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为了解江西省猪群猪传染性胸膜肺炎放线杆菌(APP)感染现状,本试验利用APP江西分离株研制了抗体间接血凝检测法(IHA)。试验将纯化的APP江西分离株培养菌液反复冻融,经超声波裂解处理后提取菌体抗原,致敏经戊二醛固定鞣酸化的兔红细胞,成功建立了APP抗体的IHA法。应用该法对猪瘟病毒、猪繁殖与呼吸综合征病毒、猪伪狂犬病病毒等病毒的阳性血清进行检测试验,结果均为阴性;对大肠杆菌、沙门氏菌、副猪嗜血杆菌阳性血清检测均为阴性。不同批次APP抗原经IHA检测,结果显示重复性好。本试验结果表明所建立的APP抗体IHA检测法有较好的特异性和重复性,为下一步血清学调查奠定基础。 相似文献
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为了建立一种快速、敏感、特异的检测犬冠状病毒(CCV)的方法,利用感染CCV的犬肾传代细胞包被酶联反应板,以辣根过氧化物酶标记的金黄色葡萄球菌A蛋白(HRP-SPA)作为第2抗体,建立了检测CCV抗体的间接ELISA方法。抗原的包被量为每孔3×104个染毒细胞,酶标抗体的工作浓度为1∶40。试验发现,包被的病毒抗原经甲醇固定30min后可以提高试验的敏感性,减少病毒抗原的使用量。与中和试验比较证实,2种抗体检测方法的测定结果呈正相关 相似文献
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间接ELISA检测鸭疫里默氏杆菌血清抗体方法的建立 总被引:2,自引:2,他引:0
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。 相似文献
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应用间接ELISA检测动物隐孢子虫病抗体 总被引:4,自引:0,他引:4
分别应用蔗糖和氯化铯密度梯度离心法纯化小型隐孢子虫( Cryp tosp oridium p arvum )卵囊,获得高纯度的抗原,建立了用间接 E L I S A 检测动物隐孢子虫病抗体的方法。抗原最适包被质量浓度是 5.0 m g/ L,待检血清最佳稀释度是 1∶100,明胶封闭体积分数和作用时间分别为 1% 和 60m in。试验证明,该法具有快速、简便、特异和重复性好等优点。 相似文献
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用东毕吸虫成虫,经葡聚糖凝胶G—100层析柱制备纯化抗原,应用ELISA间接法检测自然感染东毕吸虫绵羊的抗体,同时,配合沉淀法、孵化法和全身剖检法进行检查,显示阳性符合率为98.4%,阴性符合率为95%;除与肝片吸虫阳性血清有4.8%的交叉反应外,未发现与矛形双腔吸虫阳性血清和前后盘吸虫阳性血清有交叉反应;ELISA的阳性检出率高于沉淀法和孵化法;并证明东毕吸虫感染强度与血清抗体无相关关系。 相似文献
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ELISA间接法检测兔血清中抗牛血清白蛋白抗体 总被引:1,自引:0,他引:1
用牛血清白蛋白(BSA)免疫普通级试验兔(新西兰白兔),获得高滴度的抗BSA血清。以系列含量的BSA溶液绘制标准曲线,测定兔血清中抗BSA抗体效价。结果表明:普通级试验兔BSA4次免疫后,兔血清中抗BSA抗体效价为1:9000。ELISA间接法敏感度高,优化了酶免疫反应条件,标准曲线线性范围内r=0.9965,可初步用于抗BSA抗体含量检测。 相似文献
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间接法Dot-ELISA检测猪流行性腹泻抗体的研究 总被引:5,自引:0,他引:5
使用非洲绿猴肾(Vero)细胞增殖适应传代细胞培养的猪流行性腹泻病毒(PEDV),并经聚乙二醇(PEG)沉淀法分离纯化PEDV抗原,建立斑点酶联免疫吸附试验(Dot-ELISA)检测猪流行性腹泻(PED)抗体。在最适工作条件下,进行了敏感性和特异性试验,结果表明,该法检测PEDV抗体,敏感、特异、重复性好,且方便、快捷,适用于大批量试样的检测,可作为一种诊断PED的比较理想的方法。采用此方法分别对来自加拿大、台湾省进口的种猪和海南省和广东省内种猪群的血清样共834份进行了检测,检测得PEDV抗体阳性率达21%。 相似文献
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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。 相似文献
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The Development and Application of the Latex Agglutination Test to Detect Serum Antibodies against Japanese Encephalitis Virus 总被引:1,自引:0,他引:1
Xinglin J Huanchun C Qigai H Xiang W Bin W Dexin Q Liurong F 《Veterinary research communications》2002,26(6):495-503
The attenuated SA14-14-2 strain of Japanese encephalitis virus (JEV) was cultured in BHK-21 cells. The viral supernatant was purified and concentrated with PEG (MW 20 000). A suitable concentration of JEV antigen was used to sensitize latex to prepare the latex antigen. The specificity, sensitivity and stability of the antigen were assessed. A latex agglutination test (LAT) was developed for rapidly detecting antibody against JEV infection. The LAT and haemagglutination inhibition (HI) assay were compared by simultaneously testing 35 porcine serum samples from five farms. Ninety per cent (20/23) of the samples were seropositive by both assays. No significant difference was found between the two methods (p > 0.05). Furthermore, when 1613 porcine sera from 120 farms were tested by LAT, the number of positive sera was 652, while that of negative sera was 961, ranging from 20% to 50% positive throughout the year. These results indicate that LAT is an appropriate candidate method for epidemiological surveys for and diagnosis of Japanese encephalitis. 相似文献
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禽白血病病毒(Avian leukosis virus,ALV)是禽白血病的病原,可引起鸡的免疫抑制和肿瘤。净化种鸡群是控制ALV的主要方法之一。本研究将ALV的p27基因克隆到表达载体pGEX-6P-1,在大肠杆菌中获得了高效表达,以纯化后的p27-GST融合蛋白为抗原包被,经过条件优化,建立了检测鸡血清中抗ALV抗体的间接ELISA方法。与IFA检测结果比较,该方法比IDEXX ELISA试剂盒有更高的符合率。可用于禽白血病病毒感染根除的大规模检测,并具有低成本、易操作的特点,能同时检测到针对ALV所有亚群的抗体。 相似文献
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Britt-Louise Ljungstrm Anna Lundn Johan Hglund Gran Zakrisson 《Acta veterinaria Scandinavica》1994,35(2):213
The protozoan parasite Toxoplasma gondii is the causative agent of the zoonosis toxoplasmosis. In sheep and goats, it is one of the most prevalent causes of infectious abortion. Also in pregnant women, a primary infection can result in miscarriage. Humans acquire the infection either by ingestion of oocysts excreted by cats, the definitive host of the parasite, or by eating raw or undercooked meat from latently infected animals (Dubey & Beattie 1988). In Sweden, toxoplasmosis is a notifiable disease, and cases of clinical disease in humans as well as animals must be reported. In both veterinary and human medicine serological assays based on detecting the humoral antibody response of the host against the parasite are used as diagnostic tools. So far, solid phase assays, such as the indirect fluorescent antibody test (IFAT) and the enzyme-linked immunosorbent assay (ELISA), have been widely used to diagnose T. gondii infection in many species including cats, pigs and sheep (Dubey & Beattie 1988). However, both IFAT and ELISA require appropriate anti-species specific immunoglobulins (Ig) that must be carefully evaluated for each species prior to use. This makes these assays complicated and time consuming. Consequently, alternative, simpler methods that do not require specific antisera would be of great value. The direct agglutination test (DA), which is based on the principle that formalin-treated organisms agglutinate in the presence of specific IgG antibodies, is such an assay (Fulton & Turk 1959). The DA-test is widely used in human medicine as a screening test for T gondii infection but it has not yet been thoroughly evaluated for use in veterinary medicine (Uggla & Buxton 1990). 相似文献
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用ELISA检测猪细小病毒(PPV)血清抗体 总被引:7,自引:0,他引:7
采用差速离心、透析、聚乙二醇(PEG)浓缩方法纯化猪细小病毒(PPVDK7113)制备抗原。用纯化的PPV抗原包被聚苯乙烯微量反应板,建立了检测PPV血清抗体的间接ELISA。抗原最佳包被浓度为6.3μg/ml,被检血清最佳稀释度为1:200。用建立的ELISA检测山东、东北、广东等地的426份血清,结果阳性率为36.2%。与华中农大病毒室提供的猪细小病毒乳胶凝集试验试剂盒相比,其结果符合率为98.6%。通过阻断试验、交叉试验,说明本方法在特异性上达到了较为满意的结果,且易于操作,适用于血清学定性诊断、检疫和大规模的血清流行病学调查。 相似文献
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羊细粒棘球蚴病抗体间接ELISA检测方法的建立 总被引:1,自引:0,他引:1
本研究旨在通过建立检测羊细粒棘球蚴病抗体的间接酶联免疫吸附试验(ELISA)方法,为羊细粒棘球蚴病的检测提供快速、简便的手段.作者利用DNAStar软件对GenBank上发表的细粒棘球蚴EG95蛋白的氨基酸序列进行分析,筛选出高度亲水的优势表位区EG95s,对该区域编码基因进行克隆并表达.以纯化的重组融合蛋白为包被抗原,按常规方法建立检测羊细粒棘球蚴病抗体的间接ELISA,并对各种条件进行优化.SDS-PAGE结果表明成功获得了可溶性好、表达效率高、纯化简便的重组融合蛋白GST-1EG95s和HIS-1EG95s,Western blot检测结果表明表达产物具有较好的反应原性.间接ELISA方法优化结果显示:HIS-1EG95s作为包被抗原效果优于GST-1EG95s.经统计学分析,确定间接ELISA方法的判定标准:OD450nm值≥0.235时判定为阳性,OD450nm值≤0.191时判定为阴性,介于二者之间则为可疑.分别对采自新疆的70份羊包虫阳性血清和70份阴性血清进行检测,结果表明该方法与新西兰Wallaceville动物研究中心提供的间接ELISA方法符合率为100%,阻断试验结果显示与其他蛋白无交叉反应,批内变异系数介于3.8%~5.6%,批间变异系数介于5.7%~8.5%,结果表明该方法特异性强、敏感性高、重复性好.作者所建立的羊细粒棘球蚴病抗体的间接ELISA检测方法有望为羊细粒棘球蚴病的检测提供快速、简便的手段. 相似文献
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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。 相似文献
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Comparison of Different Commercial Serological Tests for the Detection of Toxoplasma gondii Antibodies in Serum of Naturally Exposed Pigs
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R. Steinparzer K. Reisp B. Grünberger J. Köfer F. Schmoll T. Sattler 《Zoonoses and public health》2015,62(2):119-124
Toxoplasma gondii is the aetiological agent of the zoonotic disease toxoplasmosis and transmitted among other ways by chemically and physically untreated, that is, raw pork to humans. The detection of Toxoplasma gondii is impossible by currently practiced meat inspection, but serological tests can be used to detect Toxoplasma gondii antibodies in pig herds and can consequently be helpful to identify potentially contaminated pork. Therefore, appropriate serological tests are required. In this study, serum samples of 1368 naturally exposed slaughter pigs from 73 Austrian farms were collected. Serum samples of at least 16 slaughter pigs per farm were tested. The prevalence of Toxoplasma gondii antibodies in serum was measured by a commercial available modified agglutination test (MAT) and compared to three different commercial available enzyme‐linked immunosorbent assays (ELISA). The MAT detected 6.5%, ELISA I 6.7%, ELISA II 4.8% and ELISA III 4.3% of the pigs as Toxoplasma gondii antibody positive. The agreement, according to the kappa coefficient (κ), was substantial between the MAT and ELISA I (κ = 0.62), II (κ = 0.64) and III (κ = 0.67). A better agreement was determined between ELISA I and II (κ = 0.715), ELISA I and III (κ = 0.747) and ELISA II and III (κ = 0.865). At least one pig per farm was detected Toxoplasma gondii antibody positive in 17 (23.3%) farms by the MAT, 26 (35.6%) farms by ELISA I, 16 (21.9%) farms by ELISA II and 11 (15.1%) farms by ELISA III. Pig farms with a high number of Toxoplasma gondii antibody‐positive pigs or high antibody titres were identified by all of the four used serological tests. Concerning the occurrence of Toxoplasma gondii antibodies in Austrian pig farms, a monitoring and surveillance programme would be reasonable to find high‐risk farms. 相似文献