抗牛边缘无浆体MSP5单克隆抗体的制备

为制备抗牛边缘无浆体膜表面蛋白(MSP5)单克隆抗体(MAb),以原核表达的重组MSP5蛋白(rMSP5)免疫BALB/c鼠,应用常规杂交瘤技术获得2株能稳定分泌特异性MAb的杂交瘤细胞株(1D8和2F3).间接ELISA检测腹水效价分别为5.49×105和7.83×104,亚类鉴定其重链为分别为IgG2b和1gG2a,轻链均为K型;ELISA叠加试验表明这2株MAb识别的抗原位点相同或相近;Western blot结果显示2株MAb均能与rMSP5发生反应.特异性抗MSP5的MAb的获得,为牛边缘无浆体检测奠定了基础.     

检测牛边缘无浆体抗体的竞争抑制ELISA方法的建立

为建立检测牛边缘无浆体(Anaplasma marginale)抗体的方法,本研究以牛A.marginale膜表面重组MSP5蛋白作为包被抗原,抗MSP5单克隆抗体(MAb)作为竞争抗体,建立一种用于检测牛A marginale抗体的重组MSP5蛋白竞争抑制ELISA(CI-ELISA)方法.经优化确定CI-ELISA的最佳反应条件为:抗原包被浓度为2μg/孔,封闭液为2%脱脂乳,MAb的稀释度为1:400,酶标二抗的稀释度为1:1000,阴性和阳性血清临界值分别为33%和40%;该方法具有良好的特异性和重复性;2 348份临床血清样品的检测结果表明,217份为阳性,阳性率为9.2%,与IDEXXA marginale抗体检测试剂盒的阳性符合率为95.3%,阴性符合率为100%.本实验建立的ELISA方法具有较高的特异性和重复性,可用于流行病学调查研究.     

边缘无浆体MSP5蛋白基因的克隆及原核表达

参照已发表的边缘无浆体主要表面蛋白5（MSP5）基因的核苷酸序列,设计了1对特异性引物,以边缘无浆体基因组DNA为模板,采用PCR技术扩增获得了MSP5蛋白基因.将其克隆到pGEM-T Easy载体,并进行测序分析.结果表明,克隆的MSP5基因与GenBank上登录的Florida株MSP5蛋白基因的序列同源性达98.6%,编码氨基酸的同源性为99.0%,并且该序列包含有完整的开放阅读框,大小为633 bp.将该基因亚克隆入原核表达载体pGEX-4T-1,构建了重组原核表达载体.将其转化到DH5a宿主菌中,用IPTG进行诱导表达,实现了融合表达,表达产物的分子质量为45 ku.Western-blot分析表明,此表达产物能够被抗边缘无浆体阳性血清所识别.     

Development of a recombinant Anaplasma marginale DNA probe

An Anaplasma marginale DNA probe has been developed by using an improved method for the isolation of genomic DNA. Purified genomic A. marginale DNA from the St. Croix isolate was partially digested with Sau 3A1 into fragments (greater than or equal to 5.0 kb). The restriction fragments were cloned using standard techniques in the pBR322 vector and used to transform E. coli (DH5) host cells. The recombinant A. marginale DNA library was screened by the colony lifting procedure. Colonies containing plasmids with A. marginale DNA inserts were identified by hybridization with a genomic A. marginale DNA radiolabeled probe (32P). Seven recombinant A. marginale DNA probes were evaluated by dot-blot in vitro hybridization assays to identify candidates as diagnostic tools in bovine anaplasmosis studies. Specificity and sensitivity experiments were carried out by using heterologous and homologous DNAs. The heterologous panel contained bovine DNA (WBC) and blood parasites DNA from Babesia bovis (Bb), Babesia bigemina (Bbi), Eperythrozoon suis (Es) and Eperythrozoon wenyoni (Ew). The homologous DNA panel included A. marginale DNAs of 12 different isolates which were isolated in the Caribbean, Mexico, and the U.S.A. The selected diagnostic probe was identified as pSt. Croix A1, and labeled with 32P by using in vitro nick translation and random primer techniques. The pSt. Croix A1 probe demonstrated 100% specificity and high sensitivity by hybridization in dot blotting and Southern blotting. The probe can detect 500-1000 infected erythrocytes per microliters which corresponds to a parasitemia of less than 0.01%. The A. marginale DNA insert was approximately 6.4 kb in size and a partial restriction map has been constructed.     

Molecular conservation of MSP4 and MSP5 in Anaplasma marginale and A. centrale vaccine strain

Anaplasma centrale msp4 and msp5 genes were cloned and sequenced, and the recombinant proteins were expressed. The identity between Anaplasma marginale and A. centrale MSP4 was 83% in the nucleotide sequences and 91.7% in the encoded protein sequences. A. centrale msp5 nucleotide sequences shared 86.8% identity with A. marginale msp5, and there was 92.9% homology between A. centrale and A. marginale encoded amino acids of the MSP5 protein. Southern blots hybridized with probes derived from the msp4 and msp5 central regions indicate that msp4 and msp5 of A. centrale are encoded by single copy genes. Recombinant MSP4 and MSP5 fusion proteins reacted with anti-A. marginale monoclonal antibodies ANAR76A1 and ANAF16C, respectively, demonstrating the conservation of conformation-sensitive B-cell epitopes between A. centrale and A. marginale. These data demonstrate the structural and antigenic conservation of MSP4 and MSP5 in A. centrale and A. marginale. This conservation is consistent with the cross-protective immunity between A. marginale and A. centrale and supports the development of improved vaccines based upon common outer membrane proteins.     

边缘无浆体MSP5基因序列分析及其融合蛋白的纯化

参照已发表的主要表面蛋白5（MSP5）基因的核苷酸序列,设计了一对特异性引物,以边缘无浆体基因组DNA为模版。采用PCR技术扩增获得了MSP5基因;将其克隆到pGEM—TEasy载体,并进行测序分析,结果表明,克隆的MSP5基因与GenBank上登录的Florida株MSP5基因的序列同源性达98．6％,编码氨基酸的同源性为99％,并且该序列包含有完整的开放阅读框,大小为633bp。将该基因亚克隆入原核表达载体pGEX-4T-1,构建了重组原核表达载体。将其转化到DH5a宿主茵中,用IPTG进行诱导表达,实现了融合表达。表达产物的分子质量为45ku。Western blot分析表明,此表达产物能够被抗边缘无浆体阳性血清所识别。通过裂解、洗涤、变性、复性等方法对包涵体蛋白进行处理,获得的纯化产物浓度为1mg／mL。     

Radioimmunoassay for Anaplasma marginale antibodies in cattle

A radioimmunoassay is described for use in the detection of Anaplasma marginale antibodies in cattle sera. Optimal sensitivity and specificity were obtained by using 2 antigens, an A marginale antigen and a RBC antigen (obtained before infection was established) from the same calf. In addition, sera were preabsorbed with RBC from healthy cattle and with sonicated Babesia bovis. Of 86 sera obtained from cattle with A marginale infection (as determined by blood smear examination or by results of subinoculation of blood from such infected cattle into splenectomized calves), 85 had positive results by use of this test. Of 100 sera obtained from cattle raised in an anaplasmosis-free area, 98 yielded negative results, and sera obtained from 35 cattle (97 sera) infected with B bigemina and from 18 cattle infected with Theileria orientalis yielded negative results. By use of this test, 99 of 100 sera obtained from cattle with B bovis infection were negative for A marginale. Anaplasma marginale antibodies were detected in 18 cattle that had been pastured in a Boophilus microplus-free area for 2 years after natural infection. After 3 years, 16 of these cattle were still seropositive for A marginale. Sixteen cattle pastured in a Bo microplus-infested area had detectable antibody against A marginale 27 months after initial infection with A marginale. Sensitivity and specificity of the test were assessed as 98.8% for each.     

An enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies to Anaplasma centrale and Anaplasma marginale

An enzyme-linked immunoassay (ELISA) was applied to detect antibodies to A. centrale and A. marginale using homologous and heterologous antigens. The assay was compared with the indirect fluorescent antibody (IFA) test, and although a similar degree of sensitivity was obtained, the ELISA test had several advantages. Partially purified Anaplasma initial bodies used for antigen preparations contained negligible amounts of residual erythrocytic material, and did not interfere with the specificity of the ELISA. The antigenic similarity between A. marginale and A. centrale was further substantiated by cross-reactivity obtained with heterologous antigens in both ELISA and IFA tests, and antibodies produced during natural infection with A. marginale were indistinguishable in both tests from those produced following vaccination with A. centrale.     

间接ELISA检测番鸭细小病毒抗体

鸭细小病毒病是由鸭细小病毒（Duck parvovirus,DPV）引起的一种急性病毒性传染病,以雏鸭易感,临床主要表现为气喘、脚软和渗出性肠炎,死亡率约50%～80%,在世界各养鸭地区都有分布,其预防和控制已直接关系到水禽养殖业的持续稳定发展[1]。临床和实验室试验证实番鸭细小弱毒疫苗是预防控制该病最有效措施,而对细小病毒特异性抗体的监测是评价疫苗免疫效果及鸭群合理制定免疫程序的关键[2]。目前国内已经报道[3]的检测番鸭细小病毒抗体的方法有血清微量中和试验、微量点凝试验、胶乳凝集反应、琼脂扩散试验,     

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.     

Comparison of a competitive inhibition ELISA and the card agglutination test for detection of antibodies to Anaplasma marginale and Anaplasma centrale in cattle

OBJECTIVE: To compare a recently developed recombinant MSP-5 competitive inhibition ELISA with a card agglutination test for detection of antibodies to Anaplasma marginale and Anaplasma centrale in Australian cattle. MATERIALS AND METHODS: The ELISA was compared with the card agglutination test using 208 sera from cattle in Anaplasma-free herds, 86 sera from cattle experimentally infected with A marginale or A centrale and 757 sera from cattle in areas endemic for A marginale. RESULTS: The specificity of the ELISA, based on testing 208 sera from cattle in Anaplasma-free areas, was 99.5%, and the sensitivities for detection of antibodies to A marginale and A centrale in sera from the experimentally infected cattle were 98.0% and 100%, respectively. For the same sets of sera, the specificity of the card agglutination test was 98.6% and the sensitivities for detection of antibodies to A marginale and A centrale were 98.0% and 100%, respectively. For the 757 sera collected from cattle in areas endemic for A marginale, the agreement between the ELISA and the card agglutination test depended on the positive threshold selected for the ELISA. The maximum achievable agreement was 91.5% (kappa = 0.73; 95% confidence interval 0.66, 0.79). CONCLUSION: We conclude that the competitive inhibition ELISA is a useful alternative to the card agglutination test for detection of A marginale or A centrale infection in cattle. The assay should be particularly useful for epidemiological applications such as prevalence studies and control programs.     

用间接ELISA试剂盒检测鸡传染性支气管炎抗体消长规律

自 1 930年在美国北达科他州首先发现鸡传染性支气管炎以来 ,该病给养鸡业生产造成严重的经济损失[1] ,因此对于该病的诊断以及防制引起了国内外科研工作者广泛的关注并展开了大量的研究工作。由于 IB病毒型别多 ,临床表型多样化 ,以及容易发生变异等 [2 ] ,所以如何找到有效的免疫检测方法和制定正确的免疫程序 ,成为 IB研究中的重要难题。针对这一问题 ,本试验利用具有群特异性的 IBV抗体检测试剂盒对鸡的 IBV抗体消长规律进行了探索。1　材料与方法1 .1　材料　 ( 1 )试验鸡 ,购自北京某鸡场的 1日龄雏鸡。( 2 )自制 IBV抗体检测试…     

Antigenic characterization of morphologically distinct Anaplasma marginale isolates using a panel of monoclonal antibodies

The present study, describes the antigenic characterization of a Brazilian isolate of Anaplasma marginale with appendage (tail). A panel of monoclonal antibodies (McAbs) was produced and tested by the indirect fluorescent antibody test (IFAT), ELISA and Western blotting, and used to characterize two isolates of A. marginale (one with appendage and another without appendage). Among the clones produced, eight recognized antigenic proteins, with molecular weights varying from 18.4 to 66kDa. In Western blotting, the McAb reacted against a 45kDa antigen, which was shown, by the IFAT, to be located in the tail. Immunocytochemistry confirmed the tail specificity of the monoclonal reacting against the 45kDa antigen. The panel of McAb produced has a potential use in discriminating morphologically distinct A. marginale isolates. The present study, demonstrates the occurrence of antigenic diversity among Brazilian isolates of A. marginale.     

Detection of antibodies against Brucella in the aqueous humor using ELISA

In aqueous bulbi of experimentally against Brucella abortus immunized guinea pigs and rabbits specific antibodies could be detected by ELISA-tests. This first demonstration of brucella antibodies in the aqueous shows that aqueous can be used for serological examinations. For practical use more investigations in experimental animals are necessary, e.g. on kinetics of orbital antibodies absence of interfering factors and biomechanics of proteins in aqueous.     

Anaplasma marginale: lack of cross-protection between strains that share MSP1a variable region and MSP4

In Mexico, there are no commercial alternatives for the immunoprophylaxis of bovine Anaplasmosis, a disease responsible for great economic losses. Blood derived Anaplasma marginale used for immunizing susceptible cattle has shown promising results for homologous protection and controversial results against unrelated strains. The present study examined, under controlled conditions, the cross-protective potential of an immunogen composed of blood derived A. marginale of three strains against challenge with strains not included in the immunogens. Groups 1 and 2 were immunized with blood derived Anaplasma from strains Mexico, Morelos and Yucatan, group 4 with strains Morelos, Veracruz and Yucatan, two more groups (2 and 5) of equal conditions were inoculated with an adjuvant alone. Groups 1, 4 and 5 were challenged with Mexico strain; groups 2 and 3 were challenge-inoculated with strain Veracruz; groups 3 and 5 with strains Veracruz and Mexico as controls. Only animals in group 1, immunized and challenged with strain Mexico showed adequate protection. Both groups challenged with strains not included in the immunogens developed poor protection, while all the controls had to be treated to prevent death.     

Cloning, expression, and characterization of the MSP1a and MSP1b recombinant proteins from PR1 Anaplasma marginale strain, Brazil

The Anaplasma marginale is a bacterium that has obligate intraerythrocytic multiplication in cattle causing important economic loss. The A. marginale major surface protein 1 (MSP1) complex, heterodimer composed of MSP1a and MSP1b, has been identified as adhesins for bovine erythrocytes. The objectives of this study were to sequences the msp1β gene and produce and characterize recombinant MSP1a and MSP1b from a Brazilian strain of A. marginale, PR1. The msp1α and msp1β genes from the PR1 strain were cloned and expressed in E. coli BL21 Star using the vectors pET102 and pET101/D-TOPO. Antibodies were produced against the recombinant proteins and were shown to react with rMSP1a and rMSP1b demonstrating a molecular mass of 70 kDa to 105 kDa and 100 kDa, respectively for these proteins. Bovine erythrocytes were agglutinated by BL21/rMSP1a and BL21/rMSP1b and, this agglutination was inhibited by the presence of the IgY anti-rMSP1a, confirming the adhesion function of these proteins. Additionally, using the IgY anti-rMSP1a and rMSP1b in a IFI, the presence of rMSP1a and rMSP1b was confirmed on the outer membrane of the recombinant E. coli BL21. Our results show that the msp1β gene from the PR1 strain has both the conserved region and contain the defined polymorphism regions previously described for other strains of A. marginale. The results from this study confirm adhesive functions for rMSP1a and rMSP1b from PR1 strain in bovine erythrocytes invasion.     

副猪嗜血杆菌血清5型间接血凝试验和间接ELISA抗体检测方法的建立

为建立副猪嗜血杆菌（HPS）的血清学诊断方法，通过探索HPS荚膜多糖产生的最适体外培养条件，提取了HPS血清5型菌株的荚膜多糖（CPS），并以之为抗原分别建立了间接血凝试验（IHA）和间接ELISA两种抗体检测方法，对其特异性、敏感性和符合率进行了比较研究。结果表明，两种检测方法的特异性良好，但ELISA的敏感性是IHA的5～10倍，二者的阳性符合率、阴性符合率和总符合率分别为79．7％、55．2％和65．3％。用这两种方法检测了320份临床送检猪血清，IHA和ELISA的阳性率分别为40％和59％。结果证实，这两种方法适用于不同实验室条件下HPS的诊断和流行病学调查。     

牛传染性鼻气管炎病毒重组gD蛋白间接ELISA方法的建立及应用

用大肠杆菌表达的牛传染性鼻气管炎病毒（IBRV）重组gD蛋白纯化后作为包被抗原,建立了检测牛传染性鼻气管炎病毒抗体的间接ELISA方法。交叉反应试验表明,该重组抗原与其它常见的5种牛病阳性血清不发生交叉反应;阻断反应试验表明,IBRV病毒悬液能在很大程度上阻断重组抗原与阳性血清的反应,而对阴性血清没有明显影响。在重复性试验中,批内重复的变异系数小于5%,批间重复的变异系数小于15%。与中和试验相比较,符合率、敏感性和特异性分别为84.1%、85.0%和83.4%。应用该诊断方法和本实验室已建立的以IBRV全病毒作为包被抗原的ELISA诊断方法,同时检测了采集于国内11个省份的2012份血清样本,IBRVgD-ELISA检测的平均阳性率为46.0%（926/2012）,而且相对于IBRV全病毒ELISA诊断方法的符合率、敏感性和特异性分别为91.9%、94.2%和90.2%。本研究所建立的IBRVgD-ELISA具有良好的敏感性和特异性,为国内IBR流行病学调查提供了一种快速、简便的血清学诊断方法。     

山羊痘病毒p32基因原核表达及间接ELISA抗体检测方法的建立

为了建立山羊痘病毒(GPV)抗体快速检测方法,本研究通过人工合成密码子优化的GPV p32基因,并在大肠杆菌中进行截短表达(p32-opti).表达的重组p32-opti蛋白主要以包涵体形式存在,Western blot分析表明,p32-opti与GPV标准阳性血清具有良好的抗原性.以纯化的重组p32-opti蛋白作为ELISA包被抗原,建立间接ELISA抗体检测方法.该方法检测小反刍兽疫、蓝舌病和口蹄疫阳性血清均无交叉反应;与中和试验(VNT)比较,两者的符合率为95.7%;ELISA批内和批间重复性试验显示,OD值的变异系数小于10%.上述结果表明.该ELISA检测方法具有良好的特异性、敏感性和重复性.对来自黑龙江、内蒙古和新疆自治区的390份山羊和绵羊血清进行检测,抗体阳性率为80.2%,表明这些地区的山羊和绵羊群普遍存在羊痘病毒抗体.本研究建立的间接ELISA方法可用于GPV感染的流行病学调查以及疫苗接种动物的抗体水平监测.     

以重组PvpA蛋白作为抗原的鸡毒支原体抗体间接ELISA检测方法的建立

为建立鸡毒支原体(MG)种特异性检测的间接ELISA检测方法,本研究以原核表达的PvpA蛋白作为包被抗原,初步建立了检测MG抗体的间接ELISA检测方法.特异性试验结果表明,重组PvpA蛋白与鸡新城疫病毒、鸡传染性支气管炎病毒、滑液支原体、大肠杆菌“O”抗原、禽沙门氏茵“O”抗原阳性血清均不发生交叉反应.该方法的批内变异系数小于8％,批间变异系数小于11％,表明具有较好的重复性.采用该检测方法与进口试剂盒对不同省份送检的鸡血清进行检测比较,两者阳性和阴性符合率均达到90％以上.本研究建立的间接ELISA检测方法为MG抗体检测试剂盒的研制奠定了基础.