检血清最终稀释度数为1∶64时O型试剂盒联合检出523份口蹄疫抗体阳性和102份阴性,符合率为94.70%,结果一致性为极强(Kappa值=0.82);待检血清最终稀释度数为1∶128时O型试剂盒联合检出476份口蹄疫抗体阳性和125份阴性,符合率91.06%,结果为高度一致(Kappa值=0.75);血清最终稀释度数为164时A型试剂盒联合检出498份口蹄疫抗体阳性和120份阴性,符合率为93.64%,结果一致性为极强(Kappa值=0.81),血清最终稀释度数为1∶28时A型试剂盒联合检出354份口蹄疫抗体阳性和233份阴性,符合率为88.94%,结果为高度一致(Kappa值=0.77)。在不同包被方式下,LBE试剂盒检测待检血清牛口蹄疫病毒抗体结果一致性较好。兽医实验室可应用一致性检验,结合本单位人力、财力和物力等条件,选择适宜的牛口蹄疫病毒抗体检测试剂盒。     

四种试剂盒检测猪口蹄疫O型合成肽疫苗抗体的对比试验

使用国产的4种猪口蹄疫病毒VP1结构蛋白抗体ELISA诊断试剂盒(VP1-ELISA)、猪口蹄疫O型液相阻断ELISA检测试剂盒(LB-ELISA)、猪口蹄疫正向间接血凝试剂盒、猪O型口蹄疫病毒ELISA抗体检测试剂盒(O-ELISA)同时检测猪口蹄疫O型合成肽疫苗免疫后的50份血清样品,阳性率分别为98.0%、94.0%、54.0%、90.0%。同时使用农业部规定的3种试剂盒分别检测20份血清样品的抗体滴度发现,VP1-ELISA试剂盒和LB-ELISA试剂盒阳性符合率为95%,平均抗体滴度相差0.7,与IHA试剂盒符合率为55%,平均抗体滴度相差3.8,故LB-ELISA试剂盒可以用来评价合成肽疫苗免疫的抗体,而IHA试剂盒不能用来检测合成肽疫苗免疫的抗体。     

便捷式液相阻断ELISA试剂盒检测牛O型口蹄疫抗体水平的效果试验

采用便捷式液相阻断ELISA试剂盒和传统液相阻断试剂盒,同时对117份牛血清样品进行口蹄疫O型抗体水平检测,以探讨其相关性和特点。结果表明:便捷式液相阻断ELISA试剂盒与传统液相阻断试剂盒的检测结果相比,kappa值为0.84,具有高度一致性;检测时间,便捷式液相阻断ELISA试剂盒检测为2h,传统式检测则需时5.25 h。应用便捷式液相阻断ELISA试剂盒对上海光明荷斯坦牧业有限公司下属牧场检测牛血清样品8729份,O型口蹄疫免疫抗体合格率为96.9%。     

A型口蹄疫抗体固相竞争ELISA检测方法的建立

为评价A型口蹄疫疫苗免疫水平,本研究以本实验室前期制备的口蹄疫病毒(FMDV)的单克隆抗体(MAb)3D9为捕获抗体,以HRP标记的MAb 9A9作为检测抗体,建立了基于MAb的检测A型FMDV抗体的固相竞争ELISA(SPCE)方法,并对其条件进行优化。结果显示,MAb 3D9的包被浓度为1.16μg/mL,A型FMDV抗原的最佳稀释度为1:5,HRP标记的MAb 9A9的最佳稀释度为1:5 000,当血清132稀释时,检测的临界值确定为35%。利用该方法分别检测A型、O型口蹄疫抗体阳性标准血清以及牛冠状病毒、牛轮状病毒以及猪繁殖与呼吸障碍综合征病毒、猪圆环病毒、猪瘟病毒的标准阳性血清。结果显示,除A型口蹄疫阳性标准血清为阳性结果外其余均为阴性结果,未出现交叉反应,表明本研究建立的方法特异性强。该方法对经病毒中和试验(VNT)检测为阳性的3份血清进行敏感性试验,敏感性分别为1:1 024、1:256、1:128,均高于VNT,表明其敏感性较高;重复性试验结果显示,该方法批内和批间重复试验的变异系数均小于10%,表明其重复性较好。利用该方法与液相阻断ELISA方法(LPBE)和VNT对112份血清样品同时检测,分析三者相关性。结果显示,本实验建立的SPCE方法与二者的相关系数分别为0.901和0.916,表明该方法可靠性较好且与VNT的相关性更高。进一步利用本研究建立的SPCE方法和韩国Jeno A型FMDV ELISA抗体检测试剂盒同时检测470份临床血清样品(90份羊血清、170份牛血清和210份猪血清),结果显示该方法检测羊血清、牛血清和猪血清与Jeno试剂盒总体符合率分别为90.0%、91.8%和89.0%。表明该方法的检测结果较为准确。本研究为建立A型口蹄疫检测方法奠定了基础。     

猪口蹄疫O 型抗体检测试纸条对比试验

为了解不同猪口蹄疫O 型抗体检测试纸条的性能,通过检测已知相关及非相关阳性血清样品、阴性血清样品和免疫 猪血清样品,对4种猪口蹄疫O型抗体检测试纸条的敏感性、特异性进行研究评估,并对比猪口蹄疫O型VP1结构蛋白抗体ELISA诊断试剂盒的检测结果。结果表明,仅有A试纸条的敏感性和特异性较好,且与试剂盒抗体检测结果的符合率较高（90%）;而其他试纸条的敏感性和特异性较差,与试剂盒抗体检测结果的符合率较低（40%～65%）。通过对比检测,仅有A试纸条可用于大量临床样品的快速检测和现场检测,更适合基层兽医和养殖户使用,而其他试纸条的检测性能还有待进一步优化提升。     

两种O型口蹄疫ELISA检测试剂盒的对比

为比较哪种ELISA试剂盒能够更准确、简便、快捷地检测出口蹄疫病毒抗体,本研究使用液相阻断酶联免疫吸附试验(LB-ELISA)试剂盒和固相竞争酶联免疫吸附试验(SPC-ELISA)试剂盒,对2014年广西各市县动物疫病预防控制中心送检的已免疫过口蹄疫疫苗的猪、牛、羊血清共计1121份进行了检测,对比两种试剂盒的特异性、敏感性、阳性检出率及符合率。检测结果表明,两种口蹄疫ELISA试剂盒的阳性检出率不同,LB-ELISA的阳性检出率比SPC-ELISA高2.4%;LB-ELISA试剂盒对血清抗体滴度水平的要求相比SPC-ELISA试剂盒要低,因此LB-ELISA试剂盒更适合于口蹄疫病毒感染的检测。     

三种试剂盒检测猪口蹄疫O型抗体的对比试验

使用三种猪口蹄疫O型抗体检测试剂盒分别检测接种猪口蹄疫O型合成肽疫苗和猪口蹄疫O型灭活疫苗的免疫抗体,以探讨三种抗体检测试剂盒的相关性。结果发现,猪口蹄疫病毒VP1结构蛋白抗体ELISA试剂盒和猪口蹄疫O型液相阻断ELISA试剂盒都可检测猪口蹄疫O型合成肽疫苗,两种试剂盒的相关性达90.8%,而间接血凝试剂盒不能用来检测猪口蹄疫O型合成肽疫苗;猪口蹄疫O型液相阻断ELISA试剂盒和间接血凝试剂盒可用来检测猪口蹄疫O型灭活疫苗,两种试剂盒的相关性达93.7%,而猪口蹄疫病毒VP1结构蛋白抗体ELISA试剂盒不能用来检测猪口蹄疫O型灭活疫苗。     

猪口蹄疫ELISA诊断试剂盒质量检测

利用参考血清,按试剂盒标明的使用方法,对上海优耐特生物医药有限公司提供的3批猪口蹄疫病毒VP1结构蛋白抗体和3批猪口蹄疫病毒NS非结构蛋白抗体ELISA诊断试剂盒的敏感性和特异性分别进行了检测.结果表明,2种试剂盒的敏感性和特异性均符合标准规定.     

反刍动物口蹄疫ELISA诊断试剂盒质量检测

利用参考血清,按试剂盒标明的使用方法,对上海优耐特生物医药有限公司提供的3批反刍动物口蹄疫病毒VP1结构蛋白抗体和3批反刍动物口蹄疫病毒NS非结构蛋白抗体ELISA诊断试剂盒的敏感性和特异性分别进行检测.结果表明,2种试剂盒的敏感性和特异性均符合相关标准的规定.     

Evaluation of commercial ELISA kits for the detection of antibodies against bluetongue virus

The aim of this study was to estimate the diagnostic value of different commercially available ELISA kits for the detection of bluetongue virus (BTV) antibodies in infected and vaccinated animals. The relative specificity of ELISA kits was evaluated using a panel of sera originating from healthy cattle, never vaccinated nor exposed to BTV. All ELISA kits applied had a high relative specificity (99.3 - 100%). The relative sensitivity of ELISA kits assessed using a panel of sera collected from BTV infected cattle was also high and similar for all the kits (97.3 - 100%). However, the relative sensitivity evaluated on the basis of testing vaccinated animals was different: the highest sensitivity was found for Ingenasa, PrioCHECK and ID VET ELISAs (96.5 - 98.3%). Slightly lower sensitivity was calculated for Pourquier and LSI kits (82.8% and 85.4%, respectively) and much lower sensitivity was found for VMRD ELISA kit (69.5%). The repeatability of BTV ELISA kits was expressed as a coefficient of variation (CV) of results of sera tested 5 times in the same day and in different days by the period of 2 months, by the same person, in the same conditions, and by using the same equipment. The CVs of sera tested in all ELISA kits ranged from 6.1 to 9.8% and were below 10% threshold adopted as a maximum for the acceptable repeatability of the method. In conclusion, it can be stated that the applied ELISA kits can be a valuable diagnostic tool for the serological monitoring studies in the BTV contaminated premises. All the methods are very specific and sensitive when testing BTV infected animals. Nevertheless, the Ingenasa and PrioCHECK can be the most useful in sero-surveillance of livestock following vaccination.     

非洲猪瘟病毒抗体检测间接ELISA方法的建立

以基因工程表达的非洲猪瘟病毒VP73蛋白作为包被抗原,建立了间接ELISA方法,用以检测猪血清中抗非洲猪瘟VP73蛋白的抗体。该方法对非洲猪瘟标准阳性血清的检测灵敏度可以达到1∶2 560,与同类进口ELISA试剂盒相当。此方法只特异性检出非洲猪瘟阳性血清,而对猪传染性胸膜肺炎等5种猪传染病阳性血清的检测结果均为阴性,表明其具有良好的特异性。批内和批间重复性试验结果发现,检测同一份血清的变异系数小于10%,表明其重复性较好。包被好的酶标板37℃放置5d后,对同一份血清的检测敏感性无明显变化,初步表明其稳定性较好。利用建立的间接ELISA方法和进口ELISA试剂盒分别对150份血清样品进行非洲猪瘟血清抗体检测,结果表明本方法的特异性和敏感性分别为99.1%和94.3%,2种方法检测结果的符合率为98%。以上试验表明,本试验建立的间接ELISA方法具有良好的特异性和敏感性、较好的重复性和稳定性,可以满足临床检测的需求。     

Comparison of sensitivity and specificity in three commercial foot-and-mouth disease virus non-structural protein ELISA kits with swine sera in Taiwan

Three commercialized ELISA kits for the detection of antibodies to the non-structural proteins (NSPs) of FMD virus were compared, using sera from uninfected, vaccinated, challenged and naturally infected pigs. The kinetics of the antibody response to NSPs was compared on sequential serum samples in swine from challenge studies and outbreaks. The results showed that ELISA A (UBI) and ELISA B (CEDI) had better sensitivity than that of the 3ABC recombinant protein-based ELISA C (Chekit). The peak for detection of antibodies to NSPs in ELISA C was significantly delayed in sera from natural infection and challenged swine as compared to the ELISA A and B. The sensitivity of the three ELISAs gradually declined during the 6-month post-infection as antibodies to NSP decline. ELISA kits A and B detected NSP antibody in 50% of challenged pigs by the 9-10th-day and 7-8th-day post-challenge, respectively. ELISA B and C had better specificity than ELISA A on sequential serum samples obtained from swine immunized with a type O FMD vaccine commercially available in Taiwan. Antibody to NSPs before vaccination was not detected in swine not exposed to FMD virus, however, antibody to NSPs was found in sera of some pigs after vaccination. All assays had significantly lower specificity when testing sera from repeatedly vaccinated sows and finishers in 1997 that were tested after the 1997 FMD outbreak. However, when testing sera from repeatedly vaccinated sows or finishers in 2003-2004, the specificity for ELISAs A, B and C were significantly better than those in 1997. This effect was less marked for ELISA A. The ELISA B was the best test in terms of the highest sensitivity and specificity and the lowest reactivity with residual NSP in vaccinates.     

Evaluation of two commercial PRRSV antibody ELISA kits with samples of known status and singleton reactors

Two commercial PRRSV ELISA kits (IDEXX and Bionote) were evaluated for their sensitivity and specificity using 476 PRRS-positive serum samples collected from 7 animal challenge experiments and 1,000 PRRS-negative sera. Both ELISA kits exhibited 100% sensitivity with sera collected 14 to 42 days post-infection, and the results from the kits were highly correlated (R²=0.9207). The specificity of IDEXX or Bionote kit was 99.9% or 99.7%, respectively. In addition, the Bionote ELISA kit was used to examine 100 sera that were determined to be falsely positive either by IDEXX 2XR or 3XR ELISA, and only 7 of these samples were found to be positive. These results indicate that both ELISA kits exhibited similar levels of sensitivity and specificity and would complement one another for the verification of false-positive samples.     

Evaluation of a commercially available ELISA kit for detection of antibodies to Anaplasma marginale and Anaplasma centrale in cattle in Australia and Zimbabwe

A newly available competitive inhibition ELISA kit for the serological diagnosis of anaplasmosis was evaluated in Australia and Zimbabwe. In Australia the performance of the test was compared with the card agglutination test (CAT).The assay was evaluated using negative sera collected from Anaplasma-free herds, positive sera from experimentally infected cattle and sera from Anaplasma marginale-endemic herds. The sensitivity and specificity of the ELISA in Australia were 100 % and 83,3 %, respectively, and the sensitivity and specificity of the CAT were both 100%. The agreement between the ELISA and CAT in the sera from endemic herds was 86,4 % (kappa = 0,718). The specificity of the ELISA in Zimbabwe was 100%. No meaningful estimate of sensitivity was possible in Zimbabwe because few known positive sera were available for testing, but all eight known positive sera that were available were clearly positive. We conclude that the ELISA is a useful alternative to the CAT for epidemiological studies.The ELISA kits have advantages over the CAT in that the ELISA is more robust and reagents are better standardized, but the kits are expensive.     

REV重组env蛋白间接ELISA诊断试剂盒的研制

以纯化的env蛋白为包被抗原,建立了检测REV抗体的间接ELISA（ienv-ELISA）方法。ienv-ELISA工作条件优化结果表明,最佳包被液为CB;最佳封闭液浓度为1%BSA-PBS;最佳抗原包被量为8μg/mL;血清最佳稀释度为1∶300;血清最佳作用时间为1 h;二抗的最佳稀释度为1∶5000;二抗最佳作用时间为1 h;底物最佳显色时间为25 min。重复性试验、特异性试验和对比试验结果显示,建立的ienv-ELISA方法有良好的可重复性,标准差均小于15%;与鸡MDV、ALV、NDV、H5N1、H9N1等病毒阳性抗体均无交叉反应;与REV抗体检测试剂盒（以REV全病毒为包被抗原）对比,所建立的ienv-ELISA诊断敏感性、诊断特异性以及准确率分别为90.625%9、3.75%和92.7%。     

猪伪狂犬病血清抗体gB-ELISA检测方法的建立

用纯化的猪伪狂犬病病毒gB重组蛋白为抗原,建立了检测猪伪狂犬病血清抗体的gB-ELISA方法。最佳反应条件为：抗原包被浓度为3.15μg/mL,待检血清稀释度为1∶40。该方法对猪圆环病毒病、猪瘟、猪细小病毒病、猪繁殖与呼吸综合征（猪蓝耳病）、猪乙型脑炎、猪布氏杆菌病5种疾病阳性血清和SPF猪阴性血清检测呈阴性反应。批间、批内试验变异系数均不超过8%。用该方法与HerdChek ELISA试剂盒同时对119份血清进行了平行检测,其相对敏感性、特异性和符合率分别为：75%、80.7%和79%。试验结果表明：猪伪狂犬病血清抗体gB-ELISA检测方法具有较高的敏感性和特异性,且重复性好,可用于猪伪狂犬病毒血清抗体检测。     

Differentiation of foot-and-mouth disease-infected pigs from vaccinated pigs using antibody-detecting sandwich ELISA

The presence of serum antibodies for nonstructural proteins of the foot-and-mouth disease virus (FMDV) can differentiate FMDV-infected animals from vaccinated animals. In this study, a sandwich ELISA was developed for rapid detection of the foot-and-mouth disease (FMD) antibodies; it was based on an Escherichia coli-expressed, highly conserved region of the 3ABC nonstructural protein of the FMDV O/TW/99 strain and a monoclonal antibody derived from the expressed protein. The diagnostic sensitivity of the assay was 98.4%, and the diagnostic specificity was 100% for na?ve and vaccinated pigs; the detection ability of the assay was comparable those of the PrioCHECK and UBI kits. There was 97.5, 93.4 and 66.6% agreement between the results obtained from our ELISA and those obtained from the PrioCHECK, UBI and CHEKIT kits, respectively. The kappa statistics were 0.95, 0.87 and 0.37, respectively. Moreover, antibodies for nonstructural proteins of the serotypes A, C, Asia 1, SAT 1, SAT 2 and SAT 3 were also detected in bovine sera. Furthermore, the absence of cross-reactions generated by different antibody titers against the swine vesicular disease virus and vesicular stomatitis virus (VSV) was also highlighted in this assay's specificity.