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1.
A monoclonal antibody blocking ELISA was developed for the detection of antibodies directed against either EHV1 or EHV4. For this purpose, we selected a monoclonal antibody directed against a cross-reactive, conservative and immunodominant epitope of both EHV1 and EHV4. High antibody titres were found in rabbit antisera and SPF-foal antisera infected with either EHV1 or EHV4. After experimental challenge of conventional horses with EHV1 or EHV4 significant increases in CF and ELISA titres were found, whereas VN antibodies did not always increase significantly. In 344 paired serum samples submitted for diagnostic purposes a good agreement (kappa = 0.75, confidence limits = 0.63-0.88) was found between VN test and ELISA regarding a significant increase in titres. Also, a good correlation was found between VN and ELISA titres (r = 0.76, p<0.0005). The relative sensitivity and specificity of the Mab blocking ELISA as compared with the VN test were 99.9 and 71%, respectively. The rather low relative specificity of the ELISA may be explained by a relatively low sensitivity of the VN test. The ELISA also detected increases in titre after vaccination with an EHV1 subunit vaccine, and after primary field infections in weaned foals. We concluded that the Mab blocking ELISA is more sensitive, easier to perform, more rapid and more reproducible than the VN test. We consider this test as a valuable tool for serological diagnosis of both EHV1 and EHV4 infections. 相似文献
2.
以TTMV重组蛋白作为包被抗原,通过反应条件优化,建立了检测人TTMV血清抗体的间接ELISA方法,结果显示：抗原最佳包被浓度为1．63μg／mL,37℃孵育1h后4℃过夜;血清稀释度为1：320;酶标二抗孵育时间为37℃60min;ELISA酶标板的临界值为0．211。运用该方法对上海市和辽宁省共计280份血清样品进行检测,阳性检出率分别为34．68％和39．74％。检测结果表明,建立的间接ELISA方法特异性与重复性均较好,为进一步完善TTMV临床诊断方法打下了基础。 相似文献
3.
为建立以重组PCV2Cap蛋白为包被抗原的间接ELISA检测方法,构建了猪圆环病毒2型（PCV2）ORF2基因原核表达质粒pET28a-ORF2。SDS-PAGE显示,在0.1mmol/L IPTG和37℃条件下诱导4h,重组Cap蛋白高效表达。Western blotting证实该蛋白能够被PCV2阳性血清特异性识别。以纯化的蛋白为抗原建立了检测PCV2抗体的间接ELISA方法。结果表明,抗原最适包被质量浓度为2mg/L;血清最佳稀释度为1∶100;酶标二抗最适浓度为1∶2 000,该方法的敏感性为86.96%,特异性为100%。用该方法对河南省152份猪血清样品进行检测,与间接免疫荧光（IFA）的符合率为85.53%（130/152）,与商品化的韩国金诺PCV2ELISA试剂盒的符合率为88.16%（134/152）。本试验成功建立了PCV2血清抗体间接ELISA方法,具有较高的敏感性和特异性,可用于大规模的血清学检测。 相似文献
4.
Hartley CA Wilks CR Studdert MJ Gilkerson JR 《American journal of veterinary research》2005,66(5):921-928
OBJECTIVE: To compare methods of detecting equine herpesvirus type 1 (EHV1)- and EHV4-specific antibodies in horse sera. SAMPLE POPULATION: 33 acute and convalescent serum samples from experimentally or naturally infected horses after confirmed EHV1 or EHV4 infection. PROCEDURE: For each sample, serum antibody titers against EHV1 and EHV4 were determined by use of virus neutralization (VN) and complement fixation (CF) assays. The ELISA absorbance values for each serum sample were determined against the EHV1 and EHV4 recombinant ELISA antigens. Values obtained for acute and convalescent sera in each assay were compared. RESULTS: Following experimental infection of foals, EHV1 or EHV4 antibodies that were specific for the inoculating virus were detected only by use of the ELISA. Results of VN and CF assays indicated that the foals seroconverted to EHV1 and EHV4 following infection with EHV4 only. After EHV1-induced abortion, myeloencephalitis, or respiratory tract disease, the VN and CF assay results revealed seroconversion to EHV1 and EHV4, whereas results of the ELISA revealed seroconversion to EHV1 only. Similarly, after confirmed EHV4-induced respiratory tract disease, increases in EHV4-specific antibodies were detected only by use of the ELISA with no indication of an increase in EHV1 antibodies. The CF and, to a lesser degree, VN assays revealed that seroconversion to EHV1 and EHV4 occurred between the time of obtaining acute and convalescent serum samples. CONCLUSIONS AND CLINICAL RELEVANCE: The EHV1/EHV4 type-specific antibody ELISA clearly identifies horses that have been infected with EHV1 or EHV4 by use of acute and convalescent sera. Results of VN and CF assays indicate that cross-reactive antibodies greatly limit their use. 相似文献
5.
褪黑素（MT）广泛存在于哺乳动物体内,是一种常见的吲哚类物质,可起到清除自由基、调节免疫、调节神经等作用。本文使用褪黑素-OVA作为免疫原,免疫5 只BALB/c小鼠,5 次免疫后采血检测,通过间接ELISA方法确定抗血清针对褪黑素-OVA的滴度。经ELISA筛选,最终获得5 株可稳定表达褪黑素抗体的单克隆细胞株（3G3B2、3G3C7、3G3D1、3G3F4、3G3G3）。纯化后抗体浓度为0.28 mg/mL,经测定,褪黑素单克隆抗体滴度可达1∶64 000,为进一步开发褪黑素的快速检测方法奠定了材料基础。 相似文献
6.
试验旨在制备抗阪崎肠杆菌的单克隆抗体,初步建立其ELISA检测方法。以灭活的阪崎肠杆菌全菌体为抗原免疫BALB/c小鼠,筛选血清效价高的小鼠脾细胞与SP2/0骨髓瘤细胞进行细胞融合,制备杂交瘤细胞,并用间接ELISA法选取阳性杂交瘤细胞,扩大培养后测定单克隆抗体的效价,进行特异性及抗体间的配对,使用mAb亚类检测试剂盒鉴定单克隆抗体的亚型,并利用得到的抗体建立双抗体夹心ELISA检测方法。本试验得到3株具有良好特异性能稳定分泌单克隆抗体的阳性细胞株5C10、2B6和Ab02,经两两配对,据阳性D450 nm值及P/N值选择1：20000稀释的5C10作为包被抗体,1：40000稀释的Ab02作为酶标二抗,建立ELISA检测法,应用建立的方法与荧光定量PCR方法检测动物实验室保存的20份进出口送检奶粉样品,结果显示试验结果一致。本试验成功制备阪崎肠杆菌的单克隆抗体并建立其ELISA检测法,为大批量快速检测阪崎杆菌奠定了基础。 相似文献
7.
以大肠杆菌表达、纯化的禽白血病病毒（ALV）重组P27蛋白为包被抗原,建立了检测ALV-P27抗体的间接ELISA方法,为禽白血病流行病学调查提供了一种简便的血清学检测方法.该方法与9种禽常见传染病病毒阳性血清及大肠杆菌阳性血清无交叉反应,具有较好的特异性;批内、批间重复试验变异系数均小于10％,具有良好的重复性;新建立的ELISA方法比IDEXX公司生产的ALVA、B亚群抗体检测试剂盒和J亚群抗体检测试剂盒相对于免疫荧光试验（IFA）有更高的符合率. 相似文献
8.
利用本实验室建立的昆虫细胞/杆状病毒表达系统表达犬细小病毒病毒样颗粒（CPV-VLPs）,采用硫酸铵沉淀、蔗糖密度梯度离心对表达的CPV-VLPs进行纯化,用电子显微镜、SDS-PAGE及Western-blotting方法检测纯化效果。以纯化的CPV-VLPs作为包被抗原建立CPV间接ELISA检测方法,对各反应条件进行优化并分析其特异性、敏感性、重复性。结果显示,CPV-VLPs经过纯化后纯度可达到95%以上;优化的ELISA最佳工作条件为：纯化抗原包被浓度为5.0mg/L,4℃包被过夜;1%BSA,37℃封闭2h;待检血清1∶40稀释,37℃孵育1.5h;HRP标记的酶标二抗1∶20 000稀释,37℃孵育1h;TMB室温避光显色30min;确定的血清阴性阳性临界值D450nm值为0.264。该方法可特异性检测犬细小病毒阳性血清,与犬瘟热、犬传染性肝炎、犬冠状病毒病、狂犬病阳性血清均不发生反应。该方法的敏感性为1∶640,批内重复试验变异系数小于6%,批间重复试验变异系数小于8%。42份临床血清样本的检测结果表明,与血凝抑制试验的符合率为90.48%。 相似文献
9.
牛γ-干扰素ELISA检测方法的建立与初步应用 总被引：1，自引：0，他引：1
以纯化的rHis-BoIFN-γ制备兔多抗血清,辛酸—硫酸铵两步法对体内诱生的抗rBoIFN-γ单抗腹水进行纯化,用美洲商陆素（Pokeweed mitogen,PWM）刺激奶牛全血产生的分泌性天然BoIFN-γ筛选出与之反应最佳的单抗5E11。将单抗5E11以40μg/mL浓度包被,与1∶3 000倍稀释的兔抗rHis-BoIFN-γ多抗血清（13.8μg/mL）配对,以1∶6 000稀释的商品化酶标羊抗兔IgG为指示抗体,建立了BoIFN-γ抗原捕获ELISA检测方法,该方法可以检出2.56 U/100μL（30.5 pg/100μL）的rHis-BoIFN-γ、8U/100μL的rBac-BoIFN-γ和1U/100μL的分泌性天然BoIFN-γ。以商品化试剂盒作为平行对照,将获得的奶牛临床检测血浆样品使用BoIFN-γ抗原捕获ELISA法进行检测,结果显示两种方法检测符合率达到83.9%。本研究建立的BoIFN-γ抗原捕获ELISA检测方法可有效检测分泌性IFN-γ,为进一步开发BoIFN-γELISA检测试剂盒奠定了基础。 相似文献
10.
将从水貂中分离的犬瘟热病毒（Canine distemper virus,CDV）HB株经培养和纯化后免疫小鼠,制备分泌抗CDV-HB的杂交瘤细胞株,分别命名为2E1和7F3,ELISA测定腹水效价均大于107。亚类鉴定结果表明单抗2E1的分子亚类为IgM,7F3的分子亚类为IgG1,间接免疫荧光试验表明,这两株单抗均可以与CDV-HB特异性结合。采用柠檬酸三钠还原法制备胶体金颗粒并标记2E1单克隆抗体,获得检测貂犬瘟热病毒抗原的胶体金免疫层析试纸,且鉴定证明制备的检测CDV的胶体金试纸条具有良好的的特异性。 相似文献