禽脑脊髓炎病毒单克隆抗体制备

禽脑脊髓炎病毒（ＡＥＶ）在患鸡体内含量很低,且细胞培养的复制水平也很低,故对该病毒的研究一直不够深入,国外有关ＡＥＶ单克隆抗体的报告首见于１９９４年［１］。本研究制备了３株抗ＡＥＶ单克隆抗体,为ＡＥＶ的实验室诊断提供了一种简单、快速、可靠的方法,同时...     

抗禽脑脊髓炎病毒单克隆抗体的研究——禽脑脊髓炎病毒抗原的培养和纯化

为获得用于制备AEV单克隆抗体的抗原，将禽脑脊髓炎病毒（AEV VR）接种于鸡胚成纤维细胞（CEF）盲传，通过间接免疫荧光试验（IFA）监测AEV增殖情况，以第六代CEF培养物作为种毒扩大培养，将其培养物经差速率心后的半提纯物接种6日龄SPF鸡胚、1日龄SPF鸡，再将半提纯物经密度梯度离心后进行PAGE－SDS电泳，电镜观察。结果证实，AEV VR在CEF上增殖成功。     

An in vitro assay for quantifying the virus of avian encephalomyelitis

Chicken embryo brain (CEB) cell cultures support the replication of embryo-adapted strains, vaccine strains, and field isolates of avian encephalomyelitis virus. A centrifugal force of 1,500 X g was applied during virus adsorption. Viral antigen was detected in the infected cells by using the indirect fluorescent-antibody technique (IFAT). Combining the infectivity of the virus in CEB cell culture with the ability to detect viral antigen by the IFAT resulted in the development of a virus-titration method. This in vitro assay proved to be more sensitive than the standard embryo-inoculation assay. It was concluded that the in vitro assay provides a satisfactory alternative to the embryo-inoculation assay.     

An enzyme-linked immunosorbent assay for the detection of antibodies to avian reticuloendotheliosis virus using whole cell antigen

An enzyme-linked immunosorbent assay (ELISA) using reticuloendotheliosis virus-infected chick embryo fibroblasts as coating antigen is described for the detection of antibodies to reticuloendotheliosis virus in chicken sera. The ELISA was specific and during the early stages of infection more sensitive than an indirect fluorescent antibody test.     

Comparison of precipitin antibodies and virus-neutralizing antibodies to infectious bursal disease virus

Three hundred twenty-two serum samples from commercial pullets and multiplier breeders were analyzed for agar-gel precipitin (AGP) antibodies and virus-neutralizing (VN) antibodies to infectious bursal disease virus. Two hundred thirty-four of these sera were AGP-positive, and 88 were AGP-negative. The geometric mean of the reciprocal of the VN titers for the AGP-positive sera was 208.7, and 232 (99.1%) had a VN titer of 1:16 or greater. In contrast, the geometric mean of the reciprocal of the VN titers for the AGP-negative sera was 6.1, but 53 (60.2%) had a VN titer ranging from 1:4 to 1:256. When the AGP test was compared with the VN test, the sensitivity and specificity, respectively, of the AGP test were 81.5% and 100%.     

利用鸡胚成纤维细胞培养禽脑脊髓炎病毒的研究

利用鸡胚成纤维细胞 (CEF)培养禽脑脊髓炎病毒 (AEV) ,经过六次盲传发现 :AEV在 CEF上无细胞病变 (CPE) ,但利用 CEF细胞上清接种 SPF鸡胚 ,可产生不同程度的 AE鸡胚病变。分别取不同时间的感染细胞上清 ,测定 AEV浓度 ,结合培养条件 ,进而确定 AEV培养的最佳时机。结果表明 :以 AEV在 CEF上培养 7天最好 ,病毒滴度可达 10 2 .8EID50 / 0 .2 ml。将经 CEF培养的 AEV差速离心 (浓缩约 5 0 0倍 ) ,接种 SPF鸡胚 ,可产生典型的鸡胚病变 ,其滴度为 8× 10 5.0 EID50 / 0 .2 m l。通过 Cs Cl密度梯度离心提纯病毒 ,在电镜下观察到了大小基本一致的病毒粒子 ,病毒直径约为 2 5 nm。利用 AEV感染的 CEF或通过“细胞飞片”制备荧光片 ,建立了间接免疫荧光快速检验 CEF是否感染 AEV的方法。     

A trivalent antigen for the detection of type I avian adenovirus precipitin

A double immunodiffusion antigen prepared from cell-culture-propagated CELO virus was not capable of detecting precipitin directed against all of the type I avian adenovirus (fowl adenoviruses) isolates tested. However, an antigen pool containing CELO-4, B-3, and IBH-2 (Tipton) fowl adenovirus isolates detected precipitin directed against representative isolates of 10 type I serotypes. Additionally, this antigen pool markedly improved detection of adenovirus field exposure.     

Enzyme-linked immunosorbent assay to detect subgroup-specific antibodies to avian leukosis viruses

An enzyme-linked immunosorbent assay (ELISA) was developed to detect antibodies against avian leukosis viruses (ALV), using antigens extracted from Rous-associated virus-inoculated chicken embryo fibroblast (CEF) cells by Nonidet P-40 treatments. The antigens reacted strongly to sera of chickens immunized with antigenically homologous viruses, but weakly to those of chickens immunized with heterologous viruses. Antigens extracted from noninoculated CEF cells by the same procedures did not react to either of the immune sera. Normal control sera did not react to any of the antigens. Reactivities of immune sera were decreased markedly by the sera adsorbing with homologous Rous-associated virus-inoculated CEF cells, but not with heterologous CEF cells. The ELISA-specific optimal doses (the differences between the optimal doses with antigens from ALV-inoculated and noninoculated CEF cells) were correlated strongly with the virus-neutralization titers (r = 0.876, P less than 0.01). Examination of the antibody response from ALV-inoculated chickens revealed that ELISA detected antibodies at the same time or several weeks earlier than did the virus-neutralization test.     

An enzyme-linked immunosorbent assay for detection of antibodies to avian adenovirus and avian adenovirus-associated virus in chickens

An enzyme-linked immunosorbent assay system (ELISA) was adapted for the detection of antibodies to avian adenovirus (AV) and avian adenovirus-associated virus (A-AV). Both before and after exposure, sera from chickens undergoing natural and experimental infections were assayed by ELISA, virus neutralization (VN), and immunodiffusion (ID) for antibody to both CELO virus and A-AV. The ELISA system was found to be comparable to VN for determining antibody concentrations to CELO virus and A-AV. In many cases, ELISA was found to be more sensitive than ID.     

An enzyme-linked immunosorbent assay for detection of antibodies to exogenous avian leukosis virus

A microplate enzyme-linked immunosorbent assay (ELISA) for detecting antibodies to avian leukosis virus (ALV) of subgroups A and B in infected chickens was developed with the use of Rous-associated virus (RAV)-1 (subgroup A) and RAV-2 (subgroup B) antigens purified by sucrose-gradient centrifugation. The antigen was used for ELISA after treatment with Triton X-100. In the ELISA, the subgroup viral antigen reacted strongly with homologous antiserum but also reacted with heterologous antiserum. Tests with serum absorbed with purified homologous and heterologous virus and tests for antigen-blocking by group-specific antibodies to ALV revealed that the reaction was caused mainly by subgroup-specific antibodies. The ELISA was 8 to 32 times more sensitive than the virus-neutralization (VN) test and detected antibodies to ALV earlier than the VN test in chickens infected experimentally with RAV-1 and RAV-2. In field application of the ELISA, 44.2% of 484 chicken sera were positive for RAV-1 and/or RAV-2 antigen, and 80.4% of flocks were positive. These findings indicate that ELISA is superior to the VN test in sensitivity, simplicity, rapidity, and applicability for large-scale field surveys for ALV infection.     

应用原位杂交检测禽脑脊髓炎病毒

禽脑脊髓炎病毒(AEV)是属于小RNA病毒科、肠病毒属的一种正链RNA病毒，病毒粒子具有六边形轮廓，无囊膜，大小为22～25nm，病毒基因组全长为7055nt，具有PloyA尾，它主要侵害幼鸡中枢神经系统，从而引起以非化脓性脑炎为主要病理特征的一种病毒性传染病。由于AEV致病性     

禽脑脊髓炎病毒单克隆抗体的制备与初步应用

利用提纯的禽脑脊髓炎病毒（AEV）Van Roekel株作为免疫原，免疫6周龄BALB／c小鼠，采取其脾细胞与SP2／0骨髓瘤细胞融合，用间接ELISA法筛选，间接免疫荧光法（IFA）和免疫组织化学法鉴定，经3次亚克隆得到了稳定分泌抗AEV单克隆抗体的杂交瘤细胞株F11和G2，制备了腹水，并利用该单克隆抗体初步建立了Dot—ELISA、间接ELISA和IFA等特异性检测AEV抗原的方法。     

Antigenic relatedness of pigeon herpes encephalomyelitis virus to other avian herpesviruses

Pigeon herpes encephalomyelitis virus (PHEV) was compared with seven avian herpesviruses for antigenic relatedness using monospecific antisera and the indirect fluorescent-antibody (IFA), agar-gel-immunodiffusion, and serum-neutralization tests. No antigenic relationship was detected between PHEV and Marek's disease virus, turkey herpesvirus, infectious laryngotracheitis virus, and duck enteritis virus. A common precipitating antigen was detected between the PHEV and pigeon herpesvirus (PHV), owl herpesvirus (OHV), and falcon herpesvirus (FHV). These four viruses also cross-reacted in the IFA test. Weak neutralizing activity was detected only between PHV antiserum and PHEV. These results suggest that the PHEV should be classified as a herpesvirus related to, but distinct from, the PHV-OHV-FHV group of viruses with which it shares common antigens.     

Persistence of passively acquired antibodies to hemagglutinating encephalomyelitis virus in swine

The persistence of passively acquired antibodies to hemagglutinating encephalomyelitis virus (HEV) was determined in 4 pigs in each of the litters of 10 sows. At time of delivery by the sows, the colostrum and serum samples (from the 10 sows) had hemagglutination-inhibiting antibodies to HEV. All of the pigs also had hemagglutination-inhibiting antibodies to HEV at 2 days of age. The level of circulating antibodies to HEV decreased at a nonlinear rate and persisted for about 4 to 18 (mean 10.5) weeks in the circulation of pigs. All 40 pigs were seronegative at 20 weeks of age.     

禽白血病病毒群特异性抗原单克隆抗体的制备与鉴定

为制备禽白血病病毒(ALV)群特异性抗原的单克隆抗体(MAb),本研究从ALV-J病毒株感染DF-1细胞的冻融裂解液中提取DNA,通过PCR方法扩增出ALV群特异性抗原p27基因,并将其克隆至pMD18-T载体中,酶切鉴定后进行序列测定和分析。最后将p27基因亚克隆至载体pET-30a(+)中,转化受体菌BL21,IPTG诱导表达,经SDS-PAGE检测获得融合蛋白。经过western blot检测,表达的蛋白具有良好的反应原性。将纯化的p27蛋白作为抗原,免疫7周龄BALB/c小鼠,利用淋巴细胞杂交瘤技术,获得5株能稳定分泌特异性MAb的杂交瘤细胞株。5株MAb与纯化的p27蛋白以及不同亚群ALV可以发生特异性反应。所制备的MAb为禽白血病的诊断方法研究奠定了基础。