应用酶免疫斑点检验检测猪布病的研究

本文应用酶免疫斑点试验和试管凝集试验两种方法检测血清６８１份，作布氏病普查及两种方法检测结果比较。试验表明布氏菌和０：９型耶氏菌有强烈的血清交叉反应，酶免疫斑点试验能明确区别两种交叉抗体，因而具有较高的检测准确性。     

疫苗免疫控制奶牛布氏杆菌病的效果分析

某规模养殖场奶牛群发生布氏杆菌病流行和蔓延,能繁母牛出现流产、死胎症状.根据检疫结果对布氏杆菌病阳性牛隔离淘汰处理,对布氏杆菌病阴性牛(假定健康牛)进行免疫接种.奶牛群口服接种S2株活疫苗后15d,即可检出疫苗诱导的布氏杆菌抗体,30d抗体水平达到高峰(36%),45～90d抗体阳性率呈现缓慢下降的趋势.结果表明,S2...     

检测布氏杆菌病的四种血清学方法比较及国际标准单位的应用

本研究通过布氏流产杆菌（Brucella abortus)国际标准参考血清（ISABS）的使用，对目的应用的补体结合试验（CFT）、酶联免疫吸附测定法（ELISA）、虎红平板凝集试验（RBPT）及试管凝集试验（SAT）等四种血清学检测方法的测定结果进行了校正和临界点的设定。并对42份不同抗体效价血清进行了测定，同时通过计算和应用标准参考血清校正系数，对不同方法间的测定结果如何换算成国际标准单位进行了研究和探讨。结果表明，该校准方法可对不同实验室间的布氏杆菌病检测结果校准到统一的国际标准上来，可作为血清学测定国际标准化一条途径。     

布鲁氏菌诊断学研究进展

布鲁氏菌病是一种人兽共患传染性疾病,分布广泛,严重威胁公共卫生事业和畜牧业的发展,布鲁氏菌的诊断是防控该疫病的关键。本文对细菌学、常规免疫学、酶联免疫吸附（ELISA）、免疫胶体金层析法（GICA）、荧光偏振法（FPA）、聚合酶链式反应（PCR）和基因探针等布鲁氏菌检测技术及其优缺点和应用现状进行综述,并对诊断技术的发展前景进行了展望。     

布鲁氏菌鉴定和检测方法研究进展

布鲁氏菌是重要的人畜共患病病原菌，其鉴定和检测方法受到国内外的普遍重视。本文对近年来布鲁菌DNA同源性及其内切酶图谱分析、血清学检测、PCR扩增、核酸探针杂交等鉴定、检测方法进行了综述，并展望了分子生物学技术在布鲁氏菌快速、准确鉴定、检测中的良好前景。     

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.     

不同的血清学方法对布病疫苗免疫绵羊抗体检测结果的比较研究

将国内广泛用于预防羊布病的S2和M5疫苗免疫绵羊,然后定期采血进行细菌分离和血清学检测,并比较血清学方法的检测结果。结果表明ELISA操作方便,具有较好的敏感性和特异性,建议在检疫中用敏感性较高的RBT初筛,用特异性较好的ELISA确诊,以得到较好的效果。     

Prevalence of brucellosis and infectious bovine rhinotracheitis in organized dairy farms in India

This study was carried out to investigate the prevalence of bovine brucellosis and infectious bovine rhinotracheitis (IBR) in organized dairy farms with history of abortion in India. ELISA and Rose Bengal Plate Test (RBPT) were used to detect the seropositive animals and the test results indicated that 22.18% and 13.78% animals were declared as sero-positive by ELISA and RBPT, respectively. Milk Ring Test (MRT) was carried out only in one farm and 12.82% of the tested animals were turned positive. Culture examination analysis of milk samples, two animals revealed the presence of organisms indistinguishable from Brucella spp. The organism was confirmed as brucella by morphological characteristics and biochemical tests. An overall sero-prevalence of antibodies against IBR was found to be 60.84%. None of the genital and nasal swab samples was found to be positive for presence of bovine herpesvirus -1 (BHV-1) on repeated passage in Madin-Darby Bovine Kidney (MDBK) cell lines. Brucella and IBR considered as the causal agent for abortions in these farms. The present study indicates the urgent need and the necessity for control of these infectious diseases which cause heavy economic losses to the organized farms.     

A Serological Survey of Sera from Domestic Animals on Easter Island

Animals' sera collected on Easter Island from December 1964 to February 1965 were tested by appropriate methods for the presence of antibodies to various infections. These included, ornithosis, Q-fever, brucellosis, Johne's disease, leptospirosis, toxoplasmosis and vesicular stomatitis viruses. It appeared that the cattle and sheep were exposed to the ornithosis group of agents. The sheep were also exposed to toxoplasmosis. The low-grade reactions observed on the cattle sera with the leptospira and brucella antigens were not sufficient to indicate past infection. All sera tested with Q-fever and Johne's disease antigens gave negative reactions. The results suggested that neither strain of vesicular stomatitis virus had yet been introduced into this restricted animal population.     

布鲁氏菌omp28基因的克隆、表达及反应原性分析

本研究克隆了羊种布鲁氏菌16M株、羊种布鲁氏菌M28株、犬种布鲁氏菌、绵羊附睾种布鲁氏菌、牛种布鲁氏菌A19株、猪种布鲁氏菌S2株的omp28基因并对以上不同种菌株的omp28基因序列及编码的氨基酸序列进行了比对,结果显示不同种布鲁氏菌omp28基因之间仅6个碱基不同,而且只有2个氨基酸不同,亲水性分析结果显示两处氨基酸的差异对蛋白亲水性不造成影响.将羊种布鲁氏菌16M的omp28基因亚克隆到pET32a中表达,OMP28在低温下诱导以可溶性形式高效表达.Westem-blot结果显示OMP28反应原性良好,是布鲁氏菌病诊断抗原的可能选择.     

Preliminary evidence for a diagnostic immunoglobulin G1 antibody response among culture-positive cows vaccinated with Brucella abortus strain 19 and challenge exposed with strain 2308

The sera of cows inoculated with Brucella abortus have a characteristically high titer of immunoglobulin (Ig) G1 antibodies to a soluble brucella antigen compared with sera of noninoculated vaccinated cattle. Concentrations of antigen-specific IgG1 were greater than 10-fold higher than those for IgG2, even though total IgG2 concentrations were higher than total IgG1 concentrations. Increases in IgG1 antibodies to Brucella abortus soluble antigen were detected shortly after vaccination in those cows from which strain 19 was isolated and by 28 weeks in cows from which strain 2308 was isolated. Increases in specific antibodies were not paralleled by increases in either total IgG1 or total IgG2 concentrations. Rather, there was a 15-fold to greater than 200-fold increase in specific activity, with up to 16% of the IgG1 specific for the brucella antigen used in the assay. Thus, measurement of changes in total IgG1 concentrations is not a reliable method to identify brucellosis-associated anti-Brucella abortus soluble antigen activity. Only one cow in a panel of 10 selected for detailed study showed a false-positive IgG1 titer, whereas some serologic assays showed as many as 4 or 5 false-positives. Results of the complement-fixation test, among the battery of serologic tests used for detection of brucellosis, best agreed with the occurrence of increased IgG1 antibody levels.     

Quality control criteria for quantitative enzyme-linked immunosorbent assay of porcine immunoglobulins A and M

Using radioimmunoassay methods, quality control criteria were applied to monoclonal antibodies produced to measure porcine immunoglobulins by quantitative ELISA. Porcine IgM and IgA were purified to homogeneity and were used to produce murine hybridomas that secreted antibodies against IgM, IgA, and immunoglobulin light chains. A competitive ELISA was developed to measure IgM, and a sandwich ELISA was used to quantify IgA in serum and colostrum. Both ELISA were tested for specificity, accuracy, sensitivity, and precision. Monoclonal antibodies were specific for porcine IgM or IgA in serum and colostrum, and competitive and sandwich ELISA fulfilled all validation criteria.     

新疆绵羊种布鲁氏菌HtrA基因与GroEL基因的克隆及其原核表达

根据已发表的牛流产型布鲁氏菌HtrA（High temperature requinnent A）基因、GroEL（热休克蛋白）基因设计特异性引物，从新疆绵羊种布鲁氏菌基因组中扩增出HtrA、GroEL基因片段，将HtrA、GroEL基因片段纯化后分别克隆到T载体上测序，结果表明新疆绵羊种布鲁氏菌HtrA基因片段长1542bp，编码513个氨基酸，与发表的牛种（B．abortus）、羊种（B．melitensis）、猪种（B．suis）的HtrA基因序列的同源性分别为99．68％、99．81％、99．55％。GroEL基因片段长1641bp，编码546个氨基酸，与B．melitensis、B．suis以及B．aborms GroEL基因的核苷酸序列同源性分别为99．88％、99．82％、99．88％。HtrA基因和GroEL基因与发表的B．abortus、B．melitensis、B．suis的HtrA基因和GroEL基因序列的具有很高的同源性。按正确的阅读框架分别将两基因片段定向克隆到表达载体pET．28a上，将重组质粒转化到大肠杆菌BL21菌株，经IPTG诱导表达，SDS-PAGE电泳和western blot分析表明，HtrA、GroEL基因能在大肠杆菌中成功表达，表达的蛋白分子量都约为60Ku，并能和布鲁氏菌免疫兔子产生的抗体发生特异性的结合。     

Evaluation of enzyme-linked immunosorbent assays based on monoclonal antibodies for the serology and antigen detection in canine parvovirus infections

An enzyme-linked immunosorbent assay (ELISA) system was developed for the detection of canine parvovirus (CPV) or CPV antigen in dog faeces and two other ELISA systems were developed for the detection of CPV-specific antibodies in dog sera. The ELISA's were based on the use of CPV-specific mouse monoclonal antibodies, which recognise different epitopes of the haemagglutinin of CPV and which also neutralise the virus. A double antibody sandwich (DAS) ELISA for the detection of CPV in dog faeces was compared with the haemagglutination (HA) test. The DAS-ELISA proved to be more specific, sensitive and easier to perform than the HA assay. An indirect ELISA and a competitive ELISA for the detection of CPV-specific antibodies in dog sera were compared with the haemagglutination inhibition (HI) test. Both ELISA systems proved to be specific and easy-to-use methods for the detection of CPV-specific antibodies. The indirect ELISA, specially, proved to be more sensitive than the HI test. The higher sensitivity and specificity of the ELISA's as compared to HA and HI tests, and their ease of use, make them suitable for routine use in the serology and diagnosis of CPV infections.     

Use of immunoenzyme (ELISA) diagnosis of Aujeszky's disease in pigs during the recovery period

Samples of blood and blood serums of pigs were examined for the presence of antibodies to the Aujeszky's disease virus. The virus-neutralizing (VN) test and the enzymoimmunologic (ELISA) method were used for this examination. As indicated by comparison of the average titres of antiviral antibodies determined by both methods, the ELISA method is 60 to 600 times more sensitive than the VN test. The high sensitivity of the ELISA method enabled to detect antiviral antibodies even in samples considered as negative after VN-testing. The method has been used with success for the sanitation of three swine stocks where the Aujeszky's disease was eradicated without interruption of operation.     

间接ELISA,HI及AGP检测鸡血清中抗AIV抗体的敏感性比较

用鸭源Ａ型流感病毒Ｈ９Ｎ？滴鼻、点眼辅以腹腔注射,人工感染３周龄ＳＰＦ来航鸡,分别用ＡＧＰ、ＨＩ试验及间接ＥＬＩＳＡ定期测定其抗ＡＩＶ血清效价。结果,ＨＩ试验及间接ＥＬＩＳＡ于攻毒后第７～７９天显示阳性;ＡＧＰ试验从第１３～７９天呈阳性。根据首次检出血清中抗ＡＩＶ抗体的时间,间接ＥＬＩＳＡ比ＡＧＰ、ＨＩ更灵敏、快速。     

A method for the accurate determination of the specificity of monoclonal antibodies reactive with porcine immunoglobulins

Monoclonal antibodies against porcine IgG were produced by fusion and characterized. The supernatants of microtiter wells containing fusion hybrids were first screened with an ELISA using semi-purified porcine IgG as antigen. Hybrids reactive in ELISA were cloned by limiting dilution. Further characterization of the specificity of the monoclonal antibodies was done by a combination of two methods: SDS-PAGE electroimmunoblotting and convection blotting of immunoelectrophoretic patterns (IEP-immunoblotting). Using these techniques, we identified monoclonal antibodies specific for porcine Ig gamma chains.     

Development and comparison of serologic methods for diagnosing chicken anemia virus infection.

This paper describes the development of an indirect immunoperoxidase assay (IIP) and an indirect enzyme-linked immunosorbent assay (ELISA) for detecting antibodies to chicken anemia virus (VAC). The IIP assay developed used CAV-infected MDCC-MSB1 cells for detecting antibody to CAV, whereas the ELISA utilized gradient-purified immunoadsorbed CAV as the target antigen. The IIP and ELISA were compared with the standard indirect immunofluorescent antibody (IFA) assay, which is more conventionally used to screen chicken serum for antibodies against CAV. Comparative test results of 185 field samples of chicken serum by these three methods were in agreement 84% of the time. Both IFA and IIP assays yielded fewer positive tests than did the ELISA. IFA and IIP assays were in agreement 93% of the time, as compared with 91% agreement of IIP and ELISA results, or 84% agreement for comparative IFA and ELISA results.     

Comparison of enzyme-linked immunosorbent assays and virus neutralization test for detection of antibodies to avian pneumovirus

Two different whole-virus enzyme-linked immunosorbent assays (ELISAs), developed in Ohio (OH) with APV/Minnesota/turkey/2a/97 and in Minnesota (MN) with APV/Colorado/turkey/97, and the virus neutralization (VN) test were used to test 270 turkey serum samples from 27 Minnesota turkey flocks for avian pneumovirus (APV) antibodies. In addition, 77 turkey serum samples and 128 ostrich serum samples from Ohio were tested. None of the turkey samples from Ohio had antibodies to APV by the VN test and OH ELISA. The ostrich samples were only tested with the VN test and were all negative for antibodies to APV. For the Minnesota serum samples, 107, 115, and 120 were positive by the VN test, the OH ELISA, and the MN ELISA, respectively. The Kappa values of 0.938 and 0.825 showed excellent agreement between the VN test and the OH ELISA and the MN ELISA, respectively, for detection of antibodies to the APV. The OH ELISA and MN ELISA had sensitivities of 1.0 and 0.953, specificities of 0.950 and 0.889, and accuracies of 0.970 and 0.914, respectively. Our results indicate that the 3 methods are sensitive and specific for diagnosis of the APV infection.     

An evaluation of five serological tests for the detection of antibody to bovine herpesvirus 1 in vaccinated and experimentally infected cattle

More than 300 bovine sera from a previously reported vaccination and challenge trial were tested for antibodies to bovine herpesvirus 1 (BHV1) by five serological assays: enzyme-linked immunosorbent assay (ELISA) for IgM and IgG, passive haemagglutination (PHA), and two methods of virus neutralisation (VN). In a statistical comparison of ELISA (IgG), PHA and VN results, the assays showed highly significant correlations (P less than 0.01). The sensitivities of ELISA and 24-hour neutralisation tests were similar, in contrast to passive haemagglutination and one hour neutralisation which failed to detect BHV1 antibodies in some low titre sera.