3种口蹄疫O型抗体检测试剂盒对疫苗免疫牛血清抗体检测的比较试验

为了提供检测结果准确且操作便捷的试剂盒,本试验应用两种商品化试剂盒和本实验室研制的一种基于合成肽包被抗原的间接ELISA试剂盒同时对口蹄疫疫苗免疫后14、21和28 d的150份免疫牛血清进行口蹄疫O型抗体水平的检测,并对3种试剂盒测定结果的符合程度、重复性和便捷性进行了比较。试验结果表明3种试剂盒测得的抗体水平检测结果之间高度符合,且3种试剂盒各自的检测结果重复性良好,基于合成肽包被抗原的间接ELISA试剂盒在操作便捷性方面优势明显。     

Application of non-structural protein ELISA kits in nationwide FMD surveillance in pigs to demonstrate virus circulation in Taiwan

Large scale surveillance of FMD non-structural protein (NSP) antibody in pigs was conducted to monitor for FMD virus circulation in Taiwan using Ceditest and UBI NSP ELISA kits after recurrence of FMD in 2009. A total of 53,759 serum samples were collected from pigs in the auction markets in 2009. There were 43 farms with positive FMD NSP reactors to both NSP ELISA tests in the nationwide surveillance. After tracing back, clinical examination and the NSP ELISA testing using both Ceditest and UBI on 14 follow-up serum samples from all the herds with confirmed NSP reactors in 2009, there were 4 farms classified as positive on follow-up testing criteria. In this surveillance, we have demonstrated that the NSP ELISA tests of outbreak farms followed by clinical and serological investigation could be used to detect FMD circulation in the pig population in Taiwan even while the national compulsory vaccination program is ongoing.     

Occurrence of cross reactions to foot-and-mouth disease virus in normal swine sera.

Sera from 101 swine never exposed to foot-and-mouth disease virus were tested by the plaque-reduction neutralization (PRN) and radial immunodiffusion techniques for cross-reactions to 5 types of foot-and-mouth disease viruses. Depending on the group of sera and the virus used, the percentage of sera cross-reacting at low levels varied from 0 to 50% with the PRN technique and 0 to 20% with the radial immunodiffusion technique. 5erum-neutralization tests in mice support the finding of neutralizing antibody by the PRN technique. Ultracentrifugation and 2-mercaptoethanol studies indicate that the cross-reactions are the result of immunoglobulin M or similar macroglobulins.     

Comparable sensitivity and specificity in three commercially available ELISAs to differentiate between cattle infected with or vaccinated against foot-and-mouth disease virus

Three commercially available ELISAs for the detection of antibodies to the non-structural proteins of foot-and-mouth disease virus (FMDV) were evaluated, using sera from uninfected, vaccinated, infected, inoculated, first vaccinated and subsequently infected, and first vaccinated and subsequently inoculated cattle. We compared antibody kinetics to non-structural proteins, sensitivity, and specificity. One of the ELISAs had a higher sensitivity and much lower specificity than the other two, therefore we established standardised cutoff values for the compared assays using receiver operated characteristic (ROC) curves. Using the standardised cutoff values, all three ELISAs produced comparable results with respect to sensitivity and specificity. Antibody development to non-structural proteins after infection and after vaccination/infection was not significantly different. Development of antibodies, however, both neutralising and directed to non-structural proteins, was significantly delayed after intranasal inoculation as compared to intradermolingual infection. Based on results of sera obtained after vaccination and experimental infection all three assays can be used for testing sera collected between 4 weeks and 6 months after infection. More information is needed on the prevalence of positive reactors in a situation where emergency vaccination has been used and FMD transmission was still observed.     

Detection of antibodies against serotypes 1 and 2 infectious bursal disease virus by commercial ELISA kits

Two distinct serotypes of infectious bursal disease virus (IBDV) are recognized in chicken and turkey flocks in the United States. Serologic testing of chicken flocks for serotype 1 viruses is routinely performed to monitor disease status and vaccination. Earlier studies indicated that enzyme-linked immunosorbent assay (ELISA) test detects antibodies to both serotypes of the virus, while the virus neutralization (VN) test is serotype specific. It is useful to evaluate currently available commercial ELISA kits for their ability to differentiate between antibodies elicited by the two serotypes. Three trials were performed in which chickens were orally inoculated with either a high or a low dose of serotype 1 STC or serotype 2 OH strains of IBDV. Sera collected at 0, 7, 14, and 21 days from these chickens and antisera procured from naturally infected broiler (n=20) and layer (n=30) flocks were tested with five different commercial ELISA kits and by VN. All ELISA kits detected different levels of antibodies elicited against serotype 1 of the virus and moderate and high levels of antibodies against serotype 2 virus. A correlation existed between the ELISA and the VN titers of experimentally infected chickens. All serum samples tested from the commercial layer flocks and 65% of the broiler flocks had antibodies against the OH strain. However, no correlation between the VN titers and ELISA titers was observed for the commercial broilers and layers sera by the majority of the kits. The results indicated that currently available commercial ELISA kits detect antibodies elicited by the two serotypes of IBDV. Hence, the prevalence of serotype 2 antibodies in the flocks should be considered while determining antibody profiles of the flocks against serotype 1 viruses.     

尿样中莱克多巴胺残留ELISA检测试剂盒的比对实验

酶联免疫吸附技术（ELIsA）由于其快速、简便、适合同时处理大量样品的特点，被国内外普遍应用于进行口一兴奋剂等各种兽药残留的筛选检测。国内目前销售的菜克多巴胺残留ELISA试剂盒主要有4种，据悉新的试剂盒还将推出。但国内外尚缺乏对不同莱克多巴胺ELISA试剂盒的检测性能的权威评估。由于不同试剂盒的检测效果可能存在差异，检测试剂不统一，可能导致检测结果差异较大，将不利于政府残留监控计划的实施和企业维护自身权益。本研究拟通过比对检测试验，对不同莱克多巴胺ELISA试剂盒进行评估和比对，为制定合理的莱克多巴胺残留检测技术方案和准确评判检测结果提供科学依据。     

3种ELISA试剂盒检测抗牛支原体抗体的比较

为比较3种抗牛支原体(M.bovis)血清抗体的ELISA试剂盒检测效果,本实验应用3种检测M.bovis血清抗体ELISA诊断试剂盒对38份阳性样品(自然感染19份,人工感染18份)和37份阴性样品进行检测.结果表明:本实验室制备的HVRI试剂盒与商品化试剂盒Kit 1的检测结果和综合检测结果符合率分别达到92%和96%;而商品化试剂盒Kit 2的检测结果与综合检测结果符合率仅为74.67%.一致性检验结果显示:HVRI试剂盒与Kit 1的一致性较高;Kit 2与HVRI试剂盒、Kit 2与Kit 1有中度的一致性.此外,3种试剂盒对牛传染性胸膜肺炎国际标准血清(PS2)的检测结果显示HVRI试剂盒和Kit 1均为为阴性,Kit 2为阳性.因此,HVRI试剂盒与Kit 1更适于M.bovis检测和开展流行病学调查.     

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.     

Direct contact transmission of three different foot-and-mouth disease virus strains in swine demonstrates important strain-specific differences

A novel direct contact transmission model for the study of foot-and-mouth disease virus (FMDV) infection of swine was utilized to investigate transmission characteristics of three FMDV strains belonging to serotypes A, O and Asia1. Each strain demonstrated distinct transmission characteristics and required different exposure times to achieve successful contact transmission. While a 4h exposure was sufficient for strain A24 Cruzeiro (A24Cru), both O1 Manisa and Asia1 Shamir transmission required 18h or more. Viral excretion levels from donors (for all three strains) and virus present in room air (for A24Cru and O1 Manisa) were evaluated and associated with clinical signs and observed transmission pattern. Although all directly inoculated donor animals showed acute FMD, A24Cru had the highest levels of viral shedding in saliva and nasal swabs followed by O1 Manisa and Asia1 Shamir. Virus levels in room air were higher and were detected longer for A24Cru than for O1 Manisa. These results provide direct evidence for important strain-specific variation in transmission characteristics and emphasize the need for thorough evaluation of different FMDV viral strains using a well defined contact transmission methodology. This information is critical for vaccine and biotherapeutic efficacy testing, pathogenesis and disease modeling of FMDV transmission.     

Presence of antibodies to non-structural proteins of foot-and-mouth disease virus in repeatedly vaccinated cattle

For the purpose of removing infected animals by detecting humoral immune responses to non-structural proteins of the foot-and-mouth disease (FMD) virus, antibodies induced by contaminated residual non-structural proteins contained in less pure FMD vaccine can be problematic for serological screening. The aim of the present study was to measure the possible presence of antibodies against these non-structural proteins in repeatedly vaccinated calves and beef cattle. Five imported FMD vaccines were examined using two commercial ELISA kits, UBI FMDV NS EIA and Ceditest FMDV-NS, for serological testing. After five doses of vaccination, the serum of one calf tested positive, and two vaccines induced a significant increase in anti-3ABC antibodies in calves. This finding demonstrated that a positive reaction to non-structural proteins due to impurities in the FMD vaccine was detectable using commercial tests. A low percentage of field sera sampled from beef cattle in Kinmen also tested positive, but the key factor resulting in the positive reactions could not be positively identified based on our data.     

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.     

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.     

猪口蹄疫病毒多抗原表位的原核表达与纯化

利用限制性酶切从重组质粒pShuttle-CMV-VP中得到猪O型口蹄疫病毒VP1(21-60)-(141-160)-(200-213)位氨基酸的基因。将此多抗原表位基因克隆至原核高效表达载体pET43.1 a(+),在E.coliBL21中用IPTG诱导表达了含有猪口蹄疫病毒多抗原表位的融合蛋白,并用镍柱亲和层析法获得了纯化蛋白。W estern-b lot结果表明融合蛋白可被猪O型口蹄疫病毒标准阳性血清所识别,从而为进一步研究FMDV多表位抗原的免疫特性和诊断方法奠定了基础。     

Reconstruction of a swine SLA-I protein complex and determination of binding nonameric peptides derived from the foot-and-mouth disease virus

No experimental system to date is available to identify viral T-cell epitopes in swine. In order to reconstruct the system for identification of short antigenic peptides, the swine SLA-2 gene was linked to the beta(2)m gene via (G4S)3, a linker encoding a 15-amino acid glycine-rich sequence (G4S)3, using splicing overlap extension-PCR (SOE-PCR). The maltose binding protein (MBP)-SLA-2-(G4S)3-beta(2)m fusion protein was expressed and purified in a pMAL-p2X/Escherichia coli TB1 system. The purified MBP-SLA-2-(G4S)3-beta(2)m protein was cleaved by factor Xa protease, and further purified by DEAE-Sepharose chromatography. The conformation of the SLA-2-(G4S)3-beta(2)m protein was determined by circular dichroism (CD) spectrum. In addition, the refolded SLA-2-(G4S)3-beta(2)m protein was used to bind three nonameric peptides derived from the foot-and-mouth disease virus (FMDV) O subtype VP1. The SLA-2-(G4S)3-beta(2)m-associated peptides were detected by mass spectrometry. The molecular weights and amino acid sequences of the peptides were confirmed by primary and secondary spectra, respectively. The results indicate that the SLA-2-(G4S)3-beta(2)m was 41.6kDa, and its alpha-helix, beta-sheet, turn, and random coil by CD estimation were 78 aa, 149 aa, 67 aa, and 93 aa, respectively. SLA-2-(G4S)3-beta(2)m protein was able to bind the nonameric peptides derived from the FMDV VP1 region: 26-34 (RRQHTDVSF) and 157-165 (RTLPTSFNY). The experimental system demonstrated that the reconstructed SLA-2-(G4S)3-beta(2)m protein complex can be used to identify nonameric peptides, including T-cell epitopes in swine.     

Single dilution ELISA for detection of serum antibody to foot-and-mouth disease virus in cattle

A single dilution blocking ELISA was developed and evaluated for measuring serum antibody to foot-and-mouth disease virus (FMDV). Basic parameters of the assay were established and a positive-negative threshold determined from testing 176 specific antibody negative sera from Australian cattle. Sera collected from immunised animals in Thailand were tested by ELISA and virus-neutralisation (VN) tests and the results compared. A positive correlation between ELISA and VN titres was recorded for each of the 3 FMDV serotypes endemic in Thailand, with the overall correlation coefficient being r = 0.8990. A positive correlation for each of the serotypes was also found between ELISA titre and the degree of blocking (percentage inhibition) of each test serum at a dilution of 1:16, with the overall correlation being r = 0.8704. This simplified ELISA was sensitive, specific and gave reproducible results, and had the potential to test quickly and efficiently a considerable number of sera.