琼脂凝胶扩散试验检测鸡传染性支气管炎抗体

本文对鸡传染性支气管炎琼脂凝胶扩散试验进行了研究。试验结果表明，感染鸡传染性支气管炎病毒的鸡胚绒毛尿囊膜和尿囊液都能用于制备琼扩抗原，而且以感染ＩＢＶ后２０－３０小时的绒毛尿囊膜和感染ＩＢＶ后４８－６０小时的尿囊液效果最佳。通过传染性支气管炎琼脂凝胶扩散试验的研究和对血清样品的测定，以及与美国ＫＰＬＡＢＯＲＡＴＯＲＩＥＳ的ＩＢＶ－ＥＬＩＳＡ诊断盒比较，表明所建立的琼脂凝胶扩散试验可用于鸡传染性支气     

应用间接免疫荧光法检测禽网状内皮组织增生病病毒抗体的研究

用禽网状内皮组织增生病病毒（ＲＥＶ）感染ＳＰＦ鸡胚次代成纤维细胞涂片为抗原,建立了检测ＲＥＶ抗体的间接免疫荧光法（ＩＦＡ）。确定了待检血清稀释度１∶６４和兔抗鸡ＩｇＧ荧光抗体的稀释度１∶３２的工作浓度。应用该ＩＦＡ方法对人工感染ＲＥＶ的２０只３０日龄ＳＰＦ鸡进行了检测,接种后４天时均呈阴性反应,７天时８只鸡呈阳性反应,１４天２０只全部呈阳性的反应。通过对ＮＤＶ、ＩＢＤＶ、ＡＬＶ、ＭＤＶ、ＣＩＡＶ、ＡＩＶ等标准阳性血清的交叉试验,证明该方法特异性强。应用该ＩＦＡ方法对我国辽宁、山东、黑龙江省部分鸡群进行ＲＥＶ抗体检测,证明该方法特异性强、敏感性高,适于在生产中推广应用。     

用间接荧光抗体试验检测鸡肾型传染性支气管炎抗原的研究

建立检测鸡肾型传染性支气管炎抗原的间接荧光抗体试验（ＩＦＡ）其抗体感染作时间Ｉ抗以５０～６０ｍｉｎ，Ⅱ抗以３０～４０ｍｉｎ最佳。该法具有良好的特异性，可检测出人工感染鸡肾型传染性支气管炎病毒（ＩＢＶ）Ａｕｓｔ－Ｔ，ＨＮ９３０１，ＴＪ９３０１，ＢＪ９３０１毒株后的抗原；对自然发病病例检测结果，阳性符合率高于Ｄｏｔ－ＥＬＩＳＡ和气管环中和试验法。     

应用间接免疫荧光法检测禽网状内皮组织增生病病毒抗体的研究

用禽网状内皮组织增生病病毒（ＲＥＶ）感染ＳＰＦ鸡胚次代成纤维细胞涂片为抗原，建立了检测ＲＥＶ抗体的间接免疫荧光法（ＩＦＡ）。确定了待检血清稀释度１：６４和兔抗鸡ＩｇＧ荧光抗体的稀释度１：３２的工作浓度。应用该ＩＦＡ方法对人工感染ＲＥＶ的２０只３０日龄ＳＰＦ鸡进行了检测，接种后４天时均呈阴性反应，７天时８只鸡呈阳性反应，１４天２０只全部呈阳性的反应。通过对ＮＤＶ、ＩＢＤＶ、ＡＬＶ、ＭＤＶ、ＣＩＡＶ、     

间接免疫荧光检测禽脑脊髓炎病毒抗体

ＡＥＶＶａｎＲｏｅｋｅｌ鸡胚适应株在鸡胚成纤维细胞（ＣＥＦ）和鸡胚神经胶质细胞（ＣＥＢ）传代,用盲传至第６代的ＣＥＦ、ＣＥＢ制成荧光标本。建立了ＡＥＶ荧光抗体检测方法,确定了待检血清稀释度１∶１０和荧光抗体ＦＩＴＣ羊抗鸡ＩｇＧ的稀释度１∶１０的工作浓度。通过对ＮＤＶ、ＭＤＶ、ＩＢＤＶ、ＡＩＶ、Ｒｅｏ等标准阳性血清的交叉试验,证实该法特异性强,且简单、方便、经济,适合于鸡群ＡＥＶ抗体的检测。     

间接免疫荧光检测禽脑脊髓炎病毒抗体

ＡＥＶ Ｖａｎ Ｒｏｅｋｅｌ鸡胚适应株在鸡胚成纤维细胞和鸡胚神经胶质细胞传代，用盲传至第６代的ＣＥＦ，ＣＥＢ制成荧光标本。建立了ＡＥＶ荧光抗体检测方法，确定了待检血清稀释度１：１０和荧光抗体ＦＩＴＣ羊抗鸡ＩｇＧ的稀释率１：１０的工作浓度。通过对ＮＤＶ，ＭＤＶ，ＩＢＤＶ，ＡＩＶ，Ｒｅｏ等标准阳性血清的交叉试验，证实该法特性强且简单，方便经济，适合于鸡群ＡＥＶ抗体的检测。     

应用间接免疫荧光法检测鸡传染性贫血病病毒抗体的研究

用鸡传染性贫血病病毒（ＣＩＡＶ）ＭＳＢＩ－ＴＫ５８０３毒株感染的ＭＤＣＣ－ＭＳＢ；细胞涂片为抗原，建立了检测ＣＩＡＶ抗体的间接免疫荧光法（ＩＩＦＡ）。应用该ＩＩＦＡ方法对人工感染的ＳＰＦ鸡进行了检测１５只１日龄ＳＰＦ鸡在接种后第７、１４天时均呈阴性反应；２１天时２只鸡呈弱阳性反应，２８天时均呈弱阳性反应，２１天时２只鸡呈弱阳性反应，２８天时均呈弱阳性反应，３５天时呈明显阳性反应；１４只４０日龄ＳＰ     

鸡传染性法氏囊病的快速诊断

应用鸡传染性法氏囊病病毒单克隆抗体及其荧光标记物，对接种ＩＢＤ疫苗、强毒感染和自然病例鸡组织进行直接荧光抗体试验、斑点酶联免疫吸附试验和琼脂扩散试验三种检测方法的比较，结果表明，鸡接种疫苗后１０天内ＤＦＡ和Ｄｏｔ－ＥＬＩＳＡ均呈阳性反应，而ＡＧＰ只在接种后６－８天的样品中检出抗原。２２例人工感当鸡和４０例自然发病鸡组织的检测结果表明，ＤＦＡ和Ｄｏｔ－ＥＬＩＳＡ的敏感性基本一致，且高于ＡＧＰ，它们的     

斑点免疫金银染色法检测鸡传染性支气管炎病毒抗原的研究

用建立的斑点免疫金银染色（Ｄｏｔ－ＩＧＳＳ）法检测鸡传染性支气管炎病毒（ＩＢＶ）抗原,确定兔抗ＩＢＶ血清工作浓度为１：４００,ＳＰＡ－胶体金探针的工作浓度为１：８０,该法对纯化ＩＢＶ抗原的最低检出量为０．４３１４ｎｇ／点,用Ｄｏｔ－ＩＧＳＳ与斑点酶联免疫吸附试验（Ｄｏｔ－ＥＬＩＳＡ）同时检测１５只人工感染ＩＢＶ鸡的气管,肺,肾病科,ＩＢＶ阳性检出率均为１００％,对３２份疑似ＩＢＶ感染鸡病料检测,Ｉ     

单抗介导的斑点ELISA检测鸡传染性支气管炎病毒的研究

用群特异性的单克隆抗体作第一抗体,用酶标兔抗体ＩＢＶＩｇＧ作第二抗体,建立夹心法Ｄｏｔ－ＥＬＩＳＡ程序检测鸡传染性支气管炎病毒抗原。试验表明,本程序检测ＩＢＶ抗原高度敏感性和特异性。最低抗原检出量为０．５μｇ,约１００个气管环半数感染量（１００ＴＯＣＩＤ５０）,阳性检出率为９６％。     

Comparison of a tissue-culture virus-neutralization test and the enzyme-linked immunosorbent assay for measurement of antibodies t infectious bronchitis

Two serological tests, the virus-neutralization (VN) test in tissue culture using a tissue-cell-adapted virus and the enzyme-linked immunosorbent assay (ELISA), were compared to detect antibodies against Massachusetts 41 and Connecticut 46 strains of Infectious Bronchitis Virus (IBV). The VN test was conducted in wells of microplates by the usual procedure. The two strains of IBV were adapted after 20 serial passages to induce CPE in 24 hours in chickens embryos kidney cells (CEKC). The ELISA test was carried out using partially virus following ultracentrifugation of each stain of IBV as antigen. The ELISA test detected higher geometric mean antibody titers (GMT) against both strains of IBV than did the VN test. One hundred four serum samples taken at 1, 3, 5, 9, 22, 24, and 26 weeks of age from a flock of chickens vaccinated with the Mass strain three times and the Conn strain of IBV two times during the growing period showed higher antibody titer responses to the Conn 46 than to the Mass 41 strain. Maternal antibodies in chicks one week of age were readily detected by the ELISA test, whereas low or insignificant titers were found by the VN test. Sera of vaccinated chickens collected following challenge with Mass 41 or Conn 46 strain of IBV showed that the ELISA was more sensitive and showed higher titers than did the VN test. Although the VN test showed no rise in GMT in the same sera tested with the heterologous virus, the ELISA showed a slight increase or cross-reaction. The serum samples from the unchallenged control group showed no change in GMT with either test or IBV strain.     

鸡传染性支气管炎病毒抗体间接ELISA检测方法的建立

为建立快速检测鸡传染性支气管炎病毒(IBV)的血清学方法,本试验以IBV为检测抗原,建立了一种检测IBV抗体的间接ELISA方法,并对各种检测条件进行了优化.研究结果显示,抗原的最佳包被浓度为19.2 μg/mL,最佳包被条件为37 ℃ 1 h后4 ℃过夜;血清的最佳稀释度为1:800,37 ℃孵育60 min;酶标二抗稀释度为1:7 000,37 ℃孵育60 min;底物显色为37 ℃避光作用10 min.经特异性、重复性、敏感性试验证明,该方法与鸡常见病原的阳性血清均无交叉反应,重复性较好及血清稀释至1:12 800时仍可检测到IBV抗体.结果表明,本试验所建立的间接ELISA方法具有良好的特异性、重复性和敏感性.     

Establishment of Indirect ELISA Method for Detecting Antibodies against Avain Infectious Bronchitis Virus

The purpose of this research was to establish a rapid detection serological method against avain infectious bronchitis virus (IBV).In this study,an indirect ELISA method was established using IBV as the detected antigen and a variety of testing conditions were optimized.The results showed that the optimal antigen coating concentration was 19.2 μg/mL and the optimal condition for coating was incubated at 37 ℃ for 1 h and then 4 ℃ overnight.The optimal dilutions of serum and enzyme labeled antibody were 1:800 and 1:7 000 incubated at 37 ℃ for 60 min,respectively.The optimal condition of chromogenic substrate was incubated at 37 ℃ for 10 min in the dark.The specificity,repeatability and sensitivity tests proved that the indirect ELISA did not cross-react with positive antiviral sera of other chicken diseases,had good repeatability and could detect IBV antibody when serum was diluted 1:12 800.We concluded that the established indirect ELISA was specific,repeatable and sensitive.     

Development and Application of a PEDV Nsp7-based Indirect ELISA for Antibody Detection

This study was aimed to establish an indirect ELISA to detect antibodies against different strains of porcine epidemic diarrhea virus (PEDV). Puried nonstructural protein 7(Nsp7) was used as coating antigen, and the indirect ELISA was established by optimizing the ELISA reaction conditions. The results showed that the optimal reaction conditions were as follow:The amount of coating antigen was 0.20 μg/well, and the coating condition was at 37℃ incubation for 1 h then 4℃ overnight; The working dilution of serum samples and HRP-labelled secondary antibody were 1:300 and 1:10 000, and the incubation time were 2 and 1.5 h, respectively; TMB substrate incubation time was 15 min. Serum sample was determined as positive when its S/P>0.1694 and negative when its S/P<0.1398. The ELISA was specific, reproducible and sensitive. Forty samples of suspected PEDV serum samples were tested by the established ELISA, and the coincidence rate between the ELISA and the commercial kit was 95%. The ELISA established in this study could be used clinically to detect the antibody level of different strains of PEDV, and it also had the potential for early diagnosis of PEDV, providing a basis for the development of effective measures to control PEDV.     

鸡传染性支气管炎ELISA抗体检测试剂盒的研制

采用加蔗糖垫离心纯化鸡传染性支气管炎病毒(IBV)制备抗原，用提纯的IBV抗原包被微量板，建立了检测鸡传染性支气管炎(IB)抗体的ELISA试剂盒。抗原、被检血清和酶标结合物的最佳工作浓度分别为10ug／ml、1：200和1：3200。与IDEXX试剂盒相比，其敏感性、重复性、特异性均接近国外同类产品水平。对SPF鸡血清、实验免疫与攻毒的SPF鸡血清进行检测，结果表明所建立的ELISA特异性为95．6％，与IDEXX试剂盒符合率为95．6％。用于检测抗IBV特异性IgG抗体发现在免疫接种IB弱毒苗后，第4天即可检测到IgG抗体，峰值在第3周。试验鸡在通过滴鼻、点眼途径人工感染IBV强毒后，第5天抗体滴度明显上升。我们认为，该法是目前我国SPF鸡质量监测、养鸡生产中进行IB监测较好的方法。     

Detection of antibody to turkey coronavirus by antibody-capture enzyme-linked immunosorbent assay utilizing infectious bronchitis virus antigen

An antibody-capture enzyme-linked immunosorbent assay (ELISA) for detection of antibody to turkey coronavirus (TCV) utilizing infectious bronchitis virus (IBV) antigen was developed. Anti-TCV hyperimmune turkey serum and normal turkey serum were used as positive or negative control serum for optimization of the ELISA system. Goat anti-turkey immunoglobulin G (light plus heavy chains) conjugated with horseradish peroxidase was used as detector antibody. The performance of the ELISA system was evaluated with 45 normal turkey sera and 325 turkey sera from the field and the cutoff point was determined. Serum samples of turkeys experimentally infected with TCV collected sequentially from 1 to 63 days postinfection were applied to the established antibody-capture ELISA using IBV antigens. The optimum conditions for differentiation between anti-TCV hyperimmune serum and normal turkey serum were serum dilution at 1:40 and conjugate dilution at 1:1600. Of the 325 sera from the field, 175 were positive for TCV by immunofluorescent antibody (IFA) assay. The sensitivity and specificity of the ELISA relative to IFA test were 93.1% and 96.7%, respectively, based on the results of serum samples from the field turkey flocks using the optimum cutoff point of 0.18 as determined by the logistic regression method. The ELISA values of all 45 normal turkey sera were completely separated from that of IFA-positive sera. The ELISA results of serum samples collected from turkeys experimentally infected with TCV were comparable to that of the IFA assay. Reactivity of anti-rotavirus, anti-reovirus, anti-adenovirus, or anti-enterovirus antibodies with the IBV antigens coated in the commercially available ELISA plates coated with IBV antigens could be utilized for detection of antibodies to TCV in antibody-capture ELISA.     

单抗免疫过氧化物酶技术检测鸡传染性支气管炎病毒

以抗鸡传染性支气管病毒（ＩＢＶ）核衣壳蛋白（Ｎ）的单抗株６ＤＨ８作为一抗,以辣根过氧化物酶标记的羊抗鼠ＩｇＧ作为二抗,建立了检测石蜡切片中ＩＢＶ抗原的单抗免疫过氧化物酶技术（Ｍｃ－ＩＰ）,并对人工攻毒鸡及临床ＩＢＶ感染疑似鸡进行了检测。在ＩＢＶＭ４１株人工攻毒鸡,用该技术于１～１２ｄ从气管、２～７ｄ从肾脏可以检测到ＩＢＶ抗原,阳性染色集中于气管粘膜上皮细胞及肾小管上皮细胞胞浆;临床疑为ＩＢＶ感染的病鸡,以Ｍｃ－ＩＰ技术和单抗免疫荧光试验（Ｍｃ－ＩＦＡ）同时进行检测,结果阳性率分别为９０．３％及８３．９％。     

猪脑心肌炎病毒重组抗原间接ELISA诊断方法的建立与应用

以猪脑心肌炎病毒VP1重组蛋白为抗原,建立了检测猪脑心肌炎病毒(EMCV)血清抗体的间接ELISA诊断方法。经优化后抗原最适包被浓度为2μg/mL,血清最适稀释度为1∶50,其作用时间为90 min,酶标抗体最适稀释度为1∶20 000,最适作用时间为30 min。判定标准为OD450≥0.427为阳性,OD450≤0.35为阴性,介于二者之间为可疑。该重组蛋白与PRRSV、猪瘟、PCV2、FMDV抗体反应呈阴性,证明具有良好的特异性。应用该方法检测了来自我国不同地区的26家猪场的156份临床血清,结果证明我国部分规模化猪场已有猪脑心肌炎疾病存在。     

Observations on the preparation and stability of infectious bronchitis virus hemagglutination antigen from virus propagated in chicken embryos and chicken kidney cell cultures

A total of 166 infectious bronchitis virus (IBV) hemagglutination (HA) antigen preparations were made during a 30-month period from allanto-amnionic fluid (AAF) from chicken embryos inoculated with 10 different IBV strains (Mass 41, Conn 46, H52, Florida 18288, Ark 99, JMK, T, Holte, EF, SE17). Antigens were prepared by inoculating 9- or 10-day-old embryos with 10(5.0) to 10(6.5) EID50 IBV, harvesting AAF after a 30-hour-postinoculation incubation, and phospholipase C (PLC) treatment of virus concentrated by pelleting from the AAF. Longer (48 hr) incubation times were tried, but production of H52 HA antigen was successful only from AAF harvested after 30 hours of incubation. AAF from JMK-infected embryos had lower infectivity titers and frequently yielded lower HA antigen titers than the other strains. The treatment of AAF with fluorocarbon did not enhance or diminish HA activity but did yield clearer antigens by removing extraneous material. Polyethylene glycol precipitation of virus was an acceptable alternative to pelleting virus at 39,000 X g. Inactivation of IBV with 0.1% betapropiolactone did not affect HA activity, whereas inactivation with 0.1% formalin caused a marked reduction in HA titer. Different buffer formulations including phosphate, tris, or HEPES were tried to optimize the conditions for PLC treatment of virus concentrate, but there were no apparent differences in the antigens prepared in the different buffers. The HA antigen preparations were stored and were stable at 4 C. Antigen titers of greater than or equal to 64 after storage for 20 months or longer were not uncommon. Addition of merthiolate as a preservative had no deleterious effect on HA activity. Antigen stability appeared to be enhanced by incorporating EDTA in buffer for virus pellet recovery and during enzyme treatment. Attempts to produce HA antigens from cell-culture-adapted virus propagated in chicken kidney cells were less satisfactory. An acceptable HA antigen was prepared from only two (Mass 41, SE17) of the seven strains that were tried. Virus propagation in chicken embryos is the better method of the two for IBV HA antigen production. Aside from the need to concentrate virus and treat the concentrate with PLC, there appeared to be considerable latitude in the procedures that can be used to make acceptable IBV HA antigens.     

鸡传染性支气管炎病理形态学及发病机理的研究

１５０只１８日龄雏鸡随机分为两组，试验组９０只接种传染性支气管炎病毒（ＩＢＶ－Ｍ４１）后３５天内分３０批依次于不同时间扑杀，作组织病理学、超微结构及病毒抗原位检查，对照组６０只作相同的检查。结果表明，ＩＢＶ攻击的靶器官是气管，气管的病变表现为粘膜上皮细胞的损伤和脱、残留的在皮细胞增殖形成复层上皮、粘膜固有层及粘膜下层大量淋巴细胞浸润及粘膜逐渐恢复的相互连续的病理过程。肺脏初级、次级支气管也有类似的