伪结核棒状杆菌抗体间接ELISA检测方法的建立及应用

为建立伪结核棒状杆菌(Cp)血清抗体间接ELISA检测方法,以热灭活处理的不同的浓度Cp菌株作为固相抗原包被酶标板,用不同封闭液封闭,设置不同的封闭时间,再用不同稀释度的待检血清和不同稀释度的酶标二抗与之反应,以此优化间接ELISA反应条件,确定阳性临界值。对间接ELISA方法的特异性和重复性进行试验,并在此基础上对采集的临床样本进行检测。结果显示,最佳抗原包被量为OD600 nm=0.084的菌悬液100μL,10 g/L BSA封闭时间2 h,一抗血清稀释度为1∶400,酶标二抗的稀释度为1∶5 000,血清阴阳性的OD450 nm值临界值为0.352。该方法仅对Cp阳性血清呈特异性反应,与6种常见病原阳性血清均无交叉反应,特异性较强。批内试验和批间试验的变异系数均小于9.5%,重复性较好。敏感性试验结果表明,当伪结核标准阳性血清进行1∶1 280稀释时检测仍为阳性,敏感性较强。用该方法对10份经细菌分离鉴定为阳性的血清进行Cp抗体检测,结果均为阳性。用建立的方法对临床随机采集的423份血清进行检测,结果显示阳性率为35%。研究建立的抗体间接ELISA方法为Cp抗体检测及血清学调查奠定了基础。     

Ⅰ群禽腺病毒五邻体蛋白间接ELISA检测方法的建立

为建立一种简便、快速检测Ⅰ群禽腺病毒(FAVI)抗体的间接ELISA方法,以表达和纯化的FAVI五邻体重组蛋白作为抗原,优化反应条件,建立了FAVI间接penton-ELISA抗体检测的方法。抗原最佳包被浓度为1.5μg/孔,最适包被条件为37℃,2h后4℃过夜;血清最佳稀释度为1∶100;酶标二抗的最佳工作浓度为1∶2000;ELISA阴、阳性临界值为0.335。用penton-ELISA方法检测100份鸡血清样品,阳性率为41%。     

猪瘟野毒感染抗体阻断ELISA检测方法的建立与初步评价

以猪瘟野毒E2蛋白为包被抗原、辣根过氧化物酶标记的猪瘟野毒单抗作为酶标抗体,建立检测猪瘟野毒抗体的阻断ELISA方法.猪瘟野毒E2最适包被浓度为0.03 μg/mL,待检血清最适稀释度为1∶4,酶标猪瘟野毒单抗稀释度为1∶1 000.用建立的阻断ELISA方法检测369份临床阴性血清,计算阻断率,确定临界值,阻断率＞4...     

抗绿脓杆菌外毒素A酶标抗体的制备及其应用

从病死羊体内分离到绿脓杆菌并提取出外毒素A(PEA),再以此毒素作为抗原加油佐剂制成乳化抗原免疫家兔,获得高免血清并提取免疫球蛋白G(IgG);用过碘酸钠法将过氧化物酶标记抗PEA抗体(IgG),制成酶标抗体,经检验,酶标抗体结合物中的HRP浓度和Ab(IgG)浓度分别是0.0608 mg/mL和0.336 mg/mL;HRP/Ab(IgG)克分子比值为1.724%;酶(HRP)结合率是11.15%.利用该酶标抗体以ELISA夹心法对羊体内抗PEA抗体含量进行了检测,结果证明,用酶标抗体ELISA法比用平板凝集实验法检测的抗体效价平均高出二个滴度,表明制备的PEA酶标抗体具有灵敏度高、特异性强的优点.     

猪胸膜肺炎放线杆菌间接ELISA检测方法的建立

【摘 要】用纯化的重组蛋白抗原作为ELISA包被抗原,通过对抗原包被浓度、血清稀释倍数、酶标二抗稀释倍数、抗原和血清反应时间、血清和酶标二抗反应时间、显色剂作用时间和中止液滴加量的优化,建立了检测胸膜肺炎放线杆菌抗体的间接ELISA方法。通过特异性实验证明该ELISA方法特异性较强。将建立的ELISA方法与IDEXX公司的标准试剂盒进行了比较,二者的符合率较高,说明建立的ELISA方法比较敏感,为ELISA检测方法的商品化奠定了基础。     

间接ELISA检测猪伪狂犬病血清抗体

用猪肾传代细胞IBRS－2增殖猪伪狂犬病病毒（PRV）鄂A株，病毒培养上清液经硫酸铵沉淀、聚乙二醇（Mr20000）浓缩后作为包被抗原。用纯化的猪血清IgG免疫家兔，HRP村记撮的兔抗猪IgG，制备出高效价的酶标抗体，酶标抗体工作浓度为1：50000；经各种条件的选择，建立了检测猪伪狂犬病血清抗体的间接ELISA。所建立的间接ELISA抗原包被浓度为39.2mg/L，血清最佳稀释度为1：20，与猪细小病毒、猪、O型口蹄疫、猪衣原体标准阳性血清呈阴性反应，与标准阴性血清和临床未感染PRV的猪血清呈阴性反应；与猪伪狂犬病标准阳性血清、免疫猪血清和临床发病猪血清呈明显的阳性反应；与美国进口的PRV抗体检测ELISA诊断试剂盒检测结果比较，45份猪血清的阴、阳性检出符合率均为100％。表明建立的间接ELISA具有敏感性高、特异性强、重复性好的优点，可用于猪伪狂犬病血清抗体的定性和定量检测。     

阪崎肠杆菌单克隆抗体制备及ELISA检测方法的建立

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D_{450 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。     

鸭源鸡杆菌抗体间接ELISA检测方法的建立及应用

利用鸭源鸡杆菌YU-PDS-RZ-1-SLG分离株制备超声波裂解抗原,建立了可以检测鸭源鸡杆菌多个血清型抗体的间接ELISA方法。包被抗原质量浓度为10mg/L,包被条件为37℃2h,再4℃过夜;封闭液为1%明胶,封闭条件为37℃1h;阴、阳性血清最佳稀释度为1∶100,酶标二抗工作滴度为1∶1 000;底物显色时间为15min。经交叉性试验、阻断试验和重复性试验证实建立的ELISA方法重复性好,特异性强。板内变异系数为2.01%～5.75%,板间变异系数为2.43%～6.20%。间接ELISA方法的灵敏度是微量凝集试验的25～100倍。利用所建立的ELISA方法检测了人工感染鸭源鸡杆菌的4日龄SPF鸡在感染后不同阶段感染组、同居组和空白对照组的血清抗体,并根据感染后不同阶段所测D450值绘制抗体消长曲线,其抗体水平在感染后32～47d开始上升,60d时达到高峰,但维持时间较短,2周后迅速下降。建立的间接ELISA方法可以用于临床病例的血清学快速检测,为进行鸭源鸡杆菌的血清流行病学调查提供了手段。     

检测非洲猪瘟病毒抗原的双抗体夹心ELISA方法的建立

利用非洲猪瘟病毒(ASFV)p72蛋白不同抗原表位的2株单克隆抗体,1株作为捕获抗体,另1株用辣根过氧化物酶(HRP)标记后作为检测抗体,采用方阵法对ELISA反应条件进行优化,建立了检测ASFV抗原的双抗体夹心ELISA方法。结果显示,该方法中捕获抗体包被质量浓度为1.25 mg/L,酶标单克隆抗体的最适稀释度为1∶4 000。通过试验确定该方法的临界值为0.299,当样品D₄₅₀值≥0.3,且P/N大于2时,判定为阳性。该双单抗夹心ELISA方法可特异性检测ASFV抗原,其他抗原检测结果均为阴性,特异性强;重复性好,批内和批间变异系数(CV)均小于8%。应用本研究建立的方法与荧光PCR方法同时对225份临床样品进行检测,总符合率为96.89%。结果表明,本研究建立的双抗体夹心ELISA方法可用于ASFV抗原的大量样品检测,为ASF防控提供了技术支持。     

应用夹心阻断ELISA测定血清中抗狂犬病抗体的研究

本研究建立的检测狂犬病抗体的夹心阻断ELISA,直接利用未经提纯的病毒悬液代替提纯的抗原,并仅用一种酶标抗体,测定了9种动物的血清.本方法敏感度的95%可信限为0.0013～0.0071IU/mL,比小鼠中和试验和微量免疫酶试验均敏感.按阻断50%判定血清ELISA的阴阳性,9种动物的1134份血清中有91份阳性,其中发病点的牛、猪、犬、家鼠血清的阳性率均在10%以上.根据对一定量的抗原阻断率相同时,抗体浓度一致的原理,测定了7种动物的185份血清效价,结果狂犬病抗体浓度在0.01IU/mL以上的为63份.利用夹心阻断ELISA和小鼠中和试验测定了7种动物的23份血清,阳性份数分别为12和11.两种方法均证明家鼠血清中有狂犬病抗体.本研究表明,测定狂犬病抗体的夹心阻断ELISA,简便、敏感、快速,可用于流行病学调查.     

Preparation of Cathepsin L1 Monoclonal Antibody Against Fasclala gigantica and Establishment of Double Antibody Sandwich ELISA

【Objective】 This study was intend to obtain cathepsin L1(rFgCat L1) specific monoclonal antibody and construct the double antibody sandwich ELISA.【Method】 Five BALB/c mice were immunized with 1 mg/mL rFgCat L1 protein for four times.Mouse splenocytes were isolated and fused with SP2/0 cells to construct hybridoma cells.Strong positive hybridoma cell lines were screened, 1×10⁶ cells were injected intraperitoneally per mouse to prepare monoclonal antibodies.Antibody titer and antigenic epitope were detected using ELISA method, antibody subtype and specificity were identified using Western blotting method.The double antibody sandwich ELISA was constructed by combining the anti-rFgCat L1 polyclonal antibody, and its sensitivity and specificity were tested.The positive and negative critical value was screened by 20 negative sera with positive control, and the constructed double antibody sandwich ELISA was verified by 47 goat positive sera and 47 dairy cow positive sera.【Result】 After immunization, the antibody titers in serum of 4 mice were all more than 10⁴.After isolated mouse with the highest immune response spleen cells were fused with SP2/0 cells total of 8 of them were positive cell lines were obtained after selective culture.5D5 and 7G6 were identified as strong positive strains with stable antibody secretion.After multiple subcloning screens and subcultures, the antibodies secreted in the cell supernatant were stable, with titers of 2⁹ and 2¹⁰ respectively, with ascites titers of 10⁷ and 10⁸.Western blotting and antibody subtype identification kits identified that the two antibodies were IgG1 type and the light chain was kappa type, both of which could specifically bind FgESP.According to the same antigen site was recognized by the two kinds of antibodies, the antigen titer of the two monoclonal antibodies were comparied, 7G6 was used as the coating antibody, and anti-rFgCat L1 was used as the enzyme-labeled secondary antibody.The optimized condition of method was that 7G6 was coated at a concentration of 2 μg/mL, the dilution concentration of anti-rFgCat L1 polyclonal antibody was 25 μg/mL, the dilution of Don-HRP-conjugated was 1∶4 000, 5% skimmed milk powder was selected as the blocking solution and the color development time was 25 min.The method was proved that could recognize the lowest antigen concentration of 0.625 μg/mL, also could specifically recognize antigen of Fasciola fasciatus.The constructed sandwich ELISA method was used for antigen detection of 47 dairy cow positive serum and 47 goat positive serum infective samples kept in the laboratory and the positive antigen rate were 72.3% and 78.7%, respectively.【Conclusion】 Anti-rFgCat L1 monoclonal antibody was successfully prepared and the double-sheet sandwich ELISA method for fascioliasis was constructed, which provided a good theoretical basis and material basis for the development of low-cost and rapid diagnostic kits.     

Use of the dot enzyme-linked immunosorbent assay with isolated Anaplasma marginale initial bodies for serodiagnosis of anaplasmosis in cattle

Isolated Anaplasma marginale initial bodies were successfully used in a dot ELISA for rapid detection of antibodies to Anaplasma organisms. The enzyme immunoassay used only 25 ng of antigen dotted onto nitrocellulose disks. Antigen-antibody complexes were detected by use of alkaline phosphatase-conjugated protein A, and reactions were read visually after addition of a precipitable, chromogenic substrate. The test allowed the processing of multiple sera, either for screening or for titer determination, in less than 3 hours and was found to be as sensitive as the indirect fluorescent antibody test. The overall performance of the dot ELISA, using isolated A marginale initial bodies, for 580 bovine serum samples was as follows: sensitivity, 93%; specificity, 96%; and predictive value, 95%. Cross-reactivity was not observed with sera positive to Babesia bovis and B bigemina, Trypanosoma vivax, or common bacteria or viruses infecting cattle. The antigen dotted onto nitrocellulose disks was stable when stored at -20, 4, or 25 C. Compared with the indirect fluorescent antibody test, the dot ELISA allowed easier, faster, and more objective interpretation of results. Its simplicity and low cost combined with high sensitivity and specificity indicate that this assay could effectively replace serologic assays currently used for diagnosis of anaplasmosis in cattle.     

Development of enzyme-linked immunosorbent assays for the detection of Fusobacterium necrophorum antibody in animal sera

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.     

猪圆环病毒2型ORF2基因截断表达及ELISA抗体检测方法的初步建立

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。     

Q热贝氏柯克斯体间接ELISA检测方法的建立

本研究以Q热贝氏柯克斯体弱毒株Ⅱ相全菌为包被抗原建立Q热贝氏柯克斯体间接ELISA检测方法,通过方阵滴定法对抗原包被浓度和二抗反应浓度的确定及各项反应条件的优化,确定其阴阳性临界值为0.44。用本方法与IDEXX Q热抗体检测试剂盒对393份牛临床血清进行检测,检出的血清阳性率分别为11.45%和6.10%,符合率为94.66%。结果表明,本试验建立的检测Q热贝氏柯克斯体的间接ELISA法具有较好的特异性及较强的敏感性,可初步应用于Q热贝氏柯克斯体抗体的临床检测。     

基于非洲猪瘟病毒截短p72蛋白的间接ELISA抗体检测方法的建立

【目的】建立非洲猪瘟病毒（African swine fever virus,ASFV）ELISA抗体检测方法。【方法】以重组截短p72（p72s）蛋白作为检测抗原,通过方阵滴定法,确立ELISA的抗原、待检血清和酶标抗体的最佳工作浓度,优化抗原包被、酶标板封闭、血清/酶标抗体反应及底物显色等工作条件;应用受试者工作特征（receiver operating characteristic,ROC）曲线阈值评价标准,确定方法的阴阳性临界值;检测方法的特异性、敏感性、批内与批间重复性;最后分别用建立的ELISA方法和商品试剂盒检测124份血清,比较两者的阳性检出率,并通过Western blotting验证ELISA的检测结果。【结果】ELISA方法的抗原最佳包被浓度为0.5 μg/mL,待检血清与酶标抗体的最适稀释度分别为1∶100和1∶5 000;反应条件为：抗原于37 ℃孵育1 h后经4 ℃包被酶标板过夜、于37 ℃封闭1 h或室温封闭2 h、待检血清和酶标抗体分别于37 ℃反应60和45 min及加入底物后于室温避光显色20 min;阴阳性血清的判定标准为：当待检血清的D_{450 nm}值≥0.365时,判定为阳性;当D_{450 nm}值＜0.365时,判定为阴性。该方法只与ASFV阳性血清发生特异性结合,与猪瘟病毒、伪狂犬病病毒、猪繁殖与呼吸综合征病毒等病毒抗血清均无交叉反应;能检测到ASFV抗血清中的总蛋白量为0.091~0.153 mg/mL,低于商品试剂盒的最低检测量（0.110~0.554 mg/mL）;批内和批间重复性试验的平均变异系数分别为4.70%与5.125%。对临床血清样本检测时,建立方法和商品试剂盒的阳性检出率分别为75.81%（94/124）和32.26%（40/124）。进一步验证表明,Western blotting与ELISA的检测结果完全一致。【结论】建立了基于ASFV-p72s蛋白的ELISA抗体检测方法,该方法特异、敏感和重复性稳定,能够用于ASF临床诊断与流行病学调查,具有极大的开发应用潜力。     

伊氏锥虫变异表面糖蛋白抗原双抗体夹心ELISA检测方法的建立及初步应用

A double antibody sandwich ELISA for detection of Trypanosoma evansi variant surface glucoprotein (VSG) antigen was established by using two monoclonal antibodies against Trypanosoma evansi VSG antigen.The result of sensitivity showed when positive serum was diluted to 3200 doubles, its D_{450 nm} value was still greater than the critical value of positive and negative,and specificity test result showed that there was no cross reaction with the antigens of Theileria,Toxoplasma gondii,Chlamydia and Fasciola gigantica.82 clinical sera were detected by this double antibody sandwich ELISA and its positive rate was 9.76%.The experiment results demonstrated that this double antibody sandwich ELISA for detection of Trypanosoma evansi VSG antigen had good detecting sensitivity and specificity,and could be used as an effective method for clinical detecting buffalo Trypanosoma evansi disease.     

Establishment and Preliminary Application of Indirect ELISA Based on Recombinant NS4 Protein for Detection of Bluetongue Virus Antibodies

This study was conducted to establish an indirect ELISA method for the detection serological antibody of bluetongue virus (BTV). The purified BTV recombinant NS4 protein obtained from the prokaryotic express system was used as the coated antigen, and then an indirect ELISA antibody detection method of BTV was developed by optimizing the reaction conditions. SDS-PAGE results showed that the recombinant NS4 protein with a size of about 52 kDa was obtained, which mainly existed in the supernatant. Western blot results showed that the purified recombinant NS4 protein had good antigenicity. The ELISA reaction conditions were optimized by the square matrix test. The optimal coating amount of recombinant NS4 protein antigen was determined to be 3.0 μg per well, and the optimal dilution ratio of serum to be tested was 1:200, and the optimal dilution concentration of HRP-labeled rabbit anti-cow IgG secondary antibody was 1:4 000, and the critical values were 0.29 and 0.35, respectively. The detection sensitivity of the BTV antibody was up to 1:1 600. The intra-assay repeatability and the inter-assay repeatability coefficient of variation were less than 10%. The positive coincidence ratio and negative coincidence ratio were 98% and 100% respectively. The indirect ELISA method established in this study laid a foundation for clinical serum antibody detection and serum epidemiological investigation of BTV.     

基于NS4蛋白的蓝舌病病毒间接ELISA抗体检测方法的建立及初步应用

旨在建立蓝舌病病毒（BTV）血清学ELISA抗体检测方法,本研究以原核表达并纯化的BTV NS4重组蛋白为包被抗原,通过反应条件优化,建立了一种BTV重组NS4蛋白的间接ELISA抗体检测方法。SDS-PAGE结果显示,获得大小约52 ku的NS4重组融合蛋白,主要在上清中存在,Western blot显示,纯化后的重组蛋白具有良好的抗原性。通过方阵试验进行了ELISA反应条件优化,确定了重组蛋白抗原最佳包被量为3.0 μg·孔^-1;血清最佳稀释倍数为1：200,酶标二抗最佳工作浓度为1：4 000,临界值分别为0.29和0.35。上述以NS4蛋白作为包被抗原建立的BTV抗体间接ELISA方法检测敏感性可达1：1 600;批内和批间重复性变异系数均小于10%;检测76份重庆地区牛群血清样品,阳性符合率为98%,阴性符合率为100%。本研究建立的间接ELISA方法为临床BTV血清抗体检测及BTV血清流行病学调查奠定了基础。     

犬弓形虫抗体间接ELISA检测方法的建立

试验以利用重组弓形虫SAG1基因转染蜥蜴利什曼原虫所表达获得的目的蛋白作为包被抗原,建立了一种快速、特异、可检测犬弓形虫抗体的间接ELISA方法。确定了抗原最佳包被浓度为6.75 μg/mL,血清最佳稀释度为1∶100,对已知阳性血清检测的下限可达1∶6400,批间和批内重复性试验的变异系数均小于10%,包被抗原的酶标板在4、-20 ℃环境中可保存8个月以上。建立的间接ELISA方法应用于犬弓形虫抗体的检测具有较好的敏感性及特异性。