牛病毒性腹泻病毒抗体胶体金检测试纸条的制备

采用sf9昆虫细胞-杆状病毒系统重组表达牛病毒性腹泻病毒P80蛋白抗原,经纯化鉴定后将P80蛋白用胶体金标记作为示踪抗原,未标记的P80抗原作为捕获抗原,并以羊抗牛IgG抗体作为质控抗体,建立了牛病毒性腹泻病毒抗体快速诊断试纸条。试验结果表明,该试纸条检测灵敏度高,特异性良好,与牛口蹄疫病毒、牛传染性鼻气管炎病毒无交叉反应。使用建立的胶体金试纸条和IDEXX牛病毒性腹泻病毒ELISA抗体检测试剂盒同时检测80头份牛血清,阳性结果符合率为86.7%,阴性结果符合率为100%,总符合率为95%。该试纸条可用于临床牛病毒性腹泻病毒抗体的诊断和检测。     

牛传染性鼻气管炎病毒抗体胶体金检测试纸条的制备

采用SF 9昆虫细胞-杆状病毒系统重组表达牛传染性鼻气管炎病毒gD蛋白抗原,经纯化鉴定后将gD蛋白用胶体金标记作为示踪抗原,未标记的gD抗原作为捕获抗原,并以羊抗牛IgG抗体作为质控抗体,建立了牛传染性鼻气管炎病毒双抗原夹心法胶体金检测试纸条。试验结果表明,该试纸条检测灵敏度高,特异性良好,与牛口蹄疫病毒、牛病毒性腹泻病毒等阳性血清无交叉反应。使用建立的胶体金试纸条和IDEXX牛传染性鼻气管炎病毒抗体ELISA检测试剂盒同时检测112份牛血清,阳性符合率为93.5%,阴性符合率为96.0%,总符合率为94.6%。说明该试纸条可以应用于临床牛传染性鼻气管炎病毒抗体的诊断和检测。     

牛结核病抗体胶体金快速检测技术的建立和初步运用

目的:建立一种快速检测牛结核抗体的新方法,用以诊断牛结核病。方法:利用胶体金免疫层析技术原理,用大肠杆菌表达的牛分枝杆菌重组抗原MPB83和MPB70分别作为胶体金标记抗原和检测线上的捕获抗原,制备牛结核抗体检测试纸条。结果:粒径为40nm的胶体金制备的试纸条敏感性最高;胶体金最佳标记pH值为6.0;MPB83抗原最适标记量为每毫升胶体金6.5μg;MPB70抗原的最适包被浓度为3.0mg/mL;抗MPB83蛋白IgG的最佳包被浓度为2.5mg/mL;交叉试验证明试纸条不与牛的其他非相关疾病的阳性血清反应,具有较高的特异性。比较实验证明其敏感性显著高于韩国进口试纸条。在上述试验条件下生产了一批胶体金试纸条进行临床样品检测,并与细菌分离培养、结核菌素皮内变态反应(TST)和韩国试纸条比较。本试纸条与牛分枝杆茵分离培养的符合率为85%,与TST的符合率为79.73%,与韩国试纸条的符合率为98.75%。结论:快速检测牛结核抗体的胶体金免疫层析试纸条具有敏感、特异、简便、快速的特点,适用于对牛结核病进行普查和检疫,也可作为TST的辅助诊断方法,在牛结核根除计划中具有良好的应用前景。     

牛结核病抗体胶体金快速检测技术的建立和应用

为了建立一种快速检测牛分支杆菌抗体的新方法用于诊断牛结核病,利用胶体金免疫层析技术原理,用原核诱导表达的牛分支杆菌抗原蛋白MPB83和MPB70分别作为胶体金标记抗原和检测线上的捕获抗原,制备牛结核抗体检测试纸条.结果表明,粒径为40 nm的胶体金制备的试纸条敏感性最高,胶体金最佳标记pH为6.0,MPB83抗原最适标记量为每毫升胶体金6.5 μg,MPB70抗原的最适包被浓度为3.0 mg/mL,抗MPB83蛋白IgG的最佳包被浓度为2.5 mg/mL,交叉试验证明试纸条不与牛的其他非相关疾病的阳性血清反应,具有较高的特异性.比较试验证明其敏感性显著高于韩国进口试纸条.在上述试验条件下生产了一批胶体金试纸条进行临床样品检测,并与细菌分离培养、结核菌素皮内变态反应(TST)和韩国试纸条比较.本试纸条与牛分支杆菌分离培养的符合率为85%,与TST的符合率为79.73%,与韩国试纸条的符合率为98.75%.快速检测牛结核抗体的免疫层析试纸条具有敏感、特异、简便、快速的特点,适用于对牛结核病进行普查和检疫,也可作为TST的辅助诊断方法,在牛结核病根除计划中具有良好的应用前景.     

犬狂犬病毒抗体胶体金检测试纸卡的研制

为快速检测犬狂犬病毒IgG抗体,应用狂犬病毒G蛋白作为捕获抗原,鼠抗狂犬病毒G蛋白单克隆抗体和羊抗犬IgG分别作为胶体金标记物和诊断抗体,羊抗鼠IgG作为质控线,制备胶体金免疫层析抗体检测试纸。该试纸卡具有良好的特异性、敏感性、重复性和稳定性,与快速荧光灶抑制实验检测值对比总符合率为82.1%。研究显示,本试纸卡能够快速、准确检测犬狂犬病毒IgG抗体,可为临床诊断和宠物犬狂犬疫苗免疫效果的评价提供参考。     

牛副结核胶体金免疫层析试纸条的研制

将提纯的牛副结核分支杆菌重组蛋白MAP0862-2154c作为硝酸纤维素膜检测线的包被抗原,兔抗羊IgG作为硝酸纤维素膜质控线的包被抗体,金标羊抗牛IgG点喷到玻璃纤维素膜上,制成用于检测牛副结核抗体胶体金免疫层析试纸条。用牛的副结核标准阳性血清与检测试纸条反应,在检测线处出现红色反应带,而滴加阴性血清的试纸条检测线处未出现反应条带,上述2种血清在质控线处均出现红色反应条带;试纸条可检出牛副结核抗体的最低抗原包被浓度为400μg/mL;试纸条不与牛结核病、牛布鲁氏菌病的阳性血清发生反应;试纸条37℃保存9d后的检验结果与4℃保存的检验结果相同。所制备的试纸条具有灵敏、特异的优点,稳定性良好;10min左右即出结果,操作简便,可用于临床诊断。     

PRRSV重组M蛋白表达及胶体金试纸的制备

为建立PRRSV抗体的检测方法,通过扩增PRRSV ORF6基因,将其克隆至pET-28a（＋）中,IP TG诱导重组质粒转化菌表达,经SDS-P AGE和Western blot鉴定,表明重组质粒能够表达目的蛋白;经亲和层析纯化表达蛋白,以SP A标记胶体金,用纯化M融合蛋白和羊抗猪抗体为检测线和质控线研制胶体金试纸,检测猪血清样品,比较与IDEXX ELISA试剂盒检测结果的符合率。结果表明,表达蛋白具有良好的反应性,制备的胶体金试纸与IDEXX ELISA试剂盒检测符合率为89％,该方法为P RRSV抗体的检测提供了快速简便的方法。     

快速检测猪繁殖与呼吸综合征病毒胶体金免疫层析方法的建立

为建立一种快速、简便、灵敏的检测猪繁殖与呼吸综合征病毒(PRRSV)的胶体金免疫层析方法(GICA),本研究采用柠檬酸三钠还原法制备了胶体金颗粒,标记抗PRRSVN蛋白的单克隆抗体(MAb) 2D7制备免疫检测探针,将抗PRRSVN蛋白的MAb 1G7和羊抗鼠IgG抗体印迹在硝酸纤维素膜上,分别作为检测线和质控线,经条件优化,组装成胶体金免疫层析试纸条.本研究制备的PRRSV胶体金试纸条的最低检测限度为103.0 TCID50/mL;在特异性试验中,试纸条检测PRRSV呈阳性,其它主要猪病病原均为阴性;不同批次试纸条重复检测,结果无差异;对现地猪场送检的150份病料进行PRRSV病原检测,与RT-PCR相比较,试纸条的特异性和敏感性分别为98.13％和88.37％.两种方法的一致性Kappa值为0.882.建立的PRRSV抗原胶体金免疫层析检测方法具有良好的的敏感性、特异性、重复性及现地应用性.该试纸条的研制为PRRS的快速诊断及免疫预防提供了技术手段.     

兔出血症病毒胶体金免疫层析试纸条诊断方法的建立及初步应用

为了建立一种快速、简便的兔出血症病毒(RHDV)的检测方法,用胶体金标记纯化的RHDV多克隆抗体,以杆状病毒表达系统表达的重组RHDV VP60蛋白为免疫原制备RHDV的单克隆抗体(McAh,简称单抗)A3C,将RHDV McAb和羊抗兔IgG抗体包被在硝酸纤维素膜上,分别作为检测线和质控线,经条件优化研制成RHDV免疫胶体金试纸条.该试纸条可以检出红细胞凝集试验(HA)效价为1:10的RHDV悬液,即HA检测为阴性的样品,该试纸条检测为阳性,其敏感性高于HA;特异性试验结果显示,该试纸条不与家兔其他常见病菌发生交叉反应.应用该试纸条对127份疑似兔出血症家兔肝脏样品进行初步检测,同时用HA做平行试验,阳性符合率为100％.因此,该试纸条是一种快速、灵敏、特异的兔出血症病毒检测方法,为兔出血症的现场诊断提供了有效的方法,显示出很好的临床应用前景.     

猪链球菌重组GDH蛋白间接ELISA检测方法的建立

为建立一种简便快速的胶体金免疫层析方法（GICA）以用于检测犬抗狂犬病毒抗体,本研究运用间接反应一步法原理．采用柠檬酸三钠还原法制备胶体金颗粒用以标记抗犬IgG抗体形成金一抗犬IgG抗体复合物,制成金标结合垫,加上包被有狂犬病毒糖蛋白的硝酸纤维膜,即制成胶体金免疫层析试纸条。样品中狂犬病毒抗体与金标记抗体结合后沿硝酸纤维素膜移动,     

Partial sequencing of env gene of bovine leukaemia virus from Brazilian samples and phylogenetic analysis

Analysis of the partial bovine leukaemia virus (BLV) env gp51 gene sequences obtained from three BLV strains isolated in three different regions of Brazil was carried out. The Brazilian BLV env gp51 sequences were compared with seven other corresponding sequences of BLV strains isolated in different countries and with consensus sequence as well. The obtained data point on qualitative and quantitative differences among the analysed strains as far as the occurrence of single point mutations is concerned. Two Brazilian strains show significantly higher mutation rate than other analysed strains. Amino acid analysis did not show, however, any substantial changes of the primary protein structure coded by well conserved region of BLV env gp51 gene. Based on the obtained data, the putative dendogram image of possible phylogenetic relations among the studied BLV strains is presented as well.     

Expression of the bovine leukemia virus transactivator protein p34 by a recombinant vaccinia virus

In order to characterize the bovine leukemia virus transactivator protein, a recombinant vaccinia virus (v-LOR) containing the BLV post-envelope long open reading frame was constructed. v-LOR was shown to encode a functional protein able to transactivate the BLV long terminal repeat-directed gene expression in the infected cells. The encountered level of transactivation was about one third of that measured in BLV-infected fetal lamb kidney cell lysates.     

Immunohistological demonstration of virus and tumor associated antigens in tissues experimental and spontaneous bovine leukemia virus (BLV) infection

Expression of bovine leukemia virus (BLV) antigens in vivo has not been shown. After BLV infection, however, production of antibodies directed towards BLV proteins (e.g. gp51) can be easily demonstrated. Thus, production of BLV proteins has to take place somewhere in infected cattle. Tissues and organs of experimentally infected cattle were fixed in acetone and embedded in paraffin. Monoclonal antibodies directed to gp51 were used to demonstrate BLV expression immunohistologically by the peroxidase-antiperoxidase (PAP) method. The same samples were also used to demonstrate a tumor associated antigen (TAA) employing a monoclonal antibody. Our results indicate that very few cells, found in the intestinal mucosa, produce gp51 in vivo. The expression of TAA, however, increases significantly shortly after infection with BLV and remains high throughout life.     

Use of a monoclonal antibody against envelope protein gp51 of bovine leukemia virus in a test for the diagnosis of enzootic bovine leukosis

A highly specific monoclonal antibody (mAb) directed against envelope protein gp51 and effectively bonding the antigen (Ag) on account of its high affinity from an unpurified Ag preparation was chosen for use in a double-sandwich enzyme immuno-assay (EIA) for diagnosis of bovine leukaemia virus (BLV). The epitopes recognised in bovine sera by the gp51-specific antibodies were at the same time properly exposed. Some parameters of major importance to testing were optimised (Ab and Ag quantities, dilution of bovine sera for testing). Preliminary testing of the double-sandwich EIA on selected bovine sera and comparison with both the immunodiffusion test and anti-BLV EIA confirmed its good diagnostic specificity and sensitivity. Hence, this double-sandwich EIA, developed by means of an mAB against gp51, on account of the possibility to use as Ag culture supernatant of the FLC cell line, is a sensitive, low-cost alternative to the anti-BLV EIA Dessau MTP which had so far been used. The double-sandwich EIA is recommended for use in final sanitation for its high analytical and diagnostic sensitivity.     

Induction of neutralizing antibodies against bovine leukosis virus in rabbits by vaccination with recombinant vaccinia virus expressing bovine leukosis virus envelope glycoprotein

Three kinds of recombinant vaccinia virus (RVV)--mO-HA/ATI, LO1-HA/ATI and mO-HA/7.5kD--expressing bovine leukosis virus (BLV) envelope glycoprotein (gp60) were constructed. The BLV envelope gene of RVV mO-HA/ATI and LO1-HA/ATI or of RVV mO-HA/7.5kD was expressed under control of the promoter of A-type inclusion body (ATI) protein gene of cow-pox virus or vaccinia virus 7.5-kD protein gene, respectively. The vaccinia virus strain, LC16mO, was used as vector for RVV mO-HA/ATI and mO-HA/7.5kD, and strain LO-1 was used for RVV LO1-HA/ATI. Strains LC16mO and LO-1 are attenuated vaccine virus strains originating from the Lister original vaccinia virus. All 3 kinds of constructed RVV expressed gp60 in cultured rabbit kidney cells after infection; mO-HA/ATI expressed more antigen than did mO-HA/7.5kD. Rabbits vaccinated with RVV produced considerable antibody capable of inhibiting syncytium formation, as well as antibody with virion-binding ability. The RVV that used ATI promoter induced higher antibody titer than did the RVV that used 7.5-kD promoter. Results indicate that BLV gp60 is responsible for induction of neutralizing antibodies that suppress in vitro formation of syncytia among BLV-infected cells. Applicability of RVV, especially those using ATI promoter, was evaluated in a vaccine against bovine leukosis.     

Early detection of bovine leukosis virus DNA in infected sheep using the polymerase chain reaction.

The early diagnosis of bovine leukosis virus (BLV) infection, the aetiological agent in enzootic bovine leukosis, is important for the implementation of control measures. BLV infection is currently assessed by the detection of circulating antibodies against the viral envelope protein, gp51. However, this approach has shortcomings in the time taken to detect anti-BLV antibodies (three to four weeks after infection), and in the failure to detect antibodies in some animals. Clearly a technique such as the polymerase chain reaction (PCR), which directly detects the presence of viral DNA, has advantages over methods designed to measure host antibodies. The use of PCR for the detection of proviral DNA in an affected DNA sample with as little as 10(-5) micrograms of host DNA using agarose gel electrophoresis followed by ethidium bromide staining is described here. It was possible to improve the sensitivity of this assay by using hybridisation analysis with a BLV gene probe. PCR used in combination with hybridisation analysis will provide a sensitive diagnostic assay to detect BLV when antibody tests give weakly positive or equivocal results.     

Production and characterization of monoclonal antibodies against bovine leukaemia virus using various crude antigen preparations: a comparative study

A total of 59 monoclonal antibodies (mAbs) specific against the bovine leukaemia virus (BLV) using different antigen preparations was produced. The five antigen preparations for immunizing BALB/c mice were: live cells (CEL), sonicated and ultracentrifuged cells (SOC), cell lysates (LYS), semi-purified BLV (PV), and formalin-treated cells (FOR) from two cell lines permanently infected with BLV (FLK-BLV and BLV-bat2). These viral component presentations were selected to obtain mAbs against specific BLV proteins: located on the cell surface (FOR and CEL), in free virus particles (PV) and intracellular viral proteins (SOC and LYS). Two antigen preparations (SOC and LYS) were lethal to the mice following the intravenous and intrasplenic routes. Six fusions were performed in this study that rendered specific antibodies against BLV. The highest number of hybridomas was produced with SOC; however, the majority of the hybridomas produced (> 90%) were against cellular proteins. Even though immunization with PV gave the lowest number of hybridomas, the majority of them were specific against BLV. Based on the reactivity of the mAbs in Western blot (WB), we classified the mAbs into five groups, namely anti-gp51SU (39 mAbs), anti-gp30TM (six mAbs), anti-Pr72env (nine mAbs), anti-Pr66gag-pro (one mAb) and anti-Prgag (four mAbs). A very high percentage of the mAbs produced (48 of 59) reacted with gp51SU, suggesting that this is the most immunogenic and accessible BLV protein presented in the different antigen preparations. The majority of our mAbs recognized more than one band in WB, suggesting that, aside from reacting with mature proteins, the mAbs also recognized viral precursors.     

Detection and molecular characterization of bovine leukemia virus in beef cattle presented for slaughter in Egypt

Bovine leukemia virus (BLV) is the etiological agent of enzootic bovine leukosis, the most common neoplastic disease of cattle worldwide and a serious problem for the cattle industry. Previous studies have shown the molecular prevalence of BLV and the coexistence of BLV genotype-1 and -4 in Egyptian dairy cattle; however, the molecular characteristics of BLV in Egyptian beef cattle are unknown. Therefore, we collected blood samples of 168 beef cattle from slaughterhouses in three governorates in Egypt. Based on BLV-CoCoMo-qPCR-2 targeting long terminal repeats and nested PCR targeting the env-gp51 gene, the BLV provirus infection rates were found to be 47/168 (28.0%) and 42/168 (25.0%), respectively. Phylogenetic analysis based on 501 bp of the BLV env-gp51 gene from 42 BLV isolates revealed that at least six distinctive strains (b, e, f, g, x, and z) were prevalent in cattle across the examined regions. Furthermore, phylogenetic analysis of the 420 bp sequence of the BLV env-gp51 region of the six strains against 11 known genotypes showed that the strains b, e, f, and g were clustered into genotype-1, and strains x and z were clustered into genotype-4. Our results also indicated that strains b and x exist in both dairy and beef cattle in Egypt. The present study is the first to detect and genotype BLV among beef cattle in Egypt.