A complex-trapping-blocking (CTB) ELISA, using monoclonal antibodies and detecting specifically antibodies directed against foot-and-mouth disease types A, O and C. II. Application

The complex-trapping-blocking (CTB) enzyme-linked immunosorbent assay (ELISA) was evaluated to detect antibodies directed against foot-and-mouth disease virus (FMDV) strains A10 Holland, O1 BFS, and C1 Detmold. Log10 serum titres of uninfected, unvaccinated cattle (n = 100) were less than 1.80 in the CTB-ELISA. Sera from cattle vaccinated with either monovalent or trivalent vaccines were tested in both the CTB-ELISA and the serum neutralisation test (SNT); titres in both tests correlated positively (P less than 0.001). Titres of sera from cattle, sheep, and pigs vaccinated twice with FMDV A10 Holland also correlated positively in both tests. In another experiment, cattle vaccinated with FMDV strain C1 Detmold were intradermolingually challenged 3 weeks after primary vaccination; at the same time two controls were challenged. At 8 days after challenge, serum titres of the controls were distinctly higher in the CTB-ELISA than in the SNT, whereas serum titres of the vaccinated cattle were equally high in both tests. In potency tests for monovalent vaccines against FMDV strains A10 Holland, O1 BFS or C1 Detmold, serum titres correlated strongly in both tests with protection against the homologous FMDV strain. We concluded that the CTB-ELISA is not only sensitive, but easier to perform and more rapid and reproducible than the SNT. The CTB-ELISA may be useful in evaluating the immune response in cattle during FMD vaccine potency tests.     

Enzyme-linked immunosorbent assay for the detection of antibodies to bovine viral diarrhea virus in bovine sera

A specific and sensitive enzyme-linked immunosorbent assay (ELISA) was established for the detection of antibodies to bovine viral diarrhea virus (BVDV) in bovine sera. Polyethylene-glycol concentrated, equilibrium density gradient purified BVDV was used as test antigen at an optimal amount of 1 microgram/well, whereas the optimal concentration of conjugate was at 1/2000 dilution. The standardized test encountered no non-specific reaction with test sera at a starting dilution of 1/10. A total of 50 bovine serum samples was assayed for the presence of antibodies against BVDV by ELISA and serum neutralization test (SNT). A positive correlation between the 2 tests was found. However, ELISA could be as much as 500-fold more sensitive than SNT in detecting low levels of BVDV antibodies.     

Asia I型口蹄疫疫苗效力检验替代方法的初步研究

使用三株针对AsiaⅠ型口蹄疫146S抗原的杂交瘤细胞株(1#G3C2、2#G6B9、10#C2G1)大量生产腹水单克隆抗体,粗提纯化并按一定的比例进行混合。初步建立混合腹水单克隆抗体介导的间接夹心ELISA方法,使用该方法测定AsiaⅠ型病毒灭活抗原、O型病毒灭活抗原、正常BHK21细胞培养液,结果表明该方法特异性好,所测定OD值和AsiaⅠ型抗原稀释倍数呈明显线性关系。用该方法对11批AsiaⅠ型、O型单价或双价成品疫苗破乳抗原进行测定,结果表明不同批次不同企业生产的疫苗完整病毒颗粒抗原含量有差异,进一步完善有望用于AsiaⅠ型口蹄疫成品疫苗中有效颗粒抗原含量的测定,从而发展成一种疫苗效力检验替代方法。     

基于单克隆抗体的O型口蹄疫病毒抗体固相竞争ELISA检测方法的建立

为建立评价O型口蹄疫病毒(FMDV)疫苗免疫水平的方法,本研究以单克隆抗体(MAb)3D9为捕获抗体,以HRP标记的MAb 8E8作为检测MAb,经过条件优化建立了基于MAb的检测O型FMDV抗体的固相竞争ELISA(SPCE)方法。对该方法进行了特异性、敏感性、重复性试验。结果显示,MAb 3D9的最佳稀释度为1:25 000,灭活O型FMDV抗原的最佳稀释浓度为1:3,HRP标记的MAb 8E8的最佳稀释度为15 000,当血清1:32稀释时,检测的临界值确定为45%。该方法分别检测A型FMDV抗体阳性参考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,检测结果均为阴性,未出现交叉反应。经检测,当阳性标准血清的抗体稀释度在1:512时,该方法仍具有较好的敏感性;批内和批间重复性试验的变异系数均小于10%,表明其重复性较好。并将该方法与液相阻断ELISA(LPBE)方法和病毒中和试验(VNT)的相关性进行了比较,结果显示该方法与LPBE和VNT的相关性分别为0.896和0.923。本研究为国内评价O型FMDV疫苗免疫水平建立了一种新的方法。     

Detection of Foot and Mouth Disease Virus by RT-PCR and Microplate Hydridization Assay Using Inactivated Viral Antigens

A single step RT-PCR was tested for detection of foot and mouth disease virus (FMDV) and immunoenzymatic determination of amplified products in a microplate hybridization assay. Inactivated reference strains (ELISA antigen) of all seven serotypes were used to optimize the test. Oligonucleotide primers were selected from two different genomic regions coding for RNA polymerase and VP1 protein, respectively. The RT-PCR used to amplify the polymerase gene specific RNA detected FMDV strains A, C, O, Asial and SAT1, and the identity of the fragments obtained was confirmed with a specific internal biotin-labelled capture probe. For the amplification of the VP1 genome region, two sets of oligonucleotide primers were used. One primer pair was successfully applied for the detection of serotypes A, C, O and Asial and a second one for serotypes SAT1, SAT2, SAT3. The specific probe enabled the detection of all the amplified products in a PCR ELISA test. By comparison with antigen ELISA, the PCR ELISA method allowed the detection of smaller amounts of FMDV in the inactivated material examined. The application of molecular diagnostic methods to inactivated antigens offers a good alternative procedure for developing and optimizing a sensitive method for detection of FMDV in laboratories that are not allowed to work with viable FMDV.     

口蹄疫病毒抗原胶体金试纸条的研究

利用单克隆抗体技术制备抗口蹄疫病毒的单克隆抗体,特异性试验表明其只与O、A、Asia 1型3种血清型FMDV抗原结合。进而采用胶体金标记技术,以胶体金标记的抗口蹄疫病毒单克隆抗体、多克隆血清抗体和葡萄球菌A蛋白为主要材料,研制口蹄疫快速检测试纸条。该试纸条检测O、A、Asia 1型3种血清型灭活口蹄疫病毒均为阳性,检测水疱性口炎病毒、猪水疱病病毒、蓝舌病病毒、猪蓝耳病病毒4种灭活抗原及小反刍兽疫病毒疫苗株均为阴性,试验结果与口蹄疫实时荧光定量RT-PCR方法的完全一致,表明其具有良好的特异性。敏感性试验结果是,试纸条的检测极限为1∶160稀释的样品,其敏感性相当于实时荧光定量RT-PCR方法的1/64。由于试纸条具有操作方便、检测快速等优点,因此该试纸条可以用于大量临床样品的快速检测和现场检测。     

A serological survey for antibodies against foot-and-mouth disease virus (FMDV) in domestic pigs during outbreaks in Kenya

Foot-and-mouth disease (FMD) is endemic in Kenya and has been well studied in cattle, but not in pigs, yet the role of pigs is recognised in FMD-free areas. This study investigated the presence of antibodies against FMD virus (FMDV) in pigs sampled during a countrywide random survey for FMD in cattle coinciding with SAT 1 FMDV outbreaks in cattle. A total of 191 serum samples were collected from clinically healthy pigs in 17 districts. Forty-two of the 191 sera were from pigs vaccinated against serotypes O/A/SAT 2 FMDV. Antibodies against FMDV non-structural proteins were found in sera from 30 vaccinated and 71 non-vaccinated pigs, altogether 101/191 sera (53 %), and 91 % of these (92/101) also had antibodies measurable by serotype-specific ELISAs, predominantly directed against SAT 1 with titres of 10–320. However, only five high titres against SAT 1 in vaccinated pigs were confirmed by virus neutralisation test (VNT). Due to high degree of agreement between the two ELISAs, it was concluded that positive pigs had been infected with FMDV. Implications of these results for the role of pigs in the epidemiology of FMD in Kenya are discussed, and in-depth studies are recommended.     

An enzyme-linked immunosorbent assay using nuclear antigen for detection of feline herpesvirus 1 antibody.

To detect antibody against feline herpesvirus 1 (FHV-1) in the sera of cats, the sensitivity and specificity of an enzyme-linked immunosorbent assay (ELISA) using nuclear antigen was investigated. The standardized optical density readings (ODs) of the ELISA obtained by the 1-step serum dilution (1:80) method were compared with the serum neutralization test (SNT) results, with a correlation of 0.993, and with the hemagglutination inhibition (HI) test results, with a correlation of 0.851. The ODs for the ELISA titers were obtained using the serial serum dilution method and were compared with the SNT results, with a correlation of 0.933, and with the HI test results, with a correlation of 0.987. In the experimental infection of 4 specific-pathogen-free cats, the results of different serologic tests (SNT and HI) and the ELISA using the serial serum dilution method revealed rapid production of antibodies after inoculation, whereas the ELISA using the one-step serum dilution method indicated that titers increased more slowly. These results indicate that with the present ELISA using nuclear antigen, there are fewer demands on time and labor, making the method convenient for monitoring FHV-1 infection.     

Comparison of the antibody response to transmissible gastroenteritis virus and porcine respiratory coronavirus, using monoclonal antibodies to antigenic sites A and X of the S glycoprotein.

Pigs were inoculated with various strains of transmissible gastroenteritis virus (TGEV) or with porcine respiratory coronavirus (PRCV), and antigenic site-specific antibody responses were compared. A blocking-ELISA was used to study to what extent antibodies in convalescent sera interfered with the binding of monoclonal antibodies (MAB) 57.16 or 57.110 to the attenuated TGEV/Purdue virus. Monoclonal antibody 57.16 is directed against the A site on the peplomer, neutralizes virus, and recognizes TGEV and PRCV. Monoclonal antibody 57.110 is directed against the X site on the peplomer, but does not neutralize virus, and recognizes only TGEV. Antibodies directed against TGEV and PRCV could be detected in a blocking ELISA, using MAB 57.16 as a conjugate. Antibodies directed against both viruses were detectable as early as 1 week after inoculation. Antibody titers correlated well with those in a virus-neutralization test. Antibodies against TGEV could be detected in a blocking ELISA, using MAB 57.110 as a conjugate. Such antibodies were not induced by a PRCV infection. In the blocking ELISA, using MAB 57.110 as a conjugate, antibodies were detectable as early as 2 weeks after inoculation. There was a significant difference between antibody titers reached after infection with various TGEV strains, however. This difference is ascribed to a variation of the antigenic site defined by MAB 57.110 in TGEV strains. Conditions for a differential test for TGE serodiagnosis, and for serologic discrimination between TGEV- and PRCV-infected pigs, are discussed.(ABSTRACT TRUNCATED AT 250 WORDS)     

A slide latex agglutination test for the rapid detection of antibodies in serum against porcine parvovirus

A slide latex agglutination test (LAT) was developed and evaluated to detect serum antibodies against porcine parvovirus. Porcine parvovirus antigen was obtained by 10% PEG-6000 and 0.5 mol/l sodium chloride precipitation, and inactivated by 0.1% methanal. Two per cent suspensions of latex particles (0.5-0.8 microm) were coated by adding an equal volume of porcine parvovirus antigen at 0.34 microg/ml. Repeatability of latex agglutination test was evaluated with a panel of 100 sera using the same and different antigen lots. A good agreement between LAT and haemagglutination inhibit assay was observed. Because of convenience and speed of performance, this method would be used widely in clinic examination.     

牛口蹄疫病毒VP2结构蛋白抗体间接ELISA方法的建立

为建立牛口蹄疫(FMD)抗体的检测方法,本研究将口蹄疫病毒(FMDV)的VP2基因,通过pPROEXTM HTb表达载体在大肠杆菌DH5α中表达,获得大小为35ku的重组VP2蛋白(rVP2),western blot证实rVP2可与FMDV5种血清型的牛阳性血清发生特异性反应。以纯化复性的rVP2为抗原建立了FMDVrVP2间接ELISA方法。重复性试验证实批内、批间变异系数均小于10%。特异性交叉试验表明,该抗原不与常见的其他7种牛病阳性血清发生交叉反应。检测非免疫无口蹄疫国家牛阴性血清的特异性为100%;检测感染血清敏感性为97.3%;检测O-AsiaⅠ的二价苗免疫牛血清,与4种商品化试剂盒比较,其符合率分别为69.0%、95.0%、90.4%和86.8%。实验结果表明建立的ELISA方法可以用于口蹄疫感染和免疫抗体检测。     

Rapid methodology for antigenic profiling of FMDV field strains and for the control of identity, purity and viral integrity in commercial virus vaccines using monoclonal antibodies

Monoclonal antibodies (MAbs) developed against different foot-and-mouth disease virus (FMDV) vaccine strains were extensively used to study any possible antigenic variations during vaccine production in Argentine facilities. Additionally, a typing ELISA using strain specific MAbs was developed to detect potential cross contaminations among FMDV strains in master and working seeds with high specificity and sensitivity and to confirm strains identity in formulated vaccines. This assay was carried out for the South American strains currently in use in production facilities in Argentina (A24/Cruzeiro, A/Argentina/01, O1/Campos and C3/Indaial) and for the strain O/Taiwan, produced only for export to Asia. These non-cross reactive MAbs were also used to analyze the integrity of viral particles belonging to each one of the individual strains, following isolation of 140S virions by means of sucrose density gradients from the aqueous phase of commercial polyvalent vaccines. Antigenic profiles were defined for FMDV reference strains using panels of MAbs, and a coefficient of correlation of reactivity with these panels was calculated to establish consistent identity upon serial passages of master and production seeds. A comparison of vaccine and field strain antigenic profiles performed using coefficients of correlation allowed the rapid identification of two main groups of serotype A viruses collected during the last FMD epidemic in Argentina, whose reactivity matched closely to A/Argentina/2000 and A/Argentina/2001 strains.     

Development and validation of an indirect enzyme-linked immunosorbent assay for the detection of antibodies against duck swollen head hemorrhagic disease virus

An indirect enzyme-linked immunosorbent assay (iELISA) was developed for detecting antibody to duck swollen head hemorrhagic disease virus (DSHDV) using purified whole virus by sucrose density gradient ultracentrifugation as a coating antigen. Antiserum against DSHDV strains HY-99 (hyperimmume serum) was prepared in 30-day-old ducks by vaccination with inactivated DSHDV and used as positive sera. The iELISA test was optimized with different reagents or dilutions. The validation results showed that this assay was only specific for antibodies against duck viral swollen head hemorrhagic disease. The OD450 value for positive serum diluted 1:800 was also determined to be greater than the positive threshold. The highest coefficient of variation value was 3.66% for the intra-assay and 5.79% for the interassay, which were all less than 10%. This assay has been successfully applied to the examination of the duck sera clinically. These results in this study indicate that the newly-developed iELISA offers a precise, specific, sensitive, and reproducible means of measuring DSHDV antibodies in duck sera.     

ELISA detection of bovine viral diarrhoea virus specific antibodies using recombinant antigen and monoclonal antibodies

A panel of monoclonal antibodies was prepared by immunization of BALB/c mice with Moredun (BD) virus strains. These antibodies were characterized by immunofluorescence and seroneutralization against BD, BVD and hog cholera (HC) virus strains, and radioimmunoprecipitation of BVD-infected cells extracts. The MAbs reacting with the majority of the Pestivirus strains recognize the 80 kDa antigen of the BVD cytophathic strains. The 80 kDa antigen of the BVD/Osloss virus strain has been cloned and expressed in E. coli as a fusion protein with beta-galactosidase. The fusion protein has been purified from inclusion bodies and used successfully as an antigen for ELISA detection of BVDV specific antibodies in bovine sera. A competitive ELISA using MAbs is more specific than a direct assay. These results compare well with the ones obtained with antigen extracted from BVDV-infected cells.     

Comparison of the serum neutralization test and a competitive enzyme-linked immunosorbent assay for the detection of antibodies to vesicular stomatitis virus New Jersey and vesicular stomatitis virus Indiana.

A competitive enzyme-linked immunosorbent assay (C-ELISA) for the detection of antibodies against vesicular stomatitis virus New Jersey (VSV-NJ) and vesicular stomatitis virus Indiana (VSV-IN) was compared with the serum neutralization test (SNT) using 1,106 serum samples obtained from dairy cattle on sentinel study farms in the Poás region of Costa Rica. Kappa coefficients between the C-ELISA and the SNT were 0.8871 (95% confidence interval [CI]: 0.8587-0.9155) and 0.6912 (95% CI: 0.6246-0.7577) for the VSV-NJ and VSV-IN tests, respectively. These results indicate good to excellent agreement between the 2 tests under these conditions.     

Implementation in Australia of molecular diagnostic techniques for the rapid detection of foot and mouth disease virus

OBJECTIVE: To evaluate and implement rapid molecular diagnostic techniques for the detection of foot and mouth disease virus (FMDV) suitable for use in Australia. DESIGN: Two PCR TaqMan assays targeted to the FMDV internal ribosome entry site or the 3D polymerase coding region for the rapid detection of FMDV were evaluated using non-infectious materials to determine the test most appropriate for implementation as part of Australia's national preparedness for the rapid detection and diagnosis of FMD outbreaks. RESULTS: Two published tests (PCR TaqMan assays targeted to the FMDV IRES region or the FMDV 3D polymerase coding region) were evaluated for their ability to detect FMDV genetic material in non-infectious FMDV ELISA antigen stocks held at Australian Animal Health Laboratory. Both tests were able to detect FMDV genetic material from strains O1 Manisa, O-3039, A22, A24, A Malaysia, C, Asia 1 and SAT 1, 2 and 3. With the exception of Asia 1, the TaqMan assay targeted to the FMD 3D polymerase coding region had Ct values equal to or lower than for the TaqMan assay targeted to the IRES region suggesting that this test may provide broader serotype detection and sensitivity. However, the TaqMan assay directed to the FMDV IRES is the only one to date to have undergone substantial evaluation using clinical samples collected during an outbreak. The greatest differences observed were for O-3039, SAT 1, and 3. CONCLUSION: Given the ease of setting up both tests, AAHL currently runs both tests on highly suspect FMD investigations to provide independent confirmation of the absence of FMDV because the tests are focused on two independent regions of the FMDV genome. These tests add substantially to Australia's preparedness for FMD diagnosis complementing the already well-established virus isolation and antigen capture ELISA tests for index case diagnosis of FMD in Australia.     

口蹄疫病毒2C3AB基因的原核表达及ELISA方法的建立

口蹄疫病毒(FMDV)的2C蛋白中存在对自然感染和灭活疫苗免疫动物具有鉴别诊断意义的抗原表位。为建立一种敏感的血清学鉴别诊断方法,本研究截取FMDV 2C蛋白的C端B细胞表位较为富集的区域与全长3AB基因组合,经原核表达获得分子量约为44 ku的目的蛋白。Western blot分析表明,表达产物2C3AB与FMDV感染动物的阳性血清呈特异性反应。以纯化的目的蛋白作为包被抗原建立间接ELISA方法,敏感性检测表明该方法比3ABC-ELISA具有更高的敏感性;同时检测猪圆环病毒、猪繁殖和呼吸障碍综合征病毒、猪瘟病毒标准阳性血清均无交叉反应。批内和批间重复性试验显示,OD450nm值的变异系数小于9%。采用该方法检测不同背景的临床样品,并与3ABC-ELISA及进口试剂盒比较,总符合率分别为98.5%和90%。结果表明,该ELISA检测方法具有更高的敏感性和良好的特异性、重复性。     

牛传染性鼻气管炎病毒单克隆抗体的制备及阻断ELISA方法的建立

为建立检测牛传染性鼻气管炎病毒(IBRV)血清抗体的阻断ELISA方法,本研究以经蔗糖密度梯度离心法纯化的IBRV作为免疫原制备1株单克隆抗体(MAb),命名为cp-1-1。经间接ELISA、IFA和western blot鉴定,该MAb与IBRV呈阳性反应,与牛病毒性腹泻病毒(BVDV)及牛副流感病毒3型(BPIV3)呈阴性反应,具有较强的特异性。质谱分析结果显示MAb cp-1-1识别的表位位于IBRV VP8蛋白。以纯化的IBRV作为包被抗原、MAb cp-1-1作为检测抗体,建立检测IBRV血清抗体的阻断ELISA方法。该检测方法的抗原包被量为0.89μg/孔,样品稀释度为12,检测抗体MAb量为1.3μg/孔,二抗稀释度为15000。利用50份IBRV抗体呈弱阳性的牛血清(中和抗体效价为14~116)作为标准参考血清,确定该检测方法的阻断率Cut Off值为52.06%,即阻断率高于52.06%时判为阳性,低于52.06%时判为阴性。阻断ELISA方法特异性试验显示仅IBRV阳性血清检测为阳性,而BVDV、BPIV3、牛腺病毒3型(BADV-3)和O型口蹄疫病毒(O-FMDV)阳性牛血清均检测为阴性,表明该方法具有较强的特异性;该方法可检测的最低中和抗体效价为14,与病毒中和试验的敏感性一致,表明该方法具有较高的敏感性;重复性试验显示该方法批内、批间变异系数均小于10%,显示较好的重复性。对130份现地牛血清检测结果显示,该方法与病毒中和试验的符合率为98.46%。用该方法对某牛场接种IBRV灭活疫苗的牛血清进行检测,抗体阳性率为99.51%(205/206)。另外,采用该方法对我国8个省(市、自治区)的801份牛血清进行检测,IBRV的抗体阳性率为41.6%(333/801)。本研究建立的阻断ELISA方法可以用于IBRV疫苗免疫监测和血清流行病学调查,为我国IBR的防控提供技术支持。     

Anti-3AB antibodies in the Chinese yellow cattle infected by the O/Taiwan/99 foot-and-mouth disease virus.

The O/Taiwan/99 foot-and-mouth disease virus (FMDV), a South Asian topotype of serotype O, was introduced into Taiwan in 1999. The Chinese yellow cattle infected by the virus did not develop clinical lesions under experimental and field conditions. A blocking enzyme-linked immunosorbent assay (ELISA) kit with the 3AB antigen, a polypeptide of FMDV non-structural (NS) proteins, was used to evaluate the development and duration of anti-3AB antibodies, proving active viral replication, in the Chinese yellow cattle. The specificity of the assay was 99%, as was established with negative sera from regularly vaccinated and from na?ve cattle. The sensitivity tested with sera from naturally infected animals was approximately 64% and it was lower than that obtained by serum neutralization (SN) test. Under experimental infection, the Chinese yellow cattle developed lower anti-3AB antibodies than that developed in other species. Duration of anti-3AB antibodies was traced in two herds of naturally infected animals, indicating that anti-3AB antibodies persisted for approximately 6 months after outbreaks. On the basis of this study, we propose that the Chinese yellow cattle may have natural resistance, which limits viral replication and reduces the development of anti-3AB antibodies.