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1.
衣原体单克隆抗体在牛羊血清诊断上的应用   总被引：1，自引：0，他引：1  
采用淋巴细胞亲交瘤技术，研制出的抗衣原体单克隆抗体，应用酶联免疫吸附试验（ＥＬＩＳＡ）方法，对本地区所采集的１２８份羊血清、９０份牛血清进行检测，结果，羊血清阳性检出率为２５．８％，牛血清阳性检出率为３２．２％，选择５０份羊血清、５０份牛血清用已建立的检测衣原体抗体ＥＬＩＳＡ与间接血凝试验（ＩＨＡ）进行比较，牛血清ＥＬＩＳＡ阳性检出率为３２．０％，ＩＨＡ为２４．０％，ＥＬＩＳＡ比ＩＨＡ试验高出８个百分点；羊血清ＥＬＩＳＡ阳性检出率为２６．０％，ＩＨＡ为２２．０％，ＥＬＩＳＡ比ＩＨＡ高出４个百分点。  相似文献   

2.
应用单抗间接ELISA检测兔出血症病毒的研究   总被引：2，自引：0，他引：2  
建立了用于检测脏器组织中兔出血症病毒（ＲＨＤＶ）抗原的单抗间接ＥＬＩＳＡ法，并与血凝试验（ＨＡＴ）和多克降双抗体夹心ＥＬＩＳＡ法进行比较，在被检的７８９份样品中，种方法检测结果皆为阴性的有５５１份，皆为阳性者１７３份；单抗间接ＥＬＩＳＡ和多克隆双体夹心ＥＬＩＳＡ均为阳性者３１份；公多克隆双抗体夹心ＥＬＩＳ为阳性者３１份；仅ＨＡＴ为阳性者２份；仅单抗间接ＥＬＩＳＡ为阳性者１份。实验结果证实，单抗间接  相似文献   

3.
最近,Ｓｔａｃｋ等用提取的羊布氏菌１６Ｍ菌株的显性抗原决定簇Ｍ的脂多糖作抗原,并将抗Ｍ抗原决定簇单克隆抗体与辣根过氧化物酶结合,建立了一种竞争ＥＬＩＳＡ（ｃＥＬＩＳＡ）,用于牛布病的诊断,该方法克服了补体结合试验（ＣＦＴ）的缺点。试验检测１４７份布氏菌培养阳性、６４０份培养阴性的样品,结果表明ｃＥＬＩＳＡ优于ＣＦＴ,其敏感性为９７．９%,特异性为１００%。检测保存１、２、３和４周的５０份血样,ｃＥＬＩＳＡ的阳性率分别为１４%、１５%、１５%和１５%,而ＣＦＴ却为１０%、２%、０和０。由此可见,ｃＥＬＩＳＡ检测结果…  相似文献   

4.
用醋酸缓冲液和４０％饱和硫酸铵边续沉淀法从绵羊棘球囊液（ＳＨＣＦ）中得到部分纯化抗原（ＳＰＰＣＦ），并从中分离了抗原Ａ和抗原Ｂ，分别用单克隆抗体反向间接血凝试验（Ｍ－ＲＰＨＡ）、单克隆抗体双扩散试验（Ｍ－ＤＤ）和酶联免疫吸附试验（ＥＬＩＳＡ）进行了鉴别，表明自制的三株单克隆抗体（ＭｃＡｂ）均识别ＳＨＣＦ、ＳＨＣＦ、ＳＰＰＣＦ及抗原Ｂ，而不识别抗原Ａ；用ＥＬＩＳＡ对阳、阴性血清检测，ＳＰＰＣＦ、抗原  相似文献   

5.
在内蒙古牙克石市图里河镇非马传贫注苗区，用琼脂扩散试验（ＩＤ）和酶联免疫吸附试验（ＥＬＩＳＡ）两种方法对６４匹马进行了对比检查，检出ＩＤ、ＥＬＩＳＡ双阳性４匹、ＤＬＩＳＡ单阳性马３匹。对３匹ＩＤ阴性，ＥＬＩＳＡ阳性马血清用不同浓度琼扩抗原重复检测结果为阴性，而用不同批次的ＥＬＩＳＡ抗原包被板及酶标抗体检测结果均为陧阳性，用马传盆病毒抗原对３匹单阳性马血清中和后，再进行ＥＬＩＳＡ测定，ＯＤ值均降低５  相似文献   

6.
蓝舌病ＶＰ７抗原包被板在－２０℃保存期为６个月,４℃保存１个月。抗原批内重复性试验ＣＶ＜１０％,批间重复性试验ＣＶ＜１０％。ＶＰ７－ＥＬＩＳＡ与ＡＧＩＤ对４２０份血清平行检测结果：ＶＰ７－ＥＬＩＳＡ较ＡＧＩＤ试验多检出了 １３份阳性样品, ＶＰ,－ＥＬＩＳＡ检出的阳性样品对ＡＧＩＤ阳性样品的覆盖率为９７．４％,对采自陕西等省１８１６份牛、羊血清样品进行检测,检出阳性样品数为２５７份阳性检出率为１４．２％。  相似文献   

7.
检测鸡慢性呼吸道病抗体ELISA方法的建立   总被引：12，自引：1，他引：11  
用败血支原体（ＭＧ）Ａ５９６９株制备ＥＬＩＳＡ抗原,与抗鸡ＩＧ单抗ＩＢ７酶结合物建立了检测鸡血清抗体水平的间接ＥＬＩＳＡ方法,交叉试验、阻断试验、重复性试验等表明该方法重复性好、特异性强、灵敏度高。确立了将鸡血清６４倍稀释监测ＥＬＩＳＡ效价（ＥＴ）的回收方程ｙ＝１．３８３＋０．２２４ｘ,可用于定量测定,血凝抑制试验（ＨＩ）与ＥＬＩＳＡ比较试验表明,ＥＬＩＳＡ法比ＨＩ试验敏感性高４倍以上。  相似文献   

8.
蓝舌病ＶＰ７抗原包被板在－２０℃保存期为６个月,４℃保存１个月。抗原批内重复性试验ＣＶ＜１０％,批间重复性试验ＣＶ＜１０％。ＶＰ７－ＥＬＩＳＡ与ＡＧＩＤ对４２０份血清平行检测结果：ＶＰ７－ＥＬＩＳＡ较ＡＧＩＤ试验多检出了１３份阳性样品,ＶＰ７－ＥＬＩＳＡ检出的阳性样品对ＡＧＩＤ阳性样品的覆盖率为９７．４％,对采自陕西等省１８１６份牛、羊血清样品进行检测,检出阳性样品数为２５７份,阳性检出率为１４．  相似文献   

9.
应用单克隆抗体建立夹心ELISA检测EDS—76病毒   总被引：3，自引：1，他引：2  
利用单克隆抗体建立夹心ＥＬＩＳＡ方法，对人工感染鸡带毒和排毒检测，表明第３天血、脾、输卵管带毒，第８天时各脏器几乎都带毒，第１４天仅输卵管、肾、肝检出病毒。第６天时鸡蛋带毒，泄殖腔排毒；直至１４天时仍带毒和排毒。夹心ＥＬＩＳＡ与ＨＡ、鸭胚接种相比较，在３６份样品中，ＨＡ检出１９份。ＥＬＩＳＡ检出２４份，鸭胚接种检出１２份。病毒在鸭胚和细胞上复制动态表明，２４小时后血凝试验和夹心ＥＬＩＳＡ都能检出病毒，随着时间延长，ＨＡ价和ＥＬＩＳＡ价（ＰＮ）继续上升，８４小时至９６小时到最高峰。在２４小时时，无血凝性，但ＥＬＩＳＡ检测为阳性，因此夹心ＥＬＩＳＡ可用于病毒检测。单抗夹心ＥＬＩＳＡ的建立为生产上提供了一种更简单、快速、灵敏、应用广泛的病毒检测方法。  相似文献   

10.
双抗夹心—ELISA诊断牛隐孢子虫病   总被引：15，自引：1，他引：14  
为了建立双抗体夹心—ＥＬＩＳＡ检测牛粪便中隐孢子虫卵囊抗原的方法。采用抗小球隐孢子虫（Ｃ．ｐａｒｖｕｍ）卵囊壁单克隆抗体,经对６０头份牛粪便样本分别进行抗酸染色和双抗夹心—ＥＬＩＳＡ检测,结果抗酸染色法检出１２头份有隐孢子虫卵囊,而ＥＬＩＳＡ除对抗酸染色阳性的１２份粪样判为阳性外,还对抗酸染色阴性的４份粪样判为阳性,且不与牛球虫、牛结肠小袋纤毛虫发生类属反应。此外,本试验在稀释液中加入ＥＤＴＡ,并增加了反应温度,使得试验在抗体包被板并封闭后３０ｍｉｎ结束整个检测过程。结果表明,双抗体夹心—ＥＬＩＳＡ是敏感性高、特异性强的诊断牛隐孢子虫病的方法。  相似文献   

11.
A monoclonal antibody (4H4) against the major internal protein, p24, of bovine leukemia virus (BLV) is described. It recognizes a sequence determinant on the p24-molecule and displays high affinity to its antigen. The monoclonal antibody 4H4 was applied in capture ELISA for diagnosis of enzootic bovine leukosis, using crude BLV-preparation as antigen. This test is more sensitive than the immunodiffusion test and at least as sensitive as direct ELISA.  相似文献   

12.
为建立评价O型口蹄疫病毒(FMDV)疫苗免疫水平的方法,本研究以单克隆抗体(MAb)3D9为捕获抗体,以HRP标记的MAb 8E8作为检测MAb,经过条件优化建立了基于MAb的检测O型FMDV抗体的固相竞争ELISA(SPCE)方法。对该方法进行了特异性、敏感性、重复性试验。结果显示,MAb 3D9的最佳稀释度为1:25 000,灭活O型FMDV抗原的最佳稀释浓度为1:3,HRP标记的MAb 8E8的最佳稀释度为15 000,当血清1:32稀释时,检测的临界值确定为45%。该方法分别检测A型FMDV抗体阳性参考血清以及牛冠状病毒、牛轮状病毒以及猪繁殖与呼吸障碍综合征病毒、猪圆环病毒、猪瘟病毒的标准阳性血清,检测结果均为阴性,未出现交叉反应。经检测,当阳性标准血清的抗体稀释度在1:512时,该方法仍具有较好的敏感性;批内和批间重复性试验的变异系数均小于10%,表明其重复性较好。并将该方法与液相阻断ELISA(LPBE)方法和病毒中和试验(VNT)的相关性进行了比较,结果显示该方法与LPBE和VNT的相关性分别为0.896和0.923。本研究为国内评价O型FMDV疫苗免疫水平建立了一种新的方法。  相似文献   

13.
苯乙醇胺A单克隆抗体的研制及ELISA检测方法的建立   总被引：1，自引：1，他引：0  
为制备苯乙醇胺A(phenylethanolamine A,PA)单克隆抗体,建立一种针对苯乙醇胺A的快速、简便、灵敏度高的检测方法,本试验采用重氮化法将制备的苯乙醇胺A衍生物分别与牛血清白蛋白(BSA)和卵清白蛋白(OVA)进行偶联作为免疫原和包被原。利用弗氏佐剂充分乳化免疫原(PA-BSA),按常规免疫程序免疫6～8周龄的雌性BALB/c小鼠,选择血清抗体效价较高的小鼠,取其脾细胞与SP2/0骨髓瘤细胞以PEG法进行细胞融合。结果显示,经3次细胞亚克隆筛选后,最终筛选出一株可稳定分泌抗苯乙醇胺A单克隆抗体的杂交瘤细胞株,命名为D6H8,利用此细胞以小鼠体内诱生法制备抗体。经鉴定,D6H8腹水抗体亚型为IgG1,轻链为κ链;纯化后的抗体与沙丁胺醇、盐酸克伦特罗、盐酸异丙肾上腺素和盐酸去氧肾上腺素均无明显的交叉反应(CR<0.18%),表明该抗体特异性良好。利用此抗体建立针对苯乙醇胺A药物残留检测的间接竞争ELISA方法,结果表明,腹水抗体效价为1∶12 800,猪肉中苯乙醇胺A添加浓度在5～1 000 ng/mL时线性关系良好,标准工作曲线为y=0.3861x-0.1845 (R^2=0.990),其IC50为58.88 ng/mL,LOD为3.83 ng/mL,回收率在85.96%～104.32%之间。综上所述,本试验成功建立了检测苯乙醇胺A药物残留的间接竞争ELISA方法,该方法灵敏度高、稳定性较好。  相似文献   

14.
Cells infected with bovine coronavirus (BCV) were solubilized with Triton X-100 to yield a cell lysate (CL) antigen having high hemagglutinating (HA) titers. The antigen gave high HA titers using rat erythrocytes, suggesting that it contained large amounts of hemagglutinin esterase (HE) antigen. The CL antigen, combined with an oil adjuvant, was tested for protective and antibody-inducing activities in cattle. Four groups (2 cattle/group) of cattle were inoculated with CL antigen having HA titers of 16 000, 4000, 1000, and 250. Another group served as untreated controls. Two intramuscular inoculations were given at an interval of 3 wk. The animals were challenged with virus 1 wk after the second inoculation. The groups immunized with the CL antigen having an HA titer of 4000 or 16 000 produced hemagglutination inhibition (HI) antibody titers of > 320 and serum neutralizing (SN) antibody titers of > 1280. These groups of animals showed no clinical abnormalities after challenge. In the groups immunized with CL antigen at an HA titer of 1000 or 250, HI antibody titers were 40 to 160 and SN titers were 80 to 640. The cattle with HI antibody titers of > or = 160 and the SN titers of > or = 640 showed no clinical signs, but the cattle with the HI antibody titer < 80 and the SN antibody titer < 160 developed watery diarrhea and fever after challenge. These results indicate that CL antigen with high HA titer induces antibody production in cattle that provides effective protection against winter dysentery.  相似文献   

15.
Two methods of enzyme-linked immunosorbent assay (ELISA) were developed for the diagnosis of canine coronavirus (CCV) infection in dogs. One ELISA, in which CCV-infected CRFK cell lysate is used as antigen, is for the detection and titration of antibody against CCV, and the other ELISA uses the double antibody sandwich method for the detection of CCV antigen. The first ELISA procedure demonstrated antibody responses in dogs inoculated with CCV, as did the virus neutralization test; the second ELISA detected specific CCV antigen in feces and organ homogenates of inoculated dogs.  相似文献   

16.
柱状黄杆菌双抗体夹心ELISA检测方法的建立   总被引：1，自引：1，他引：0  
The aim of this study was to develop a rapid method for the detection of Flavobacterium columnaris based on a double antibody sandwich ELISA （DAS-ELISA）. Purified monoclonal antibody against Flavobacterium columnaris was used as the capture antibody, while polyclonal antibody was used as the detection antibody. The optimal conditions for the ELISA were as follows: monoclonal antibody with 0.08 μg per well was added to coat overnight at 4 ℃; the plate was blocked by 30 g/L bovin serum albumin for 90 min at 37 ℃; incubation concentration of polyclonal antibody was 0.11 μg per well; the incubation time for detection antigen, polyclonal antibody and enzyme labeled antibody was 1 h at 37 ℃ for each; the value of D492 nm was obtained after 15 min coloration. Judging with P/N≥2.1 and D492 nm≥0.776 as positive criteria. This method had no cross reaction with Edwardsiella tarda, E. coli, Aeromonas hydrophila, Vibrio anguillarum, Vibrio alginolyticus, Vibrio parahaemolyticus and Vibrio harveyi. Its minimum detectable limit was 1×103 CFU. Therefore, this study provided a specific and sensitive detection method for Flavobacterium columnaris for the first time.  相似文献   

17.
Three serological assays were compared for detection of antibodies to bovine herpes-virus type 1. These were virus neutralization (VN), enhanced complement fixation (CF) and enzyme-linked immunosorbent assay (ELISA). The ELISA was developed using an infected cell lysate antigen and purified virus and was optimized in relation to antigen and antisera dilutions. The CF assay was enhanced by the addition of bovine complement. These 3 assays were compared for detection of: specific virus antibody titers; sero-conversions; early antibody response in experimentally-infected cattle. Both ELISA end-point titers and single dilution values were found to be more sensitive than the CF or VN assays for specific antibody level quantitation. With a single dilution ELISA test procedure a correlation was obtained between ELISA values and VN titers. Using the single dilution ELISA test the assay also detected antibodies in experimentally-infected cattle before either the VN or CF assays, and agreed with the VN test in 35/38 seroconversions found by 4-fold or more VN changes between acute and convalescent paired sera from naturally-infected animals. The single dilution ELISA was a rapid and sensitive test for routine antibody detection in bovine sera.  相似文献   

18.
为建立检测牛传染性鼻气管炎病毒(IBRV)血清抗体的阻断ELISA方法,本研究以经蔗糖密度梯度离心法纯化的IBRV作为免疫原制备1株单克隆抗体(MAb),命名为cp-1-1。经间接ELISA、IFA和western blot鉴定,该MAb与IBRV呈阳性反应,与牛病毒性腹泻病毒(BVDV)及牛副流感病毒3型(BPIV3)呈阴性反应,具有较强的特异性。质谱分析结果显示MAb cp-1-1识别的表位位于IBRV VP8蛋白。以纯化的IBRV作为包被抗原、MAb cp-1-1作为检测抗体,建立检测IBRV血清抗体的阻断ELISA方法。该检测方法的抗原包被量为0.89μg/孔,样品稀释度为12,检测抗体MAb量为1.3μg/孔,二抗稀释度为15000。利用50份IBRV抗体呈弱阳性的牛血清(中和抗体效价为14~116)作为标准参考血清,确定该检测方法的阻断率Cut Off值为52.06%,即阻断率高于52.06%时判为阳性,低于52.06%时判为阴性。阻断ELISA方法特异性试验显示仅IBRV阳性血清检测为阳性,而BVDV、BPIV3、牛腺病毒3型(BADV-3)和O型口蹄疫病毒(O-FMDV)阳性牛血清均检测为阴性,表明该方法具有较强的特异性;该方法可检测的最低中和抗体效价为14,与病毒中和试验的敏感性一致,表明该方法具有较高的敏感性;重复性试验显示该方法批内、批间变异系数均小于10%,显示较好的重复性。对130份现地牛血清检测结果显示,该方法与病毒中和试验的符合率为98.46%。用该方法对某牛场接种IBRV灭活疫苗的牛血清进行检测,抗体阳性率为99.51%(205/206)。另外,采用该方法对我国8个省(市、自治区)的801份牛血清进行检测,IBRV的抗体阳性率为41.6%(333/801)。本研究建立的阻断ELISA方法可以用于IBRV疫苗免疫监测和血清流行病学调查,为我国IBR的防控提供技术支持。  相似文献   

19.
Enzyme-linked immunosorbent assays (ELISA) for the detection of Fusobacterium necrophorum antibody in the sera of rabbits, cattle, and sheep were developed, using a ribosome-rich extract (RRE) from F necrophorum as the antigen. Test character, including optimal antigen dilution and substrate incubation periods, was established, using rabbit, bovine, and ovine antisera produced against RRE from isolates of F necrophorum. Rabbit antisera produced against 7 other species of bacteria were used to test the specificity of the F necrophorum RRE antigen. Cross-reactivity was not detected. Sera from 50 feedlot cattle were examined with the bovine ELISA. Of the 50 samples, 43 (88%) were positive for F necrophorum antibody. The ELISA developed in this study were sensitive and specific and appear to be readily adaptable to serologic investigations of F necrophorum.  相似文献   

20.
Summary

An enzyme‐linked immunosorbent assay (ELISA) is presented for the detection of the K99 antigen of Escherichia coli in calf faeces. False‐positive reactions were not observed with K99‐negative strains and with several viral antigens. Only bovine coronavirus caused slight positive reactions which could be eliminated by a blocking test. As compared with the conventional procedure for the detection of the K99 antigen, ELISA seemed to be at least as sensitive and had the advantage that samples could be stored at —20° C before testing. In addition many samples could be handled at the same time and the results became available quickly. By carrying out the assay as a blocking test, specific antibody against K99 in serum or colostrum could be detected and titrated.  相似文献   

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