猪伪狂犬病血清抗体gE-ELISA检测方法的建立

以纯化的猪伪狂犬病病毒gE蛋白为抗原建立了检测猪伪狂犬病血清抗体的间接gE-ELISA方法。最佳反应条件为，抗原包被浓度为1．7μg／mL，待检测血清1：40稀释。与伪狂犬病阳性血清反应为阳性，与猪瘟、猪细小病毒病、猪繁殖与呼吸综合征（猪蓝耳病）、猪乙型脑炎、猪布氏杆菌病5种疾病阳性血清和猪伪狂犬病gE缺失疫苗接种的猪免疫血清及SPF猪阴性血清均无交叉反应。批间、批内试验变异系数分别不超过5％和9％。用该方法与Ingezim ELISA试剂盒和HerdChek ELISA试剂盒同时对172份血清进行了平行检测，总符合率分别达93．6％和83．7％。试验结果表明：猪伪狂犬病血清抗体间接gE-ELISA检测方法具有较高的敏感性和特异性，且重复性好，可用于猪伪狂犬病野毒感染猪的血清抗体检测。     

猪流行性腹泻病毒重组N蛋白间接ELISA检测方法的建立

为建立检测猪流行性腹泻病毒(PEDV)血清抗体的间接ELISA方法,本研究以原核表达的重组N蛋白作为检测抗原,优化ELISA反应条件,建立了PEDV间接ELISA抗体检测方法。该方法仅对PEDV血清检测为阳性,对猪传染性胃肠炎、猪轮状病毒感染猪的阳性血清检测结果均为阴性;其批内和批间重复性试验的变异系数均小于10%。采用建立的ELISA方法检测了130份临床样品,其中78份血清样品为PEDV抗体阳性。表明建立的以重组N蛋白为包被抗原检测PEDV血清抗体的方法可以用于检测PEDV感染及相关的流行病学调查。     

猪传染性胃肠炎病毒重组N蛋白抗原间接ELISA抗体检测方法的建立

本研究以纯化的原核表达猪传染性胃肠炎病毒N蛋白为诊断抗原，建立了猪传染性胃肠炎病毒抗体检测的间接ELISA诊断方法，将其命名为mTGE-ELISA。该抗原不与其他常见10种猪病的阳性血清发生交叉反应。批内和批间重复性试验的变异系数均小于15％；对仔猪免疫后不同时间的血清检测结果表明mTGE-ELISA与纯化病毒ELISA符合率达95．0％；mTGE-ELISA相对于VN试验的敏感性为96．3％、特异性为92．2％：现地试验中，mTGE-ELISA与Svanova TGEV／PRCV antibody diagnosis Kit的符合率达87．0％，通过中和试验复核结果表明，mTGE-ELISA的假阳性低于Svanova TGEV／PRCV antibody diagnosis Kit。本试验建立的mTGE-ELISA诊断方法具有良好的敏感性和特异性，为免疫猪群抗体监测和TGE流行病学调查提供了一种快速、简便的血清学诊断方法。     

猪细小病毒病血清抗体VP2-ELISA检测方法的建立

利用纯化的猪细小病毒VP2蛋白为抗原,建立检测猪细小病毒血清抗体的间接VP2-ELISA。最佳反应条件为:抗原包被浓度300 ng/孔,4℃过夜,待检测血清1:50稀释。与猪细小病毒病阳性血清呈阳性反应,与猪瘟、猪伪狂犬病、猪繁殖与呼吸综合征、猪日本脑炎和猪圆环病毒病等5种疾病的阳性血清均无交叉反应。批间、批内试验变异系数均小于10%。用该方法与Ceditest PPV猪细小病毒抗体检测试剂盒对102份血清进行平行检测和比较,间接VP2-ELISA的敏感性为93.8%,特异性为81.1%,符合率为89.2%。结果表明,猪细小病毒血清抗体间接VP2-ELISA检测方法具有较高的敏感性和特异性,且重复性好,可用于猪细小病毒感染的血清抗体检测。     

猪肝和猪尿中沙丁胺醇和克伦特罗残留的酶联免疫吸附检测法研究

采用混合酸酐法将沙丁胺醇与牛血清白蛋白偶联，并免疫家兔制备抗沙丁胺醇抗血清。以间接ELISA法测定血清效价，测得抗血清最佳工作浓度为1：6400，该抗沙丁胺醇抗血清对克伦特罗有110％的交叉反应性。建立了能够同时检测SAL和CL的间接竞争ELISA方法，该方法对沙丁胺醇的检测范围为0．48ng／mL～8．0μg／mL，对克伦特罗的检测范围为0．43ng／mL～7．3μg／mL，对沙丁胺醇的检测灵敏度为o．48ng／mL，对克伦特罗的检测灵敏度为0．43ng／mL。在猪肝和猪尿中添加1～50ng／g(ng／mL)的沙丁胺醇，添加回收率为72．2％～84．8％，变异系数为4．2％～28．3％，在猪肝脏和猪尿中添加1～50ng／g(ng／mL)的克伦特罗，添加回收率为84．3％～103．4％，变异系数为O．3％～35．5％。     

猪乙型脑炎病毒间接酶联免疫吸附试验抗体检测试剂盒的研制及其应用

为研制一种快速猪乙型脑炎病毒抗体检测试剂盒，本研究参照已发表的JEV（乙型脑炎病毒）基因组序列，RT—PCR扩增了长约1000bp的E基因片段，连接pET30a表达载体后获得了以包涵体形式表达的重组E蛋白，以该蛋白作为诊断抗原，研制了检测乙型脑炎病毒抗体的间接ELISA（酶联免疫吸附试验）诊断试剂盒。该试剂盒与其它7种常见猪病病毒（圆环病毒2型、伪狂犬病病毒、蓝耳病病毒、细小病毒、流行性腹泻病毒、传染性胃肠炎病毒、猪瘟病毒）的阳性血清不发生交叉反应；批内重复性试验的变异系数小于5％，批间重复性试验的变异系数小于10％；与HI（血凝抑制试验）相比，符合率、敏感性和特异性分别为83．1％、84.3％和80．0％。本研究制备的重组蛋白具有良好的反应活性，以其作为包被抗原研制的JEVE—ELISA诊断试剂盒具有良好的重复性、敏感性和特异性，为JEV的快速诊断、免疫猪群抗体监测和JEV流行病学调查提供了一种快速、简便的血清学诊断试剂盒。     

猪圆环病毒2型ELISA抗体检测试剂盒的研制及初步应用

用PCV2 Cap蛋白单克隆抗体预包被酶标板,将杆状病毒表达系统表达的PCV2 Cap蛋白作为检测用抗原,利用捕获包被法建立了间接ELISA法用于猪圆环病毒2型抗体的检测.通过优化ELISA条件,研制试剂盒并应用于圆环病毒2型疫苗的免疫效果检验.利用研制的试剂盒与IFA、商品化进口和国产同类试剂盒对178份猪血清进行平行检测,总符合率分别为96.63％、97.75％和84.27％,与其他几种常见猪病毒抗体阳性血清无交叉反应.试剂盒批内、批间变异系数分别为2.35％ ～4.06％和4.87％ ～ 6.54％,2～8℃保存15个月稳定,适合于对不同来源猪圆环病毒2型疫苗人工免疫猪血清抗体进行检测.     

甘肃省猪传染性胸膜肺炎病和猪弓形体病的血清学调查

1998～2000年，对全省11个地市22个猪场24批826份猪血清，应用郑州兽医研究所生产的间接血凝诊断液进行了猪传染性胸膜肺炎病的抗体检测。结果表明，抗体阳性率全省平均为87.16％，其中生产母猪群、种公猪群、肥育猪群和后备猪群的抗体阳性率分别为97．40％、85.71％、77．63％和70．09％。同时对全省7个地市140份猪血清进行了猪弓形体病的间接血凝抗体检测，结果在所童猪血清中未见一例阳性。     

猪轮状病毒重组VP7蛋白抗原间接ELISA诊断方法的建立

本研究以纯化的原核表达的猪轮状病毒VP7抗原表位区域为抗原,建立了检测猪轮状病毒抗体的间接ELISA诊断方法。特异性试验表明,该抗原与其他7种常见猪病病毒（TGEV、PEDV、CSFV、PCV2、PRRSV、PPV、PrV）的阳性血清不发生交叉反应,批内和批间重复性试验的变异系数均小于10％;对来自不同猪场的血清的检测结果表明,该ELISA方法与中和试验检测结果符合率达94．8％。本试验建立的ELISA诊断方法具有良好的重复性、敏感性和特异性,为PRV的快速诊断、免疫猪群抗体监测和轮状病毒流行病学调查提供了一种快速、简便的血清学诊断方法。     

猪乳汁中抗猪传染性胃肠炎病毒IgA抗体间接ELISA检测方法的建立

为建立检测猪乳汁中抗猪染性胃肠炎病毒(TGEV)Ig A抗体的间接ELISA方法,本研究以TGEV重组N蛋白为包被抗原,以辣根过氧化物酶标记羊抗猪Ig A为检测抗体,并采用方阵法确定包被抗原和待检乳汁的最佳工作浓度,对各种反应条件进行优化,建立了猪乳汁中TGEV Ig A抗体的间接ELISA检测方法。该方法检测稀释160倍的阳性乳清仍呈阳性;与猪流行腹泻和猪轮状病毒感染阳性乳清无交叉反应;批内和批间变异系数均小于10%。利用建立的ELISA方法与间接免疫荧光方法分别对134份临床样品进行检测,阳性检出率分别为58.2%(78/134)和59.7%(80/134),总符合率为85.6%。本研究建立的检测方法能够有效评估乳汁中TGEV Ig A抗体的水平。     

Comparison of monoclonal antibody-based competitive and indirect enzyme-linked immunosorbent assays for the diagnosis of swine trichinosis

A competitive enzyme-linked immunosorbent assay (ELISA) for the detection of swine trichinosis has been developed using a biotinylated monoclonal antibody and an avidin-enzyme conjugate. The assay is based on competitive binding between swine serum antibodies and a monoclonal antibody specific for an antigenic determinant present on proteins from Trichinella spiralis excretory-secretory products with molecular weights of 45,000, 49,000, and 53,000. The competitive ELISA reliably detected pigs infected experimentally with T. spiralis and eliminated false-positive reactions in pigs infected with other swine nematodes, particularly Trichurus suis. When the competitive ELISA and an indirect ELISA using affinity-isolated antigen were compared using serum from pigs with naturally-acquired infections of T. spiralis, both tests were highly effective in detecting infected animals.     

Enzyme-linked immunosorbent assay for the detection of toxoplasmosis in swine: interpreting assay results and comparing with other serologic tests

An enzyme-linked immunosorbent assay (ELISA) was developed for the serodiagnosis of swine toxoplasmosis. The results of the ELISA were interpreted as percentage (% ELISA) and ELISA titer and were compared with results of the dye test, indirect fluorescent antibody test, and indirect hemagglutination test. The results obtained from both the % ELISA and ELISA titer directly correlated with the dye test results. The ELISA is a rapid, sensitive, and readily performed serologic test that should be ideal for examining (screening) swine for toxoplasmosis.     

Comparison of the enzyme-linked immunosorbent assay and the indirect hemagglutination and complement fixation tests for detecting antibodies to Mycoplasma hyopneumoniae.

Caesarean-derived, colostrum-deprived swine were exposed to a broth culture of a low passage field isolate of Mycoplasma hyopneumoniae by intranasal inoculation. The intranasal-inoculated swine subsequently were commingled with their litter-mates to effect transmission via contact-exposure. Sera were collected from the swine at two to four week intervals for approximately one year postexposure and evaluated by the enzyme-linked immunosorbent assay (ELISA), indirect hemagglutination and complement fixation tests. The intranasal-exposed swine seroconverted earlier, developed higher titers and remained indirect hemagglutination and complement fixation positive longer than the contact-exposed swine. It was concluded that the antibody response of intranasal-exposed swine was artificially high and that sera from such swine were not suitable for evaluating the sensitivity of mycoplasmal pneumonia of swine serodiagnostic tests. The indirect hemagglutination test was relatively insensitive and technically cumbersome and the least promising as a practical field test. The complement fixation test appeared to be slightly more sensitive in detecting early antibody production (especially in contact-exposed swine) but it was the least sensitive in detecting late antibodies. The ELISA was generally the most sensitive procedure. Individual high ELISA titers were from ten to 32 times greater than maximum complement fixation and indirect hemagglutination titers. The most striking difference among the three tests was the persistence of high ELISA titers late in the study. All swine were ELISA positive at necropsy approximately one year postexposure despite the fact that lungs were devoid of lesions and culturally and immunofluorescent negative for M. hyopneumoniae.     

牛奶中替米考星间接竞争酶联免疫吸附检测法的建立

本研究通过棋盘法优化替米考星抗体、包被原浓度,并考察了最佳包被条件,最佳反应温度、时间和酶标二抗最佳反应时间等,建立了替米考星残留的间接竞争酶联免疫吸附试验(ELISA)并应用到牛奶样本的检测。建立的间接竞争ELISA方法半数抑制浓度(IC₅₀)为2.1 ng/mL,牛奶中替米考星的最低检测限(LOD)为2 μg/L。在5、10和20 μg/L添加浓度下,回收率为79.2%~90.1%,变异系数不高于9.0%。与其他常见的大环内酯类药物无交叉反应。本研究建立的替米考星间接竞争ELISA方法灵敏度达到了残留限量标准要求,为开发可用于牛奶中替米考星的快速检测试剂盒奠定基础。     

Establishment of Indirect Competitive Enzyme-linked Immunosorbent Assay for Tilmicosin in Milk

An indirect competitive enzyme-linked immunosorbent assay (ELISA) for detecting tilmicosin residue in milk was developed and applied to milk samples after the determination of dilution of coating antigen and antibody to timicosin by using checkerboard method,and the optimization of coating condition,reaction time and temperature,and reaction time of enzyme-labled secondary antibody.The results showed that the 50% inhibition concentration (IC₅₀) of the indirect competitive ELISA was 2.1 ng/mL and the limit of detection (LOD) of tilmicosin in milk was 2 μg/L.The recoveries of tilmicosin added in milk at 5,10 and 20 μg/L ranged from 79.2% to 90.1% with the coefficients of variation (CV) not higher than 9.0%.The indirect competitive ELISA would not react with other common macrolide drugs.The sensitivity of indirect competitive ELISA developed in the study complied to the maximum residue limit of tilmicosin set by China,and laid the foundation for the test kit of the rapid detection of tilmicosin in milk.     

Evaluation of an enzyme-linked immunosorbent assay for detection of Eperythrozoon suis antibodies in swine.

An ELISA was developed and tested to detect antibodies to Eperythrozoon suis in swine. Results were compared with those of the indirect hemagglutination (IHA) test. Antigen isolated from swine heavily infected with E suis was used for both tests. Comparison of the ELISA with the IHA test revealed a significant (P less than 0.001) correlation between results. Of 114 samples obtained from 9 swine infected with E suis, 87.7% were seropositive (titer greater than or equal to 200) via the ELISA, and 80.7% were seropositive (titer greater than or equal to 20) via the IHA test. The sensitivity of the ELISA was greater than that of the IHA test. All blood samples obtained from specific-pathogen-free swine tested negative for E suis antibody. Cross-reactions were not observed between E suis antigen and antisera against various swine and cattle disease agents using ELISA. We concluded that the ELISA may be used for rapid and effective diagnosis of infection with E suis in swine.     

Development and evaluation of enzyme-linked immunosorbent assay based on recombinant inorganic pyrophosphatase gene antigen for the detection of Mycoplasma suis antibodies

The recombinant ppa protein of Mycoplasma suis migrated to 21 kDa. Using this antigen, an ELISA system to detect the antibody against M. suis infection in swine was established. The rELISA demonstrated 98.5% specificities among negative samples and 96.9% sensitivity among positive samples with M. suis infection. A comparison of this ELISA system with an indirect hemagglutination assay (IHA) test using 132 swine samples revealed that the positive rate was 34.0% in ELISA and 28.0% in IHA. Compared with IHA, the present rELISA system using recombinant ppa antigen significantly improves the specificity, sensitivity, and stability for serodiagnosis of M. suis infection in swine.     

Development of an Indirect ELISA for the Detection of Antibodies against Peste-des-petits-ruminants Virus in Small Ruminants

Peste des petits ruminants (PPR) is an acute, febrile, highly contagious and economically important viral disease of small ruminants. A polyclonal antibody based indirect ELISA was developed for detection of antibodies to PPR virus in the serum samples of goats and sheep using purified PPR viral antigen propogated in Vero cell culture. A threshold (cut-off) value was set as twice the mean of the negative population based on the distribution of known negative serum samples in respect of PPR virus antibodies in the test. A total of 1544 serum samples from goats and sheep were screened by indirect ELISA and competitive ELISA. The indirect ELISA compared very well with competitive ELISA, with a high degree of specificity (95.09%) and sensitivity (90.81%). When compared with virus neutralization test, the present assay had 100% specificity and 80% sensitivity. With serum samples, the assay could clearly differentiate animals from the infected population from uninfected ones. These results suggest that the indirect ELISA may be a good alternative tool to competitive ELISA for seroepidemiological surveys.     

Establishment of an efficient enzyme-linked immunosorbent assay for the detection of Eperythrozoon sius antibody in swine

The Eperythrozoon suis (E. suis) antigen was purified using a Sephadex G-200 chromatograph, and thereby, a high-affinity, specific E. suis antigen was collected and confirmed with Western blotting. Using this antigen, an enzyme-linked immunosorbent assay (ELISA) system to detect the antibody against E. suis in swine was established. There was no cross-reaction with swine sera, which were affected with Mycoplasmal pneumonia, swine fever, swine colibacillosis, or toxoplasmosis. A comparison of this ELISA system with an indirect hemagglutination (IHA) test using 78 swine samples revealed that the ELISA system significantly improved the sensitivity, specificity, and stability for the serodiagnosis of swine E. suis.     

猪瘟病毒石门株NS3蛋白的原核表达及其抗体检测间接ELISA的建立

为配合猪瘟新型疫苗的研发,建立了猪瘟病毒NS3蛋白抗体检测间接ELISA,以期达到有效区分新型疫苗免疫猪与自然感染猪(包括常规疫苗接种猪)的目的。以接种猪瘟病毒(CSFV)石门株的PK-15细胞为模板提取总RNA,经特异性PCR扩增获得长度为2 049bp的CSFV NS3基因,将其克隆至插入了具有自聚集自切割功能短肽的原核表达载体pET-32a(+),在大肠埃希菌Rosetta(DE3)中优化表达CSFV石门株NS3基因。Western blot分析表明重组蛋白NS3具有反应原性。将纯化的重组蛋白NS3作为包被抗原建立检测CSFV NS3抗体的间接ELISA,以美国爱德士(Idexx)猪瘟病毒抗体检测试剂盒抗体检测结果为标准,对502份血清样品进行检测。结果表明,所建立方法的特异性为96.9%,敏感性为89.7%,总符合率为95.8%,为猪瘟新型疫苗的推广应用提供了血清学检测方法。