鸡抗传染性法氏囊病卵黄抗体的ELISA及琼脂扩散检测

鸡传染性法氏囊病(IBD)是目前危害养鸡业的主要疾病之一。卵黄抗体(IgY)是母鸡血清中的IgG转移至卵黄中并积累形成，且与血清中抗体水平相当，因此通过检测卵黄抗体可监测鸡群的抗体水平，为鸡传染性疾病(如：IBD、新城疫、禽流感等)免疫程序的建立提供参考。研究使用间接ELISA(酶联免疫吸附试验)快速检测卵黄抗体的方法，同时对卵黄抗体的生产制备具有指导意义。     

青海高原地区鸡流行病血清学调查分析

随着青海高原地区禽类及其产品市场流通日益频繁,鸡病发生及危害不断增加,为随时掌握鸡病疫情动态和观察鸡病免疫效果,全面防控疫病,我们从2000年至2004年对该地区鸡流行病进行了连续几年的血清学检测和分析。1方法与材料1.1方法:禽流感、鸡传染性法氏囊病、鸡白痢、用琼脂扩散免疫试验,检测血清中特异性抗体。鸡新城疫用血凝抑制试验,即微量血凝试验(HI),按常规微量法检测血清中HI抗体效价。禽白血病用酶联免疫吸附试验(ELISA)。1.2材料:禽流感、鸡白痢、鸡传染性法氏囊病琼脂扩散试剂,标准抗原、阳性血清、阴性血清由哈尔滨兽医研究所…     

抗H9亚型禽流感病毒独特型抗体的制备与应用

用纯化的鸡抗H9亚型禽流感病毒（AIV）免疫球蛋白G（IgG）作为抗原免疫家兔，制备兔源多克隆抗独特型抗体（Pab2），同时免疫小鼠，应用细胞融合技术建立了分泌抗AIV独特型单克隆抗体（Mab2）的杂交瘤细胞系。经鉴定，2类抗独特型抗体均能竞争性地抑制鸡抗AIVIgG与AIV的结合。保护试验证实，兔源多克隆抗独特型抗体疫苗较单克隆抗独特型抗体疫苗具有更高的保护作用。     

鸡传染性支气管炎血凝抑制试验方法标准化的研究

鸡传染性支气管炎病毒（ＩＢＶ）抗体的血凝抑制（ＨＩ）试验方法经标准化后，检测ＳＰＦ鸡血清ＩＢＶ感染鸡血清以及野外样品的结果表明，具有快速、特异、敏感、稳定等优点。标准化的实现主要通过抗原冻干和鸡红细胞醛化，从而取得较为满意的结果。     

鸡传染性支气管炎血凝抑制试验方法标准化的研究

鸡传染性支气管炎病毒（ＩＢＶ）抗体的血凝抑制（ＨＩ）试验方法经标准化后，检测ＳＰＦ鸡血清ＩＢＶ感染鸡血清以及野外样品的结果表明，具有快速，特异，敏感，稳定等优点。标准化的实现主要通过抗原冻干和鸡红细胞醛化，从而取得较为满意的结果。     

应用进口ELISA试剂盒检测鸡传染性支气管炎抗体

鸡传染性支气管炎（IB）是由传染性支气管炎病毒引起鸡的一种急性高度接触性的呼吸道传染病，是严重危害我国乃至世界养鸡业的重要疾病之一。已有多种血清学方法可用于IB抗体检测，我国目前常用的方法有琼脂扩散试验（AGP）、血清中和试验（SN）、间接血凝试验（IHA）等。国内很多学者先后建立了ELISA方法检测IB抗体均取得理想结果，但仅限于实验室检测，在基层推广应用还有一定困难。作者在加04年7月至2004年11月应用美国Affinitech-IB抗体检测试剂盒对山东几个规模化养鸡场送检的血清样品进行了检测，现报告如下。     

检测禽流感病毒抗体的重组核蛋白间接ELISA方法的建立

以大肠杆菌系统表达的H9N2亚型禽流感病毒（AIV）核蛋白（NP）为抗原，建立了禽流感间接酶联免疫吸附试验抗体检测技术（NP—ELISA）。对263份待检血清（包括临床收集的243份血清和20份H9N2亚型AIV免疫鸡阳性血清）进行检测，NP—ELISA与琼脂免疫扩散试验（AGP）的总符合率为83．3％，与血凝抑制试验（HI）的总符合率为92％。特异性试验表明，NP—ELISA方法可以检测H5、H7和H9亚型AIV特异性抗体，检测为阳性的血清样品能够被阳性鸡胚尿囊液阻断。敏感性试验证实，NP—ELISA最早可以检测鸡感染后7d的血清样品，并于感染后10d确定100％血清阳性，而AGP检测直到首免后21～28d才出现部分血清阳性，HI检测直到10～14d才出现部分血清阳性，并且NP-ELISA要比HI敏感4～40倍。试验证明，NP—ELISA是检测AIV血清型特异性抗体的一种特异、敏感、快速、经济的血清学检测技术。     

齐齐哈尔地区禽流感免疫抗体监测

2007年5-7月，采用微量法血凝抑制试验对来自于齐齐哈尔地区的11份鸡和46份鹅的卵抗和血清样品进行了禽流感抗体的血清学调查，以对齐齐哈尔地区禽流感免疫抗体进行监测。结果表明，在未免疫家禽血清中，均未检测到禽流感抗体。     

免疫层析试纸条检测口蹄疫非结构蛋白3ABC抗体的研究

为建立口蹄疫(FMD)快速检测方法,本研究以纯化的FMD病毒(FMDV)非结构蛋白(NSP)3ABC作为捕捉抗原.金标FMDV抗原作为金标试剂,建立了检测FMD NSP 3ABC抗体的胶体金免疫层析试纸条(ICS).应用FMD ICS试验与UBI FMD NSP酶联免疫吸附试验(ELISA)试剂盒对反刍动物、猪的FMD标准阳性血清的不同滴度进行测定,结果表明ICS在检测反刍动物血清时的检测下限(1:64)与ELISA(1:128)接近,而检测猪血清时的检测下限(1:8)低于UBI NSP-ELISA试剂盒(1:64).同时用ICS和UBINSP-ELISA检测313份牛血清和111份羊血清,结果ICS检测牛血清时的敏感性和特异性分别为81.81%和96.69%,检测羊血清时敏感性和特异性分别为88.88%和95.10%;ICS与UBI NSP-ELISA用于检测牛血清时符合率为95.52%,用于检测羊血清时的符合率为94.59%.结果表明ICS适合在基层单位或养殖场进行自行检测.     

用卵黄浸提液对三种鸡病抗体检测试验

本试验用卵黄聚乙二醇浸提液（卵黄液）进行鸡白痢（PD）、传染性囊病（IBD）和新城疫（ND）的抗体检测，并与相应血清进行对比试验。结果表明卵黄液PD琼脂扩散（AGP）试验与血清试验结果符合率100％，IBDAGP试验和NDMt抑制（HI）试验与血清试验结果基本一致（差异显著性检验P＞0．05），可以代替血清进行其抗体检测；且卵黄液制备方法简单，省时省力，有实际应用价值。     

An enzyme-linked immunosorbent assay for the detection of antibody against avian influenza virus

An enzyme-linked immunosorbent assay (ELISA) was developed for detecting antibody to type A avian influenza (AI) virus. The sensitivity and group specificity of the AI-ELISA were compared with those of the agar-gel-precipitin test (AGPT) and the hemagglutination-inhibition (HI) test under conditions of both controlled and field exposure. During the course of temporal experimental infection (0-76 days) of specific-pathogen-free (SPF) chickens with AI subtype Hav9N2, the AI-ELISA was able to detect specific AI antibody as early as 8 days postinoculation (PI), and it measured rising levels of antibody through 35 days PI, at which time the chickens were re-exposed to AI virus. Conversely, AGP tests were negative through 35 days PI, and HI tests began to detect low levels of AI antibody only at 21 days PI. Following a secondary infection at 35 days PI with the same AI subtype, all tests measured rising levels of AI-specific antibody (35-76 days PI). However, the AGP test was positive at only the 7- and 14-day samplings postsecondary immunization. Under field conditions, the AI-ELISA was able to detect serum AI antibody in flocks from which highly pathogenic AI was isolated, but the AGP tests of these sera were negative.     

Development and evaluation of an immunochromatographic strip for detection of Streptococcus suis type 2 antibody.

In this study, an immunochromatographic strip (ICS) was developed for the detection of antibody against Streptococcus suis serotype 2 (SS2). Colloidal gold particles labeled with staphylococcal protein A (SPA), which can bind to the F(C) fragment of mammalian immunoglobulin, were used as the detector reagent. The capsular polysaccharide (CPS) of SS2 and affinity-purified IgG from a healthy naive pig were immobilized on test and control regions of a nitrocellulose membrane, respectively. The ICS was used to 1) detect anti-CPS antibody in 14 sera taken from 4 SS2-infected pigs, 24 sera from pigs hyperimmunized with SS2, and 68 sera from pigs inoculated or infected with bacteria other than SS2; 2) determine anti-CPS antibody titers of 20 positive sera for comparison with enzyme-linked immunosorbent assay (ELISA); and 3) detect anti-CPS antibody in 226 clinical sera taken from diseased pigs also for comparison with ELISA. An ELISA used as a reference test determined the specificity and sensitivity of the ICS to be 97.1% and 86.3%, respectively. There was excellent agreement between the results obtained by ELISA and the ICS (kappa = 0.843). Additionally, there was strong agreement between the results of bacterial isolation from pig tonsils and ICS test (kappa = 0.658). Because it is rapid and easy to use, the test is suitable for the serological surveillance of SS2 at farms.     

An adhesion-hemadsorption inhibition test for the detection of serum antibody to Mycoplasma gallisepticum

A simple adhesion-hemadsorption inhibition (AHAI) test was developed for the detection of antibodies to Mycoplasma gallisepticum in the chicken sera. The AHAI antibody was detected simultaneously with HI antibody from sera of chickens intratracheally inoculated with viable cells of M. gallisepticum. A good correlation between HI and AHAI antibody titers was obtained with 382 (84.7%) of 451 sera from chickens reared on farms spontaneously contaminated with M. gallisepticum, whereas the remainder, 69 sera, was positive for HI but negative for AHAI test. It was not apparent whether the latters exhibited a non-specific reaction or the discrepancy was due to the lower sensitivity of AHAI reaction. The AHAI test does not require a great amount of antigen, special reagents or instruments, or pre-absorption treatment of test sera, and, therefore, it may serve as a simple serological test for detecting antibodies to M. gallisepticum.     

Development of Indirect Enzyme-linked Immunosorbent Assay with Nucleoprotein as Antigen for Detection and Quantification of Antibodies against Avian Influenza Virus

Avian influenza (AI) is a serious infectious disease caused by avian influenza virus (AIV) belonging to type A Orthomyxovirus. In the present study, we developed an indirect enzyme-linked immunosorbent assay (ELISA) employing E. coli-expressed full-length nucleoprotein (NP) of H9N2 avian influenza virus for the detection and quantification of antibodies against AIV nucleoprotein. The NP-ELISA was compared with the AI agar gel propagation (AGP) test, haemagglutination inhibition (HI) test, and IDEXX-FlockChek ELISA using 263 sera. The NP-ELISA was significantly more sensitive than the AGP and HI tests, and showed 96.2% agreement ratio with IDEXX-FlockChek ELISA. With results obtained using the NP-ELISA, an ELISA titre (ET) prediction equation, with which the ELISA titres of a flock or individual chickens can be determined, was derived from a positive/negative (P/N) ratio standard curve. The NP-ELISA enables an alternative rapid serological diagnosis and is suitable for influenza A antibody screening, especially in species that harbour several influenza subtypes.     

Hemagglutination-inhibition test applied to the study of akabane virus infection in domestic animals

Some factors affecting the hemagglutination-inhibition (HI) reaction with Akabane virus were investigated and an HI test developed. The test was proven to be useful in studies of antibody responses in cattle and other domestic animals infected with Akabane virus. HI antibody titers of individual animals were shown to be closely correlated with their neutralizing antibody titers and to remain undiminished for a relatively long time. In some early sera from domestic animals infected with Akabane virus, HI antibody sensitive to 2-mercaptoethanol was demonstrated.     

A survey for haemagglutination-inhibiting antibody to West Nile virus in human and animal sera in Nigeria

A survey for West Nile Virus (WNV) haemagglutination-inhibition (HI) antibody was carried out in humans and domestic animals. Human sera were collected from Ibadan, while the animal sera were collected from both Ibadan and Maiduguri. Out of 304 human sera tested, 123 were positive (40%). There was a higher prevalence of HI antibody in adults than children. Sex distribution of positive sera showed that 37% of males and 43% of females had WNV HI antibody. There was no significant difference in the prevalence of HI antibody in both sexes. On the 123 WNV HI positive sera tested, 104 (85%) and 78 (75%) had yellow fever and Potiskum HI antibody respectively. Monotypic WNV virus reactions were frequently found in children while polytypic reactions were frequently found in adults. A total of 200 animal sera were examined, 50 camels, 50 goats, 49 cattle and 51 sheep. The highest prevalence of HI antibody was found in camels (26%), followed by sheep (20%). Percentage of positive sera in other species were: goat (18%) and cattle (6%). Of the 35 WNV HI positive animal sera, 26 and 20% reacted with Yellow fever and Potiskum virus antigens respectively.     

Immunofluorescence in the serological diagnosis of parainfluenza type 3 and respiratory syncytial virus infection in calves

The fluorescent antibody (FA) test is compared with the haemagglutination inhibition (HI) test for parainfluenza virus type 3 (PI-3) and virus neutralisation (VN) test for respiratory syncytial (RS) virus for detection and titration of virus-specific antibodies. In experimentally inoculated calves PI-3 and RS virus FA tests detected seroconversion at the same time as HI and VN tests, however, in serially diluted sera, the FA test was positive to higher dilution. In studies with paired samples from calves from four farms with respiratory problems, the FA test gave similar results to PI-3 HI and RS virus VN tests. Large increases in antibody titre to RS virus detected by FA and VN tests indicated this was the problem on two of the farms. Individual animals showed large rises to PI-3 by FA and HI test on three farms. It is concluded that the FA test provides a rapid and sensitive alternative to the more conventional serological tests for respiratory viruses.     

鸡新城疫、传染性支气管炎、减蛋综合征、传染性脑脊髓炎四联灭活疫苗IB部分效力试验研究

采用《进口兽药质量标准》中传染性支气管炎灭活疫苗效力检验方法,对ND-IB-EDS76-AE四联灭活疫苗IB部分进行了效力检验。先用IBH120活疫苗作基础免疫,免疫后3～4周采血,然后用四联苗加强免疫,四联苗免疫后3～4周采血,将二次血清分别作IBHI试验。结果显示,IBH120活疫苗免后3～4周,其IBHI效价为3．6～4．610g2（1：12—1：24）,用四联苗加强免疫3～4周后,IBHI达6．2—8．610g2（1：74～1：388）,四联苗免后的IBm抗体滴度比免前提高了4．9～26倍,均大于3倍的标准。试验证明,该方法用于四联苗内IB部分效力检验是可行的。     

Detection of serum antibodies against Mycoplasma gallisepticum and Mycoplasma synoviae by a dot-immunobinding technique

Both Mycoplasma gallisepticum (MG) and M. synoviae (MS) antigens prepared for the routine haemagglutination inhibition (HI) test were diluted and absorbed to the separate pieces of durapore membrane for the measurement of dot-immunobinding (DIB) titers of test sera. Besides, durapore strips bearing both antigens were employed for a DIB test with chicken sera definitely diluted 100-fold. Shortening of reaction time of chicken sera with antigens as well as with the secondary serum markedly eliminated non-specific DIB reactions exhibited at low dilutions although the same condition was not so effective on the elimination of non-specific reactions among rabbit hyperimmune sera. Rapid and specific development of DIB antibody which continued at high titer up to 1:640 for 10 weeks postinoculation was proved in the sera of SPF chickens inoculated with MG or MS, while DIB titers of sera from uninoculated chickens remained 1:20 or lower. Non-specific reactions, which occurred in the routine serum plate agglutination test with a part of sera from the inoculated chickens, were not exhibited in the DIB as well as in the HI test with the same sera. Results of the DIB test with serum samples from 287 conventionally reared chickens definitely diluted 100-fold coincided with the results of HI test at a level of 90% with MG and 89% with MS antigen. This technique seems to be useful for a rapid, simple and specific diagnosis of avian mycoplasmosis.     

Development of enzyme-linked immunosorbent assay with nucleoprotein as antigen for detection of antibodies to avian influenza virus

During the avian influenza outbreak of 2003-04 in Southeast Asia, two avian influenza viruses (AIV), one of H5N1 subtype and the other H9N2 subtype, were isolated and identified from local farms. The nudeoprotein (NP) gene of the H5N1 AI isolate was cloned, and the segment encoding amino acid 47-384, which covers its major antigenic domains, was subcloned and expressed in E. coli. Subsequently, the NP (47-384) expression product was purified and used as the diagnostic antigen to develop a NP-based type-specific indirect enzyme-linked immunosorbent assay (ELISA) for detecting antibodies to AI from chicken sera. The ELISA is shown to be specific for AIV and does not cross-react with chicken sera that has antibodies to other avian viruses. The NP(47-384)-ELISA was compared with a hemagglutination inhibition test and a commercial AIV ELISA kit in evaluating 150 sera samples from experimentally AIV-infected or vaccinated specific-pathogen-free (SPF) chickens. Our NP(47-384)-ELISA was more sensitive than the two tests and showed an 82% agreement ratio with the HI test and an 80.67% agreement ratio with the commercial kit. The NP(47-384)-ELISA and the commercial AIV ELISA were used to evaluate 448 field sera samples from diseased chickens or vaccinated chickens during the 2003-04 AI outbreak in China. The two ELISA tests had a 95% agreement ratio. We conclude that the NP(47-384)-ELISA developed in our laboratory was specific and sensitive and it has great application potential in China's long-term prevention and control of AI.