Equine Infectious Anemia: Activity of Liquid Antigen Extracts in the Agar-gel Immunodiffusion and Complement-fixation Tests

Twenty-nine lots of acetone-ether extracted liquid antigen were prepared from the pulp of 11 spleens collected from horses at the acute phase of experimental infection. The lots prepared from the highly reactive pulp resulted in general in a liquid antigen of greater activity than those extracted from weakly reactive pulps. Some variations in activity between lots of antigen prepared from the same spleen were also observed. No matter what the results, given a wide enough variation, all results were reproducible. The procedure permitted production of a greater number of antigen test doses from reactive spleens and rendered usable the spleens which failed to give sufficient reactivity when used as pulp antigen in the agar-gel immunodiffusion test. The activity of each lot of liquid antigen was standardized, first by the complement-fixation test and finally by matching with a reference antiserum in the agar-gel immunodiffusion test.     

Equine Infectious Anemia: Preparation of a Liquid Antigen Extract for the Agar-gel Immunodiffusion and Complement-fixation Tests

An agar-gel immunodiffusion test recommended for the diagnosis of equine infectious anemia was evaluated. Our preliminary observations confirmed those of Coggins concerning the mechanism of the test and the results obtained. Furthermore, emphasis was put on the difficulties encountered in the production of spleen antigens with an optimum amount of reactivity. Acetone-ether extraction procedures for the preparation of a liquid antigen extract are described. This type of antigen was reactive in the complement-fixation test in 1:8 or greater dilution and it is proposed to use the complement-fixation test in assessing and standardizing the liquid antigen extract activity to be used in the immunodiffusion test. This antigen can also be concentrated or diluted, if required, to meet the reactivity of a standard antigen used in the test.     

Equine Infectious Anemia: Preliminary Investigation of the Complement-Fixation Test for the Demonstration of Antibodies and Antigen

Clinical field cases of equine infectious anemia were studied and the disease was reproduced experimentally in horses. Attempts were made to adapt the complement-fixation test to the detection of antibodies in the serum of infected animals and to the demonstration of antigens in tissue extracts.

A moderate complement-fixing antibody response was demonstrated in the serum of horses shortly after primary exposure to the infectious agent. However, this reactivity was of short duration and occurred with normal as well as with infected saline tissue extracts. It was therefore concluded that this reaction was not specific for equine infectious anemia. Possibly it is due to the appearance of auto-tissue antibodies. The value of this reaction in the diagnosis of the infection was limited because of its short duration and absence in chronic infection and following re-exposure to the infectious agent.

     

3种检验方法测定马传贫免疫抗体的效果观察

我国马属动物普遍进行了马传贫免疫接种,使得马传贫得到了控制.应用何种检验方法在免疫效价测定、抗体维持时间的测定时更准确,为此,我们对目前常用的补体结合反应(CF)、琼脂扩散反应(ID)、斑点试验(DB)3种方法进行了比较试验.     

不同ELISA和AGID试剂盒检测马传染性贫血病毒抗体的比较

为比较商品化ELISA试剂盒和AGID试剂盒检测马传染性贫血病毒（EIAV）抗体的效果,并评价不同ELISA试剂盒的敏感性、特异性和一致性,将EIAV强阳性血清进行倍比稀释,分别用4种商品化ELISA试剂盒和3种AGID试剂盒进行检测,比较最低检出限;此外,以28份临床马血清作为样品盘,以AGID结果作为标准,通过计算符合率、Kappa值、敏感性、特异性和约登指数,综合评价4种ELISA试剂盒的检测效果。灵敏性结果显示：4种ELISA试剂盒可检出的最大稀释度分别为1:1 024、1:256、1:32和1:128,而3种AGID试剂盒可检出的最大稀释度均为1:16。临床样本检测结果显示：4种ELISA试剂盒与AGID检测结果的符合率均超过89%,其中有3个品牌的ELISA试剂盒一致性较高;4种ELISA试剂盒敏感性为75.00%~100%,特异性为87.50%~100%,约登指数为0.75~1.00。结果表明,ELISA试剂盒的检测灵敏度普遍高于AGID试剂盒,且与AGID试剂盒的检测结果符合率高,是EIAV检测的可靠手段之一,但不同ELISA试剂盒敏感性和特异性存在一定差异,应根据实际情况合理选择使用。本研究为制定科学合理的EIAV检测方案和试剂盒筛选提供了数据支撑。     

乳胶凝集试验检测鸡传染性鼻炎血清抗体的研究

将Ａ型和Ｃ型鸡副嗜血杆菌培养后,菌落计数,离心计数,离心后用０．０１ＭｐＨ７．４磷酸盐缓冲液洗涤菌体２次。经过对致敏温度、致敏时间、致敏乳胶的菌体浓度及菌体的处理方式等条件的选择,建立了检测鸡传染性鼻炎血清抗体的乳胶凝集试验。与琼脂扩散试验相比,特异性一致,但更加敏感,且操作简便、快速。结果说明,该方法有较好的应用前景,尤其适用于本病的现场快速诊断。     

重组马传染性贫血病病毒核心蛋白抗原在 A GID 和ELISA中应用的评价

用昆虫杆状病毒表达系统获得的马传染性贫血病病毒（ Ｅ Ｉ Ａ Ｖ） 核心蛋白（ Ｇａｇ） 和 Ｐ２６ 蛋白, 作为免疫琼脂双扩散（ Ａ Ｇ Ｉ Ｄ） 和酶联免疫吸附试验（ Ｅ Ｌ Ｉ Ｓ Ａ） 抗原。对７６ 份已知马传贫非特异性血清进行检查, 同时与市售 Ａ Ｇ Ｉ Ｄ 和 Ｅ Ｌ Ｉ Ｓ Ａ 试剂盒作比较。证明, 用表达蛋白作抗原的 Ａ Ｇ Ｉ Ｄ 和 Ｅ Ｌ Ｉ Ｓ Ａ 检测结果均为阴性反应, 而用市售 Ａ Ｇ Ｉ Ｄ 试剂盒检查有５４ 份马血清出现非特异性反应, 市售 Ｅ Ｌ Ｉ Ｓ Ａ 试剂盒检查也出现了非特异性反应, Ｏ Ｄ 值比表达抗原 Ｅ Ｌ Ｉ Ｓ Ａ 高３５ 倍。初步证明在 Ａ Ｇ Ｉ Ｄ 和 Ｅ Ｌ Ｉ Ｓ Ａ 法中, 表达抗原优于常规马传贫病毒抗原。     

传染性支气管炎病毒抗体间接ELISA检测方法的建立

为了快速检测针对传染性支气管炎病毒(IBV)抗体,本研究建立相应的间接ELISA(iELISA)检测方法。以本实验室分离的临床毒株DS10为检测抗原,经过差速离心纯化获得DS10病毒作为包被抗原,利用方阵试验,优化一系列条件,最终确定了最佳的ELISA反应条件。抗原最佳包被浓度为0.11mg/mL,血清最适稀释度为1:100,封闭液为含1%BSA的PBST,封闭时间为60min,一抗作用时间90min,HRP标记的兔抗鸡IgG稀释倍数为1:3500,作用时间为60min,显色时间为10min。用iELISA方法和血凝抑制试验(HI)同时检测145份血清样品的结果表明,建立的iELISA方法与常规的HI检测的符合率为92.41%,iELISA和HI的阳性检出率分别为92.41%和90.34%,iELISA的敏感性略优于HI。     

应用间接酶联免疫吸附试验检测鸡传染性鼻炎抗体

本文应用聚苯乙烯塑料微量滴定板作为固液载体，辣根过氧物酶标记的羊抗鸡IgG为第二抗体，邻苯二胺为底物建立检测鸡传染性鼻炎抗体的间接酶联免疫吸附试验。对大庆市A、B、C三个种禽场的鸡群抽样采血检测，结果在被检的154份血清样品中，IC抗体阳性血样为35份，阳性检出率为22．8％。该方法具有快速、简便、敏感、特异等优点，适合于临床诊断和大批量样品检验。     

琼脂扩散法与酶标斑点试验结合鉴别诊断马传贫病

马传染性贫血病是一种马属家畜传染病，曾在天水部分县(区)流行，给当地畜牧业生产造成很大损失，经过广大畜牧兽医科技人员多年不懈努力，全市现已达到部颁无疫区标准。为巩固已取得的防制成果，我们在市场、运输、产地等环节积极开展了血清学疫情监测，从1994年至今累计监测马类家畜66526头(匹)，曾两次在市场采集血清中，检出琼脂扩散性马5匹，弄清是人工注苗免疫还是自然感染马，对     

四川省马传染性贫血病防控的回顾与思考

马传染性贫血病（简称马传贫）是严重危害马属动物的一种传染病。四川省于1989年在凉山州越西、昭觉两县发现了阳性马匹,经确诊后,即采取了普查、检疫、免疫注射、扑杀等综合性防控措施,1998年全省达到稳定控制马传贫标准,2003年达到消灭马传贫标准。从2003年至今,各项防控措施不停,监测表明均未发现阳性病例,成效显著。     

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。     

鸡传染性法氏囊病血清抗体间接ELISA检测方法的建立

为建立可检测鸡传染性法氏囊病血清抗体的间接ELISA检测方法,本研究经RT-PCR扩增IBDV VP2（1nt-708nt）基因片段,并应用pET28a载体进行原核表达,亲和层析纯化重组蛋白作为包被抗原,建立检测IBDV血清抗体的间接ELISA方法。应用所建方法和IDEXX试剂盒,对采自不同地区的156份鸡血清样品进行平行检测和比较。结果表明,RT-PCR扩增获得708bp的VP2基因片段;含重组表达质粒pET28a-VP2的大肠杆菌BL21经IPTG诱导后,表达了约27kDa的VP2重组蛋白,表达量约占菌体蛋白的30.1%;建立的间接ELISA检测方法与IDEXX试剂盒比较,其特异性、敏感性和符合率分别达到90.24%、95.65%和94.23%。由此可见,本研究所建立的间接ELISA方法敏感、特异,适用于IBDV血清抗体的检测。     

间接ELISA检测鸭疫里默氏杆菌血清抗体方法的建立

应用1型鸭疫里默氏杆菌(云南株)超声粉碎物作为包被抗原,建立检测鸭疫里默氏杆菌血清抗体的间接ELISA方法。用倍比稀释法确定HRP标记羊抗鸭二抗最佳稀释倍数为1∶3500,并用棋盘测定法确定抗原的最佳包被浓度为2.7mg/mL,血清最佳稀释倍数为1∶300,阴性血清临界值为0.381;阻断试验结果表明,1型鸭疫里默氏杆菌与其抗血清阻断阳性,与鸭大肠杆菌O78、O132菌株和FJ4型鸭疫里默氏杆菌阻断阴性(无交叉反应)。重复性试验结果表明,该ELISA方法批内变异系数≤0.246,批间变易系数≤0.889。结果表明,该ELISA方法特异性强,重复性好,可用于1型鸭疫里默氏杆菌(云南株)血清抗体的检测。     

Comparison of Latex Agglutination, Indirect Hemagglutination, and ELISA Techniques for the Detection of Toxoplasma gondii-specific Antibodies in the Serum of Cats

The primary purpose of this study was to determine whether commercially available latex agglutination and indirect hemagglutination kits for the detection of Toxoplasma gondii-specific antibodies were capable of detecting T. gondii-specific immunoglobulin M (IgM) in the serum of cats. Serum samples from 35 cats containing either T. gondii-specific IgM, T. gondii-specific immunoglobulin G (IgG), or both were collected. Each serum sample was assayed using a latex agglutination kit, an indirect hemagglutination kit, an enzyme-linked immunosorbent assay (ELISA) for the detection of T. gondii-specific IgG, and an ELISA for the detection of T. gondii-specific IgM. When serum samples containing only T. gondii-specific IgM as determined by ELISA were assayed, the latex agglutination kit and the indirect hemagglutination kit detected antibodies in 33.3% and 13.3%, respectively. When T. gondii-specific IgG was present in a serum sample, the results from the latex agglutination kit, the indirect hemagglutination kit, and the IgG-ELISA were similar; however, there was a wide variation in titer magnitude results between the three assays. It was concluded that the latex agglutination kit and the indirect hemagglutination kit did not adequately detect T. gondii-specific IgM in feline serum.     

A Micro-passive Hemagglutination Test for the Rapid Detection of Antibodies to Infectious Bovine Rhinotracheitis Virus

Serum antibodies to infectious bovine rhinotracheitis virus are most commonly detected using the serum-virus-neutralization test, a time-consuming and expensive procedure. A passive hemagglutination test was developed for the rapid detection of antibodies using the Microtiter System. Two cloned strains of the virus were used in the investigation, namely the BF strain and the Los Angeles strain. Erythrocytes from several species were used. The best results were obtained using the Los Angeles strain of virus adsorbed to sheep cells treated with formalin and tannic acid. Sera from field cases of the disease as well as hyperimmune bovine and rabbit sera were tested using both the micro-passive hemagglutination test and the standard plaque-neutralization procedures. Comparable titers were obtained in the two tests. The micro-passive hemagglutination test is rapid, accurate and economical.     

间接ELISA检测禽多杀性巴氏杆菌抗体中包被抗原的选择

间接ELISA已在抗体检测中广泛应用,而抗原包被作为间接ELISA中必不可少的一步可直接影响试验的准确性。本试验分别使用超声波裂解抗原、脂多糖蛋白抗原和全茵抗原作为包被抗原,建立相应的检测方法并进行重复性试验。试验证明全菌抗原建立的禽多杀性巴氏杆菌ELISA检测方法在敏感度、特异性、稳定性方面均优于其他两种抗原。     

Evaluation of a Macroscopic Plate Test and an Indirect Immunofluorescence Test to Detect Leptospiral Antibodies in Bovine Serum

A macroscopic plate test was found to be reliable and convenient for detection of Leptospira serotype pomona antibodies in bovine sera. However, the procedure was unreliable for detecting L. serotype canicola antibodies because of false positive reactions. An indirect immunofluorescence test and the microscopic agglutination test provided comparable results and they effectively detected serotype pomona antibodies in bovine sera.