高敏荧光免疫层析分析法检测牛分枝杆菌抗体

为探究高敏荧光免疫层析分析法检测牛分枝杆菌（M. bovis）抗体的可行性，对该方法的特异性、灵敏性、稳定性及临床适用性进行了分析。特异性试验结果显示，该方法能够检出M. bovis抗体阳性血清参考品，而对布鲁氏菌阳性血清、O型口蹄疫阳性血清、A型口蹄疫阳性血清、样品稀释液、结核菌素皮内变态反应（TST）阴性牛血清样品等均为阴性；灵敏性试验结果显示，该方法最低可检测到3 200倍稀释的牛结核（bTB）强阳性血清参考品；稳定性试验结果显示，该方法检测bTB强阳性血清和阳性血清的变异系数分别为5.48%和5.83%；临床样本检测结果显示，该方法与IDEXX M. bovis ELISA抗体检测试剂盒的符合率为86.36%，具有较高的一致性，且差异不显著。以上结果表明，高敏荧光免疫层析分析法特异性强、灵敏度高、结果可靠，可作为TST检疫的补充抗体检测方法，用于bTB的诊断、检疫和净化。     

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。     

比较两种ELISA试剂盒在猪伪狂犬病血清抗体检测中的一致性

比较两种ELISA试剂盒检测结果的一致性,为流行病学调查和临床诊断筛选适合的商品化试剂盒。采用来自两个厂家的野毒抗体(gE)和免疫抗体(gB)ELISA检测试剂盒分别检测362份和392份猪血清,应用Kappa检验比较试验数据的一致性。结果显示:两种猪伪狂犬病抗体(g E)检测试剂盒联合检测出95份抗体阳性和256份阴性,符合率97.24%,结果一致性为极强(Kappa值0.932);两种猪伪狂犬病抗体(gB)检测试剂盒联合检测出351份抗体阳性和9份阴性,符合率91.84%,一致性为弱(Kappa值0.347)。以上结果说明,两种猪伪狂犬病抗体(gE)检测试剂盒都适用于PRV gE抗体检测,而两种猪伪狂犬病抗体(gB)检测试剂金差异较大,需慎重选择。     

不同ELISA试剂盒检测牛结核病效果比较

本研究对自行研制的检测牛γ干扰素的双夹心ELISA试剂盒与商品化试剂盒进行了比较。用自行研制的双夹心ELISA试剂盒对1255份经牛PPD刺激的牛全血培养上清样品进行了检测,结果发现94份阳性样品,检测阳性率为7.5%。同时用进口的ELISA试剂盒对其中的472份样品进行了对比检测,两者阳性符合率为97.6%,阴性符合率为96.4%,总符合率为96.6%。比较检测结果证明,自行研制的检测牛γ干扰素的双夹心ELISA试剂盒有很好的应用前景。     

牛传染性鼻气管炎病毒血清抗体检测试验及结果分析

采用SYNBIOTICS公司酶联免疫吸附试验(ELISA)检测试剂盒对来自新疆15个不同地区(包括4个养牛场)共635份牛血清进行了牛传染性鼻气管炎病毒血清抗体检测,共检出阳性血清329份,最高感染率达90%,最低感染率为5%,平均感染率为51.8%,结果显示该病已在全疆普遍存在,感染没有明确的地域分布规律。对其中的106份血清用Institute Pourquier公司ELISA试剂盒重新检测,发现两试剂盒的符合率为90.6%,Institute Pourquier公司ELISA检测试剂盒敏感性稍高。从该106份血清中再次随机抽取其中的35份用病毒中和实验(VN)检测方法进行了检测,检测结果与SYNBIOTICS公司ELISA检测试剂盒相比较,同为阳性样品24份,同为阴性样品7份,两者符合率88.6%;与Institute Pourquier公司ELISA检测试剂盒相比较,同为阳性样品24份,同为阴性样品6份,两者符合率85.7%。试验结果表明,与VN相比,ELISA检测试剂盒具有操作简便,敏感性高等优点。     

猪瘟抗体阴性猪筛选方法的比较

评价ELISA法用于筛选猪瘟抗体阴性猪的可行性,分别采用两种商品化猪瘟抗体ELISA检测试剂盒检测了61份猪血清样品,并与兔体中和试验方法进行了比较.兔体中和试验法检测出4份阳性、2份可疑、55份阴性,而两种ELISA试剂盒均检测出6份阳性、55份阴性;两种ELISA方法与兔体中和试验检测结果阴性符合率均为100%(55/55).结果表明,ELISA法更加敏感,可以替代兔体中和试验方法用于筛选猪瘟抗体阴性猪.     

筛选猪瘟抗体阴性猪检测方法的比较

评价ELISA法用于筛选猪瘟抗体阴性猪的可行性,分别采用两种商品化猪瘟抗体ELISA检测试剂盒检测了61份猪血清样品,并与兔体中和试验方法进行了比较。兔体中和试验法检测出4份阳性、2份可疑、55份阴性,而两种ELISA试剂盒均检测出6份阳性、55份阴性;两种ELISA方法与兔体中和试验检测结果阴性符合率均为100%（55/55）。结果表明,ELISA法更加敏感,可以替代兔体中和试验方法用于筛选猪瘟抗体阴性猪。     

口蹄疫病毒A型竞争ELISA抗体检测试剂盒在流行病学调查中的应用

为评价口蹄疫病毒A型竞争ELISA(cELISA)抗体检测试剂盒在流行病学调查中的应用前景,对2017年从福建省三明市采集的336份黄牛、奶牛、羊和猪血清样品,用A型cELISA抗体检测试剂盒进行抗体检测。结果显示,92份黄牛血清、92份羊血清、92份猪血清、60份奶牛血清的A型抗体阳性率分别为13.04%、11.96%、20.65%、86.67%。从上述4种血清中,各挑选10份血清(阴性、阳性各5份)共40份,采用口蹄疫病毒液相阻断ELISA(LPB-ELISA)抗体检测试剂盒进行验证。结果显示:cELISA检测为阳性的20份血清中,用LPB-ELISA检出阳性19份;cELISA检测为阴性的20份血清中,用LPB-ELISA检出阴性17份;两种方法的κ值为0.8,总符合率为90.00%。结果表明,A型cELISA试剂盒与LPB-ELISA试剂盒的符合率和一致性均较高,可用于口蹄疫流行病学调查和血清学监测。     

副猪嗜血杆菌间接ELISA检测方法的建立

为建立一种快速检测副猪嗜血杆菌(HPS)的血清学方法,本实验采用重组表达的HPS细胞致死膨胀毒素B亚基蛋白(r Cdt B)作为包被抗原,并对各反应条件进行优化,建立了检测HPS血清抗体的间接ELISA方法。以该方法检测HPS阳性血清和其他猪病病原阳性血清,除了HPS阳性血清呈阳性外,其余均为阴性,表明该方法特异性良好。批内重复和批间重复的最大变异系数分别小于7%和8%。此外,将Cdt B-ELISA方法与商品化试剂盒Bio Chek HPS-Opp A间接ELISA抗体检测方法对背景明确的116份HPS阳性血清和112份HPS阴性血清进行检测,商品化试剂盒阳性符合率为25%,阴性血清符合率为100%,总符合率为62%,其中Cdt B-ELISA方法对阳性血清的检出率高于商品化试剂盒。应用Cdt B-ELISA方法检测330份临床猪血清样品,结果显示,HPS抗体阳性检出率为11.8%。该方法可以用于HPS的临床检测,为HPS流行病学调查和防控提供了一种快速有效的检测方法。     

巴州部分山区牦牛新孢子虫病的rELISA检测

奶牛新孢子虫病是由犬新孢子虫（Neospora caninum）引起的多种家畜共患的一种原虫病。该病对牛的危害尤其严重,主要造成孕畜流产、死胎以及新生儿的运动神经系统疾病。本研究采集巴州部分山区牦牛的210份牦牛血清样本,用重组酶联免疫吸附试验（rELISA）检测N.caninum血清杭体。结果发现,巴州部分山区牦牛N.caninum抗体阳性率为18.2%,抗体阳性血清样本刚地弓形虫（T.gondii）抗体检测的结果为阴性。被检的样品经过商品化试剂盒（Ncaninum Antiboby Test Kit,IDEXX公司产）检测对比,其符合率为99%。说明该地方山区牦牛中存在新孢子虫感染,应用重组rELISA试剂盒对其进行检测,其检出率与商品化试剂盒相符合。     

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.     

Agar gel immunodiffusion test for the detection of bovine leukemia virus antibodies: lack of trans-Atlantic standardization.

Two agar gel immunodiffusion (AGID) kits for the serodiagnosis of bovine leukemia virus (BLV) were imported from Europe and were compared with North American kits. The BLV AGID kits from North America and from Europe differed significantly. The punches were different, as were the pattern distribution in the agar of the reference and the test sera, resulting in differences in the reading of the immunoprecipitation lines. Based on the testing of 1200 serum samples from cattle, the European kits gave a good correlation with the American kits, as indicated by their respective kappa values. However, the European kits were found to be less sensitive when evaluated against weakly positive samples from field specimens or following a dilution trial. Only 65% and 50% of the weakly positive samples detected by the American kit #1 were detected by the European kits #2 and #3, respectively. The American kit was also capable of detecting BLV antibodies in 45% of strongly positive samples diluted 1/50 in negative sera, while antibodies were detected in only 15% of the samples with the European kit #2 and in none of the samples with the European kit #3. False negatives were also detected with the European kits. Among the false negatives, the degree of expected reactions was weak (European kit #2) or of varying degrees of positivity (European kit #3). Besides the differences in format and performance, the BLV-AGID kits in Europe are evaluated with the National Standard Serum E4 while a proficiency panel composed of a quadruplicate set of 10 reference sera is used in Canada to monitor the kits. Based on the overall observations, we noted a lack of standardization between the BLV-AGID kits used in North America and in Europe.     

Comparison of results of three commercial heartworm antigen test kits in dogs with low heartworm burdens

OBJECTIVE: To compare results of 3 commercial heartworm antigen test kits performed on serum samples from dogs infected with low numbers of adult female heartworms. DESIGN: Blinded laboratory evaluation. Sample Population-Serum samples from dogs (n = 208) proven at necropsy to be infected with 1 to 4 adult female heartworms and from dogs (32) without heartworms. PROCEDURE: Samples were sequentially tested with each test kit, following the manufacturers' instructions, by licensed veterinary technicians in private practice who were not aware of infection status of the dogs. The order of test kit evaluations was randomly chosen. For each test kit, sensitivity, specificity, accuracy, positive predictive value, and negative predictive value were evaluated. RESULTS: All tests yielded some false-negative results, and there were significant differences among tests in regard to ability to detect low heartworm burdens. Sensitivity of the test kits ranged from 78 to 84%. For all test kits, sensitivity increased as number of female heartworms increased. All 3 test kits had high specificity (97%). CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that sensitivity of the 3 commercially available heartworm antigen test kits ranged from 78 to 84% when used to test serum samples from dogs with low heartworm burdens, and that sensitivity varied among test kits. For all 3 test kits, specificity was 97%. All 3 test kits yielded false-positive and false-negative results for some dogs with low heartworm burdens.     

Development and evaluation of a MAb based competitive-ELISA using helicase domain of NS3 protein for sero-diagnosis of bovine viral diarrhea in cattle and buffaloes

The aim of this study was to develop a competitive inhibition ELISA (CI-ELISA) for detection of antibodies to bovine viral diarrhea virus (BVDV) using the helicase domain of NS3 (non-structural) protein and monoclonal antibody (MAb) against it and to estimate its sensitivity and specificity using two commercial ELISA kits as independent references. The 45-kDa helicase domain of NS3 protein of BVDV was expressed in Escherichia coli and 18MAbs were developed against it. MAb-11G8 was selected for use in CI-ELISA on the basis of maximum inhibition (90%) obtained with BVDV type 1 infected calf serum. Based on the distribution of percent inhibition of known negative sera (n=166), a cut-off value was set at 40% inhibition. In testing 914 field serum samples of cattle (810) and buffaloes (104), the CI-ELISA showed a relative specificity of 95.75% and 97.38% and sensitivity of 96% and 94.43% with Ingenesa kit and Institut Pourquier kit, respectively. This study proved that the use of helicase domain of NS3 (45-kDa) is equally good as the whole NS3 protein (80-kDa) used in commercial kits for detection of BVDV antibodies in cattle and buffaloes.     

Development of an antibody-detection ELISA for Fasciola hepatica and its evaluation against a commercially available test

An ELISA was developed for the detection of Fasciola hepatica antibody in serum of cattle. The assay was applied to sera from 258 naturally infected cattle, 256 non-infected cattle and six calves experimentally infected with F. hepatica. The diagnostic sensitivity and specificity of the ELISA test was 98% (95% confidence intervals, 96-100%) and 96% (95% confidence intervals, 93-98%) respectively at a cut-off value of 15% positivity. The results using sera from the experimentally infected calves showed that antibodies were first detected 2-4 weeks after infection. The ELISA test was also compared to the commercially available Bio-X bovine F. hepatica ELISA kit. A subset of 39 positive sera and 47 negative sera were selected from the samples used to evaluate the in-house test. The results indicated that the agreement between the two tests was almost perfect (k statistic=0.82).     

Comparison of two commercial radial immunodiffusion assays for detection of bovine immunoglobulin G in newborn calves.

Bovine failure of passive transfer (FPT), defined as inadequate transfer of colostral immunoglobulins from the dam to the calf, has been associated with increased risk in neonatal mortality. Currently, radial immunodiffusion (RID) assay is considered to be the gold standard in determining FPT in serum samples from calves. There are 2 commercial RID assays routinely used for serodiagnosis of FPT in calves: VET-RID and SRID. Discrepancies between results of these RID assays were observed in the authors' laboratory. The objective of this study was to compare 2 commercial RID assays by testing a paired panel of 30 blood samples collected from newborn Holsteins at birth before, and 24 hr after, ingestion of colostrum, a commercial bovine reference serum, and a panel of different concentrations of 2 purified bovine immunoglobulin G (IgG) products. Overall, the results of this study showed a high level of discrepancy and poor agreement between the 2 RID kits. The interassay precision study revealed lower between-run coefficients of variation for the VET-RID kit compared with the SRID kit. The spiking and recovery study using purified bovine IgG products demonstrated that the VET-RID kit more closely approximates the expected concentrations of the purified bovine IgG products, whereas the SRID kit consistently overestimates the concentration of purified bovine IgG products. It was concluded that this may be due to inaccuracies in the internal standards of the SRID kit.     

Optimization of in-house ELISA based on recombinant major sperm protein (rMSP) of Dictyocaulus viviparus for the detection of lungworm infection in cattle

The aim of this study was to optimize an in-house ELISA based on a recombinant version of the major sperm protein (MSP) of Dictyocaulus viviparus for routine diagnosis of lungworm infection in cattle. A recombinant MSP (rMSP) was cloned into pGEX-6P-1 vector and expressed as a glutathione-S-transferase (GST) fusion protein in Escherichia coli BL21 (DE3) chemically competent cells. The product was then employed as capture antigen in an ELISA, and validated against 304 samples of known status (216 negative and 88 positive) in which the antibody levels in sera had also been measured earlier with a commercial ELISA kit (Ceditest® lungworm ELISA). The receiver operating characteristic (ROC) curve analysis of the ELISA results estimated the optimized diagnostic sensitivity and specificity as 97.7% (95% confidence interval [CI]: 91.9–99.7%) and 98.1% (CI: 95.3–99.5%), respectively. The results from the in-house rMSP-based ELISA were compared with results obtained on both fecal examination and the Ceditest® lungworm ELISA. Rising antibody levels in sera of experimentally infected calves were observed between 21 and 28 days post infection, when patency was also confirmed by the presence of larvae in feces. Notably, using the in-house rMSP-based ELISA infection was confirmed in calves shedding larvae approximately 3–4 weeks post inoculation, while using the Ceditest® lungworm ELISA those animals remained negative. Additionally, 251 sera samples from calves naturally exposed to the parasites on pasture were used to evaluate the test. In in-house rMSP-based ELISA no cross-reactions were observed with sera from calves infected with the gastrointestinal nematodes (Ostertagia ostertagi and Cooperia oncophora), even though the presence of eggs in the feces was confirmed. Overall, the in-house rMSP-based ELISA optimized in this study showed excellent diagnostic performance for detection of lungworm infection in cattle.     

口蹄疫O型固相竞争ELISA抗体检测试剂盒的临床应用

用口蹄疫O型固相竞争ELISA检测血清1 150份,口蹄疫O型免疫血清820份,敏感性为91.83%;试剂盒特异性评价检测血清180份,其特异性为89.4%;固相竞争ELISA和液相阻断ELISA共同检测血清100份,其相关性分析为0.964 1,R2为0.929 5,属于高度相关。3批试剂盒检测血清样品50份,检测结果差异不显著,口蹄疫O型固相竞争ELISA抗体检测试剂盒检测结果比较稳定,具有良好的批间可重复性和稳定性。     

重组N蛋白抗原检测PRRSV抗体ELISA的研究Ⅳ.与IDEXX ELISA试剂盒及Western blot的比较

利用重组N蛋白抗原建立了检测猪繁殖与呼吸综合征病毒（PRRSV）抗体的ELISA方法并组装成试剂盒，将试制的3批试剂盒分别与IDEXX生产的试剂盒及Western—blot进行了符合率试验。试验结果表明，所研制的试剂盒与Western blot的符合率达97．67％以上。对460份临床血清分别用自制的试剂盒和IDEXX公司生产的试剂盒进行检测，其中有37份不相符合，用Western blot对这37份血清进行验证，有35份血清检测结果与自制的试刺盒检测结果一致。由此表明，自行研制的试剂盒，其特异性和敏感性均能满足目前临床上该疫病的流行病学分析或免疫抗体检测。     

Detection of foot-and-mouth disease virus infection in vaccinated cattle

The aim of this study was to evaluate the value of commercially available kits for the detection of foot-and-mouth disease (FMD) virus infection in vaccinated cattle. The cattle were vaccinated with a commercial aqueous FMD vaccine type A24 and subsequently challenged 28 days post vaccination with homologous FMD virus. Seven of eight animals were protected from clinical disease and all became carriers. They were bled sequentially for up to 130 days post infection and samples of sera were tested with three ELISA kits: CHEKIT FMD-3ABC, Ceditest FMDV-NS and SVANOIR FMDV 3ABC-Ab ELISA. The Ceditest kit appears to be relatively higher sensitive than the others. When examined with this ELISA, all cattle developed of FMDV nonstructural proteins (NSPs) antibodies and remained positive throughout the period of the experiment. The response of antibodies against 3ABC antigen delayed in two cattle challenged with FMDV A24 virus. One of the cattle reacted negatively in Svanoir ELISA kit and sera from two animals were found negative in CHEKIT ELISA. It can be concluded that all tested kits can be a promising tool for FMD control and eradication campaigns in situation where emergency vaccination was applied.