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1.
采用辛酸-硫酸铵法提取兔抗牛病毒性腹泻-黏膜病毒(BVDV)高免血清中的免疫球蛋白IgG,辣根过氧化物酶标记提纯后IgG,建立了检测BVDV抗原的双抗体夹心斑点酶联免疫吸附试验(Dot-ELISA)法。结果显示,抗体最佳包被量为300μg/mL,酶标记抗体的最适浓度为1∶50倍稀释,以5%牛血清作为封闭液、封闭45min效果最佳,抗原最小检出量是1.34μg/mL。应用建立的检测方法对河北省内78份腹泻奶牛血样进行了检测,阳性检出率为57.69%;经卡方检验分析,建立的Dot-ELISA与琼脂扩散(AGP)法相比较,阳性检出率差异显著。试验证明,该方法具有简便快速、特异性强、重复性高的优点,适合于基层兽医的检测诊断。 相似文献
2.
为了制备兔抗鸽IgG抗体-HRP,建立斑点酶联免疫吸附试验定性检测鸽毛滴虫抗原的检测方法,将纯化的鸽IgG加入佐剂免疫兔子,获取纯化的兔抗鸽IgG抗体;采用简易过碘酸钠法标记兔抗鸽IgG,获取兔抗鸽IgG抗体-HRP;同时将鸽毛滴虫用超声波粉碎加入佐剂后多次免疫健康鸽获取鸽抗鸽毛滴虫高免血清(一抗);然后在2×106个/mL的鸽毛滴虫抗原量下,用不同浓度的一抗抗体、兔抗鸽IgG抗体-HRP进行试验;并对70个临床样品进行检测。结果显示,在2×106个/mL的鸽毛滴虫抗原量下,一抗与兔抗鸽IgG-HRP最佳工作浓度分别为1∶100和1∶500;70个样品中,48个镜检为阳性的样品用Dot-ELISA检测有44个呈阳性,阳性符合率为91.7%;22个镜检为阴性的样品用Dot-ELISA检测有12个呈阴性,阴性符合率为54.5%;Dot-ELISA检测与镜检结果的总符合率为80%。结果表明,本试验成功地建立了鸽毛滴虫感染的Dot-ELISA检测方法。 相似文献
3.
本文利用提纯的副结核分枝杆菌胞浆特异性抗原,建立了检测牛副结核抗体的Dot-ELISA方法。用该方法对粪便培养阳杜的32头份牛副结核病血清检测,检出27头,阳性检出率为84.4％,其敏感性与ELISA相似。与10头OT变态反应阳性牛血清检测,无交叉反应。M.phlci.M.fortuitum.M.kansasii人工高免血清经两次用M.phlci悬液吸收,用建立的Dot-ELISA方法也无交叉反应。表明设立的Dot-ELISA具良好的敏感性特异性。 相似文献
4.
为建立一种快速的兔病毒性出血症病毒抗体检测方法,本研究以原核表达的重组VP60蛋白作为诊断抗原,建立了检测兔病毒性出血症病毒抗体的VP60-ELISA诊断方法。该方法检测3种常见兔病(兔轮状病毒、仙台病毒和魏氏梭菌)的阳性血清均为阴性;检测灵敏度为1:12800;批内、批间重复性试验的变异系数分别小于4.9%和6.9%。本研究建立的RHDV VP60-ELISA检测方法具有良好的特异性、敏感性和重复性,为RHDV的抗体检测及流行病学调查等快速诊断提供了一种技术手段。 相似文献
5.
以原头节可溶性粗抗原经Sephadex G-200层析纯化抗原为包被抗原，兔抗羊IgG-HRP结合物为显色剂，建立检测羊脑多头蚴病血清抗体的Dot-ELISA，并以ELISA作平行对照。结果，粗抗原和层析纯化抗原检测86头份羊脑多头蚴病阳性血清，其敏感性分别为94．18％和93．02％，两种抗原的敏感性无显著差异；检测122头份绦虫蚴病阴性血清，18头份棘球蚴病阳性血清，35头份细颈囊尾蚴病阳性血清，其特异性分别为90．29％和95．43％。两种抗原的特异性差异显著。Dot-ELISA和ELISA两种方法的符合率为100％。层析纯化抗原比粗抗原的特异性有了明显提高，而敏感性没有降低。层析纯化抗原和操作术式都具有良好的重复性，Dot-ELISA和ELISA对比试验结果相近，且具简便、快速及不需要复杂设备等优点，是一种检测羊脑多头蚴病血清抗体的理想方法。 相似文献
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7.
以琼脂糖免疫扩散法检测实验感染葡萄球菌的免,其血清沉淀抗体在感染后13～41天阳转;自然流行群中的兔血清,阳性率为41.5％,群养兔为17.40％,散养兔为10.20％。兔巴氏菌抗原、波氏菌抗原、链球菌抗原对葡萄球菌感染兔血清沉淀抗体不发生交叉夏应。葡萄球菌抗原对巴氏菌高免兔血清、巴氏菌抗体阳性兔血清、波氏菌阳性兔血清及健康兔血清不发生反应。 相似文献
8.
Dot-ELISA检测H9亚型禽流感病毒的研究 总被引：1，自引：0，他引：1
本研究采用兔抗H9亚型禽流感病毒（AIV）IgG包被于硝酸纤维素膜,酶标羊抗兔IgG作二抗,建立检测H9亚型AIV的Dot-ELISA法。经方阵试验确定兔抗AIV IgG工作浓度为1∶400,酶标羊抗兔IgG的工作浓度为1∶400。作者建立的Dot-ELISA对AIV的最小检测量为3.35×10-9g。Dot-ELISA与HA和HI、AGP及病毒分离法相比,检测63份临床疑似H9亚型AIV病料,Dot-ELISA检出32份(57.14%),HA和HI检出15份(23.81%),AGP检出11份(17.46%),病毒分离检出38份(60.30%)。用抗H9亚型AIV阳性血清可以阻断Dot-ELISA阳性反应,诊断膜片与鸡新城疫病毒、鸡传染性法氏囊病病毒、鸡传染性支气管炎病毒、产蛋下降综合征病毒不出现阳性反应,证明Dot-ELISA特异性好。分别置室温(25 ℃左右)、4 ℃和-20 ℃下保存1个月后,膜片诊断效果不变,对照反应均成立,该方法重复性好（重复符合率为93.9%）,操作简便（3 h内可完成）,不需要特殊检测仪器,结果客观,肉眼易于判断,是微生物和传染病及寄生虫病诊断标准化的新技术之一。 相似文献
9.
《中国预防兽医学报》2018,(10)
为快速检测猪传染性胃肠炎病毒(TGEV)抗体,本研究以原核表达系统串联表达的含有TGEV S蛋白抗原表位C的重组蛋白r-(C_1C_2)6为包被抗原建立TGEV血清抗体的Dot-ELISA检测方法。经反应条件优化结果显示,抗原最适包被量为62.5 ng/点;血清最佳工作浓度和工作时间分别为1∶80和45 min;羊抗猪IgG-HRP最佳工作浓度和工作时间分别为1∶800和45 min;最适封闭条件分别为5%脱脂乳37℃,45 min;血清和酶标抗体最适反应温度均为37℃;最佳显色时间7.5 min。结果显示该方法与猪轮状病毒和猪流行性腹泻病毒阳性血清无交叉反应,表明其具有良好的特异性。该方法对TGEV阳性血清最低检测限为1∶1 280。采用建立的Dot-ELISA对27份临床猪血清样品进行检测,结果显示Dot-ELISA与TGEV/PRCV抗体检测试剂盒检测结果的符合率为92.6%。本研究建立的Dot-ELISA检测方法简便、快速、灵敏、特异,适合用于TGE快速诊断。 相似文献
10.
Dot-PPA-ELISA检测仔猪副伤寒血清抗体方法的研究 总被引：1，自引：0，他引：1
以沙门氏菌的改良H.E.抗原为膜载抗原,建立了检测仔猪副伤寒血清抗体的Dot-PPA-ELISA方法.研究选取了特异性良好的抗原,确定了Dot-PPA-ELISA的最佳工作条件,初步确定了感染仔猪副伤寒的Dot-PPA-ELISA抗体阳性标准为1:32.本研究制备的诊断膜片特异性强、敏感性高,能够检测到1:2 048稀释的动物试验阳性血清;该方法操作简便快捷、结果客观、易于判定、各种试剂材料均可做到标准化,适合规模化养猪场仔猪副伤寒的抗体监测以及流行病学调查之用. 相似文献
11.
以醋酸纤维素膜作为固相载体，辣根过氧化物酶标记鸡抗传染性法氏囊病病毒抗体（ＩＢＤ—ＩｇＧ），饱和二氨基联苯胺为底物显色，建立了鸡传染性法氏囊病双抗体夹心Ｄｏｔ—ＥＬＩＳＡ诊断法。经方阵实验确定最佳反应条件为：ＩｇＧ的包被液为０．０５ｍｏｌ／Ｌ（ｐＨ９．６）碳酸盐缓冲液，包被浓度为１：５０；酶标抗体的工作浓度为１：１００；洗涤液为含０．０５％吐温—８０的０．０２ｍｏｌ／Ｌ（ｐＨ７．４）磷酸盐缓冲液；封闭液为含０．２％明胶的洗涤液；封闭时间、抗原及酶标抗体的作用时间均为３７℃３０ｍｉｎ。应用本方法和琼扩试验同步检测２０份已知阳性病料、１２０份待检病料和胚毒尿囊液、１０份正常鸡样品，结果表明，Ｄｏｔ—ＥＬＩＳＡ阳性率为９０％，而琼扩试验为４０％；凡琼扩试验阳性者，Ｄｏｔ—ＥＬＩＳＡ均呈强阳性，而在Ｄｏｔ—ＥＬＩＳＡ阳性样品中，只有４４％呈琼扩试验阳性，Ｄｏｔ—ＥＬＩＳＡ的敏感度为琼扩试验的１００倍。 相似文献
12.
Gürtürk K Ekin IH Aksakal A Solmaz H 《Journal of veterinary medicine. B, Infectious diseases and veterinary public health》2002,49(3):146-151
In this study, a dot-enzyme-linked immunosorbent assay (Dot-ELISA) was evaluated in comparison with a complement fixation test (CFT) for the detection of Campylobacter antibodies in sheep sera. Acid glycine extracts (AGE) of both Campylobacter fetus ssp. fetus and Campylobacter jejuni strains that had been isolated from the gall-bladder of slaughtered sheep was used as antigen in both tests. A total of 153 sheep sera from aborted (74) and slaughtered (79) sheep were examined by both Dot-ELISA and CFT. Twenty-two sera showed anti-complementary activity were not suitable for CFT. Of the 22 sera showing anti-complementary activity, two sera were found to be positive in Dot-ELISA. Eighty-eight (67.2%) of the remaining 131 sera were negative by both Dot-ELISA and CFT using AGE of both Campylobacter strains whereas 43 sera (32.8%) gave different reaction patterns in Dot-ELISA and CFT with the extracts of both Campylobacter strains. Twelve sera were positive by both tests using AGE of C. fetus ssp. fetus but CFT failed to detect antibodies in nine of these sera when AGE of C. jejuni was used. Twelve sera were positive by both tests only when AGE of C. fetus ssp. fetus was used. Eleven sera were positive only by CFT. Seven of these reacted only with the AGE of C. fetus ssp. fetus and four sera were positive by using AGE of both Campylobacter strains. The remaining eight sera were found to be positive only by dot-immunobinding assay either with the AGE of both Campylobacter strains or with the AGE of one of the Campylobacter strains. It is concluded that Dot-ELISA using AGE from C. fetus ssp. fetus could be employed for the detection of Campylobacter antibodies in sheep sera and the additional use of AGE from C. jejuni as antigen appeared not to be profitable for this purpose. 相似文献
13.
本研究旨在建立一种适用于快速、现场检测要求的庆大霉素残留Dot-ELISA检测方法。将硝酸纤维素膜用激光切割成小圆片,粘贴到激光打孔的塑料胶条上,制成8个圆片组成的NC膜检测条。通过条件优化,建立了基于NC膜条的庆大霉素残留直接竞争Dot-ELISA检测方法。结果表明,该方法可以快速检测牛奶等食品中的庆大霉素残留,检测灵敏度达到60 ng/mL,可用于庆大霉素残留的筛查。与微孔板ELISA相比,Dot-ELISA可以大大节省检测时间。直接竞争Dot-ELISA具有简便、快速、灵敏、直观的特点,具有推广和应用价值。 相似文献
14.
A M Tenter 《Veterinary parasitology》1988,29(2-3):89-104
The dot enzyme-linked immunosorbent assay (Dot-ELISA) was used for the detection of IgG antibodies to Sarcocystis muris and compared with the enzyme-linked immunosorbent assay (ELISA) and the indirect fluorescent antibody test (IFAT). In experimentally infected mice, first positive reactions occurred in the Dot-ELISA between 18 and 32 days after infection (dpi), in the ELISA between 18 and 49 dpi, and in the IFAT between 11 and 25 dpi. Maximum titers were 1:40 960 in the Dot-ELISA, 1:1280 in the ELISA and 1:2560 in the IFAT. High titers persisted until the end of the examination period (182 dpi) in all 3 tests. In immunized mice, all 3 tests detected antibodies 7 days after the first injection of protein antigen. The highest titers of 1:5120 and 1:10 240 were recorded in the Dot-ELISA after 35 days; titers of 1:1280 and 1:2560 were observed in the ELISA, and titers of 1:160 and 1:320 in the IFAT after 42 days. No false-positive reactions occurred in the Dot-ELISA and in the IFAT when 177 sera from non-infected mice were examined, but 1% (2/177) of the sera reacted positively in the ELISA. Sixty-three percent (94/150) of sera from mice infected experimentally with Toxoplasma gondii showed slight positive reactions up to 1:40 in the Dot-ELISA; 9% (13/150) of the sera reacted positively up to 1:40 in the IFAT and 4% (6/150) up to 1:20 in the ELISA. The Dot-ELISA appears to be a good alternative to the ELISA and the IFAT in the serodiagnosis of sarcosporidiosis and should be further evaluated for the serodiagnosis of other parasitic diseases. 相似文献
15.
Dot-ELISA检测猪胴体中沙门氏菌的研究 总被引：10，自引：0，他引：10
应用Dot-ELISA法对西宁某猪屠宰点 85份猪胴体进行了沙门氏菌的检测 ,同时采用常规分离培养鉴定技术作对照实验。结果在 85份肉样中 ,Dot-ELISA检出沙门氏菌阳性 6 5份 ,阳性率为 76 .4 7% (6 5 /85 ) ;而常规分离培养鉴定技术检出沙门氏菌阳性 6 7份 ,阳性率为 78.82 % (6 7/85 ) ,此两种方法的阳性符合率为 86 .5 7%。经统计分析 ,两种方法差异不显著 (P >0 .0 5 )。 相似文献
16.
Native cathepsin-L cysteine proteinase (28 kDa) was purified from the excretory secretory products of Fasciola gigantica and was used for sero-diagnosis of F. gigantica infection in buffaloes by Dot-enzyme-linked immunosorbent assay (Dot-ELISA). The test detected F. gigantica field infection in these animals with a sensitivity of ～ 90%. No specific IgG antibody binding was displayed by sera obtained from 76 buffaloes considered to be Fasciola and other parasite-free by microscopic examination of faeces and necropsy examination of liver, rumen and intestine. Additionally, sera from 156 Fasciola-free buffaloes, yet infected with Gigantocotyle explanatum, Paramphistomum epiclitum, Gastrothylax spp., Strongyloides papillosus and hydatid cyst were all negative, indicating that F. gigantica cathepsin-L cysteine proteinase does not cross-react with these helminth parasites in natural infection of the host. The data indicated that cathepsin-L cysteine proteinase based Dot-ELISA reached ～ 90% sensitivity and 100% specificity with relation to above parasites in the detection of bubaline fasciolosis. The present Dot-ELISA diagnostic assay is relevant to the field diagnosis of F. gigantica infection in buffaloes. 相似文献
17.
Dot-ELISA法检测致病性嗜水气单胞菌 总被引：3，自引：0，他引：3
在鱼类致病性气单胞菌诊断试剂盒的基础上,用斑点酶联免疫吸附试验(Dot-ELISA)检测致病性嗜水气单胞菌(Aeromonas hydrophila,Ah)胞外蛋白酶ECPase54,同时用脱脂奶平板、PCR特异性扩增气溶素基因aer和16S rRNA基因检测72株气单胞菌分离株.结果显示致病性嗜水气单胞菌检测阳性率分别如下:Dot-ELISA法90.3％(65/72)、脱脂奶平板法75％(54/72)、aer基因PCR法94.4％(68/72)、16S rRNA PCR法81.9％(59/72),Dot-ELISA与其他3种方法的符合率分别为79.2％(57/72)、91.6％(66/72)、81.9％(59/72).在ECPase54兔抗血清制备后的2、4、6、12、18个月,用Dot-ELISA检测72株分离株,检测结果重复性好.结果表明Dot-ELISA法敏感、特异、实用,可用于鱼类致病性Ah的临床诊断. 相似文献
18.
Nie YC Lu CP 《Zentralblatt für Veterin?rmedizin. Reihe B. Journal of veterinary medicine. Series B》1999,46(10):731-734
Seventy-six serum samples of Chinese soft-shelled turtles (Trionyx sinensis) were collected at Jiangsu Province, China. The neutralization test (NT) was performed with the sera, Testudo herpesvirus (THV) and turtle heart cells (THC). Neutralizing antibodies were detected in five of 76 samples and the titres were 1:10-1:20. Having optimized the conditions, the dot-enzyme-linked immunosorbent assay (Dot-ELISA) was developed and eight serum samples exhibited positive results. Five samples were positive by both NT and Dot-ELISA. The percentage of positive samples was only 6.6% (NT) and 11% (ELISA). It is suggested that THV infection is not a serious problem for the Chinese soft-shelled turtle culture in this region. 相似文献
19.
The dot enzyme-linked immunosorbent assay (Dot-ELISA), standard ELISA and the complement fixation (CF) tests were compared in the serodiagnosis of African visceral leishmaniasis (kala-azar). Assay sensitivity was determined using sera from 44 patients with parasitologically confirmed kala-azar. Using the Dot-ELISA, 42 of 44 patients (95%) were positive at a reciprocal titer of greater than or equal to 32 (titer range 512-524 288). In the standard ELISA technique, 43 of 44 patients (98%) were positive (titer range 32-32 768). At a reciprocal titer of greater than or equal to 8 in the CF test, 35 patients (80%) were positive, 1 (2%) was negative and 8 patients (18%) showed anticomplementary (AC) activity (titer range 8-2048). Specificity, determined using 33 sera from healthy individuals not living in endemic areas, was 97% in both the Dot-ELISA and the standard ELISA (32 of 33 sera); in he CF test, all sera were negative except 1 (3%) which showed AC activity. Sera from patients with Chagas' disease cross-reacted in the dot-ELISA up to a titer of 512. In the standard ELISA, cross-reactions occurred mainly using sera from patients with Chagas' disease, malaria and syphilis, and to a lesser extent with sera from amebiasis, schistosomiasis and trichinosis patients. Overall titer agreement in replicate experiments was highest in the Dot-ELISA (89%), followed by the standard ELISA (80%) and the CF test (72%). 相似文献