鸡传染性支气管炎病毒抗体间接ELISA检测方法的建立

为建立快速检测鸡传染性支气管炎病毒(IBV)的血清学方法,本试验以IBV为检测抗原,建立了一种检测IBV抗体的间接ELISA方法,并对各种检测条件进行了优化.研究结果显示,抗原的最佳包被浓度为19.2 μg/mL,最佳包被条件为37 ℃ 1 h后4 ℃过夜;血清的最佳稀释度为1:800,37 ℃孵育60 min;酶标二抗稀释度为1:7 000,37 ℃孵育60 min;底物显色为37 ℃避光作用10 min.经特异性、重复性、敏感性试验证明,该方法与鸡常见病原的阳性血清均无交叉反应,重复性较好及血清稀释至1:12 800时仍可检测到IBV抗体.结果表明,本试验所建立的间接ELISA方法具有良好的特异性、重复性和敏感性.     

鹅副黏病毒NP蛋白间接ELISA检测方法的建立及免疫鹅抗体水平的检测

以纯化的鹅副黏病毒NP蛋白为包被抗原,建立了检测鹅副黏病毒NP蛋白抗体的间接ELISA方法,并确定了间接ELISA的最适反应条件：抗原包被浓度为1.0μg/mL,血清稀释度为1∶200,兔抗鹅IgG辣根过氧化物酶标记抗体稀释度为1∶2 000,抗原和血清、血清和二抗均于37℃反应1 h,底物于37℃显色15 min。此间接ELISA方法的特异性强、重复性好。应用该方法对试验鹅血清进行检测,以HI试验为参照。经统计学处理后,比较了两方法测得的抗体效价,分别建立了各组鹅群的回归方程。显著性检验证明,这两种方法检测的抗体效价呈显著相关关系。     

骆驼斯氏副柔线虫病间接ELISA检测方法的建立

为建立骆驼斯氏副柔线虫病间接ELISA(iELISA)诊断方法,本试验对骆驼斯氏副柔线虫半胱氨酸蛋白酶CPR基因进行重组表达,将获取的重组蛋白(rCPR)进行纯化和Western blotting检测,然后以纯化好的重组蛋白为抗原,通过棋盘滴定试验优化了抗原包被浓度、包被条件、抗体稀释度、酶标二抗稀释度、封闭液和封闭时间等,建立了骆驼斯氏副柔线虫病iELISA诊断方法,并对建立的iELISA检测方法进行了重复性、敏感性、特异性试验和临床检测。结果显示,抗原最佳包被浓度为8μg/孔,血清最佳稀释度为1∶50,酶标二抗最佳稀释度为1∶5 000,最佳包被条件为4℃包被过夜,最佳封闭条件为3%BSA封闭2 h。临界值为0.235,待检血清D_(450 nm)值0.235则确定为阳性。重复性试验中变异系数均10%,重复性较好;用该方法检测阳性血清的敏感性为96.3%;此方法仅与骆驼斯氏副柔线虫病阳性血清发生特异性反应,与感染了其他寄生虫的阳性血清无交叉反应,特异性为100%;对140份临床血清进行检测,阳性率为86.4%。综上可知,本试验成功建立了一种快速有效诊断骆驼斯氏副柔线虫病的iELISA方法。     

副猪嗜血杆菌抗体间接ELISA检测方法的建立与应用

本试验采用121 ℃高压处理副猪嗜血杆菌4型和5型耐热蛋白,混合作为包被抗原,建立了检测副猪嗜血杆菌抗体的间接ELISA方法。通过对试验条件进行筛选优化,确定了最佳反应条件:抗原包被浓度为10 μg/mL,37 ℃包被2 h;封闭液选择含20 g/L脱脂奶粉的PBST,封闭30 min;血清的稀释度为1∶80;抗原抗体反应时间为45 min;酶标二抗稀释度为1∶12000,作用时间为30 min;底物显色时间为15 min。特异性、重复性和敏感性试验及对200份送检血清的检测结果表明,建立的间接ELISA方法特异性和重复性良好,敏感性比间接血凝试验高,对已知阴阳性血清的临床样本检测结果与国外ELISA试剂盒一致,可用于副猪嗜血杆菌的血清抗体检测和血清流行病学调查。     

骆驼斯氏副柔线虫病间接ELISA检测方法的建立

为建立骆驼斯氏副柔线虫病间接ELISA （iELISA）诊断方法,本试验对骆驼斯氏副柔线虫半胱氨酸蛋白酶CPR基因进行重组表达,将获取的重组蛋白（rCPR）进行纯化和Western blotting检测,然后以纯化好的重组蛋白为抗原,通过棋盘滴定试验优化了抗原包被浓度、包被条件、抗体稀释度、酶标二抗稀释度、封闭液和封闭时间等,建立了骆驼斯氏副柔线虫病iELISA诊断方法,并对建立的iELISA检测方法进行了重复性、敏感性、特异性试验和临床检测。结果显示,抗原最佳包被浓度为8 μg/孔,血清最佳稀释度为1：50,酶标二抗最佳稀释度为1：5 000,最佳包被条件为4℃包被过夜,最佳封闭条件为3% BSA封闭2 h。临界值为0.235,待检血清D_{450 nm}值>0.235则确定为阳性。重复性试验中变异系数均<10%,重复性较好;用该方法检测阳性血清的敏感性为96.3%;此方法仅与骆驼斯氏副柔线虫病阳性血清发生特异性反应,与感染了其他寄生虫的阳性血清无交叉反应,特异性为100%;对140份临床血清进行检测,阳性率为86.4%。综上可知,本试验成功建立了一种快速有效诊断骆驼斯氏副柔线虫病的iELISA方法。     

严重发热伴血小板减少综合征病毒(SFTSV)总抗体检测试剂盒(ELISA)的研制

为建立检测严重发热伴血小板减少综合征病毒(SFTSV)血清总抗体的双抗原夹心ELISA方法并进行初步试用.通过原核表达得到SFTSV-NP重组蛋白,以该蛋白作为ELISA的包被和酶标记抗原,确定抗原的最佳包被浓度和血清稀释度,优化各项试验条件,在此基础上研制试剂盒,并对试剂盒的重复性、特异性等方面进行研究.结果发现抗原最适包被浓度为4.0μg/ml,血清的最佳稀释浓度为1:40,重复性试验表明批内和批间的变异性都低于10﹪.该试剂盒敏感性高、特异性强、重复性良好,可应用于SFTSV的研究和临床检测.     

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。     

利用重组E2蛋白检测BVDV血清抗体间接Dot-ELISA方法的建立

利用大肠杆菌表达的牛病毒性腹泻病毒(BVDV)重组E2蛋白作为抗原,建立了检测BVDV血清抗体的间接Dot-ELISA.最佳工作条件为:E2蛋白抗原的最适包被质量浓度2.0 mg/L(2.0 ng/点),酶标抗体的工作浓度为1:500,血清稀释度为1:100,3%明胶-TBS作为封闭液,封闭45 min效果最佳.通过重复性试验、交叉试验、特异性试验和稳定性试验等证明,该方法重复性好、特异性强、灵敏度高;与用BVDV全病毒为抗原的IDEXX ELISA试剂盒相比,特异性96.67%,灵敏度90%,符合率95%.应用建立的检测方法对河北省4个奶牛场采集的178份腹泻奶牛血清样本进行检测,结果BVDV血清抗体阳性率40.45%,与IDEXX ELISA试剂盒的检出率无明显差异.     

间接ELISA检测犬瘟热血清抗体方法的建立

以重组表达的犬瘟热病毒融合蛋白(fusion protein,F)作为包被抗原,建立了检测犬瘟热病毒血清抗体的间接ELISA方法。所建立的间接ELISA抗原包被浓度为125ng/孔,血清最佳稀释度为1∶160。经阻断试验、重复性试验等表明该方法特异性强、重复性较好,可用于犬瘟热血清抗体的定量和定性检测。     

大豆抗原蛋白血清抗体间接ELISA检测方法的建立

旨在建立检测血清大豆抗原蛋白抗体的间接ELISA方法。经琼脂糖凝胶层析纯化大豆抗原蛋白,以不同剂量皮下注射免疫小鼠,采用方阵滴定法确定最佳抗原包被浓度及血清稀释度,并对其他条件进行优化,最终建立检测血清大豆抗原蛋白抗体的间接ELISA方法,利用该方法检测小鼠免疫后血清抗体水平。通过方阵滴定法确定11S蛋白最佳包被浓度为5.0μg/mL,血清稀释倍数为1∶800;7S蛋白抗原最佳包被浓度为2.5μg/mL,血清稀释倍数为1∶1 600;两者的批内、批间系数均小于10%,重复性较好,通过ELISA法确定11S和7S蛋白的最佳免疫次数为2次,免疫剂量为1 000μg/kg。结果表明本试验初步建立大豆抗原蛋白抗体检测间接ELISA方法,具有很强的特异性、敏感性和重复性,可用于大豆抗原蛋白过敏反应的临床检测。     

Large-scale ELISA testing of Spanish red deer for paratuberculosis

A role of wildlife species as paratuberculosis reservoirs is strongly suspected based on field and molecular epidemiologic evidence. This paper presents the first large-scale data on enzyme-linked immunosorbent assay (ELISA) against Mycobacterium avium subspecies paratuberculosis (MAP) antibodies in red deer from Spain, and tests the effect of host and environmental risk factors on antibody levels. A total of 257 out of 852 serum samples tested positive, yielding a total seroprevalence of 30.16% (95% CI 27.08-33.24). Sampling locality, presence of cattle and increasing age explained the variation in the individual ELISA optical density (OD) results. Data presented in this study strongly suggest that Spanish red deer are exposed to MAP. While contact with cattle was statistically significant, some wild populations showed the highest positivity to the ELISA. The results support the need of a careful study of MAP prevalence based on culture and molecular tools in order to clarify if deer play a significant role as paratuberculosis reservoirs for livestock, and if deer paratuberculosis is affecting hunting harvest, trophy quality, or wild animal welfare in Spain.     

Evidence of paratuberculosis in Ohio's white-tailed deer, as determined by an enzyme-linked immunosorbent assay

An enzyme-linked immunosorbent assay (ELISA) was developed and used to detect antibodies to Mycobacterium paratuberculosis in serum samples obtained in December of 1983 from 954 hunter-killed white-tailed deer (Odocoileus virginianus) in 13 Ohio counties. Positive or negative status was determined by calculating a signal-to-noise ratio, a ratio between the optical density of the test serum and negative reference sera; a ratio of greater than or equal to 3.0 was considered positive. Twenty-four samples (2.5%) were found to be assay positive, using this method. A statistically significant difference among age groups was found, with those less than or equal to 6 months of age having a lower proportion of positives. Differences by sex were not observed. To determine the validity of the ELISA in deer, serum samples from 46 fallow (Dama dama) and axis deer (Axis axis) harvested from a known infected population were tested by ELISA and agar-gel immunodiffusion. The agar-gel immunodiffusion test showed evidence of exposure of the deer to M paratuberculosis or a related antigen. The ELISA closely approximated the prevalence of paratuberculosis infection as previously determined by fecal culture in this population. As a result of these tests, it was concluded that free-ranging Ohio deer have been infected with M paratuberculosis or exposed to a closely related antigen.     

应用TaqMan荧光定量PCR快速检测鹿血中副结核分枝杆菌

为建立快速检测鹿血中副结核分枝杆菌(Mycobacterium avium subsp. paratuberculosis,MAP)的荧光定量PCR方法,本研究根据GenBank中登录的MAPf57基因序列设计并合成引物及探针,并检测该方法的特异性和敏感性。试验结果显示,该方法具有良好的特异性,对MAP的检测灵敏度可以达到单个菌细胞。对长春地区采集的549份血清样品进行检测,结果显示阳性血清101份,阳性率达到18.4%。本研究结果表明,荧光定量PCR用于动物性产品MAP的检测具有快速、准确的特点。     

Evaluation of enzyme-linked immunosorbent assays performed on milk and serum samples for detection of paratuberculosis in lactating dairy cows

OBJECTIVE: To determine whether results obtained for milk and serum samples with ELISAs intended for diagnosis of paratuberculosis in dairy cows were comparable to results obtained by means of mycobacterial culture of fecal samples. DESIGN: Cross-sectional study. ANIMALS: 689 lactating dairy cows in 9 Ontario herds. PROCEDURE: Milk, serum, and fecal samples were obtained from all cows. Fecal samples were submitted for mycobacterial culture. Serum samples were tested with a commercially available ELISA for antibodies against Mycobacterium paratuberculosis, and preserved milk samples were tested with an indirect ELISA for antibodies against M paratuberculosis. RESULTS: Results were positive for 130 of the 689 (18.9%) serum samples, 77 of the 689 (11.1%) milk samples, and 72 of the 689 (10.4%) fecal samples. The level of agreement between results for milk and serum samples was only moderate. Proportions of positive results for serum and fecal samples were significantly different, but proportions of positive results for milk and fecal samples were not significantly different. In addition, results for milk samples had a higher level of agreement with results of mycobacterial culture than did results for serum samples. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that the indirect ELISA used on milk samples may be a convenient method of detecting paratuberculosis in dairy herds.     

Comparison of dot immunobinding assay, enzyme-linked immunosorbent assay and immunodiffusion for serodiagnosis of paratuberculosis.

A rapid, simple and inexpensive dot immunobinding assay (DIA) was evaluated for the serodiagnosis of paratuberculosis in cattle. The assay was performed on nitrocellulose strips which were dotted with purified protoplasmic antigen of Mycobacterium paratuberculosis. After incubation with test serum samples, the bound antibodies were detected using an enzyme-amplified immunostaining procedure. The efficacy of DIA as a screening test for paratuberculosis was compared to that of an enzyme-linked immunosorbent assay (ELISA), a modified agar gel immunodiffusion (mAGID) test, and an AGID test using 329 serum samples from cattle which were examined for M. paratuberculosis infection by a sensitive fecal culture technique. The DIA and ELISA had comparable results and both of the enzyme immunoassays had higher sensitivity than tests based on AGID. The sensitivity of all four tests was influenced by the intensity of fecal bacterial shedding. Preabsorption of sera with Mycobacterium phlei increased the sensitivity of both enzyme immunoassays. the specificity but reduced the sensitivity of both enzyme immunoassays.     

An ELISA for porcine reproductive and respiratory syndrome: production of antigen of high quality.

A reliable method was developed to produce a viral antigen preparation from porcine reproductive and respiratory syndrome virus (PRRSV) infected MARC-145 cells by solubilizing the virus with Triton X-100. This method eliminated problems previously encountered with high background reactions associated with PRRSV antigen or cell control antigen. With this new antigen, an indirect enzyme-linked immunosorbent assay (ELISA) was adapted to detect swine serum anti-body against PRRSV. In the ELISA, non-specific reactions associated with test serum samples have been eliminated by utilizing an effective blocking serum diluent. The ELISA is more sensitive than an indirect immunofluorescent assay (IFA), particularly with late-infection sera, while maintaining a high diagnostic specificity. In a comparison of IFA and ELISA using sera collected from 250 pigs of various ages from 5 herds that had PRRS histories, IFA revealed 178 positive samples and 72 negative samples. All of the IFA-positive sera were proven to be ELISA reactors. However, nearly one-half (34/72) of the IFA-negative samples were also ELISA reactors. The diagnostic specificity and sensitivity of the ELISA were 100% and 96.6% with 257 serum samples collected from known healthy PRRS-negative swine herds and 57 sera collected from infected swine at 6 to 56 days after infection, respectively. The ELISA is technically superior to IFA, time-efficient and cost-effective, and suitable for testing of a large number of samples over a short period of time.     

Development and comparison of serologic methods for diagnosing chicken anemia virus infection.

This paper describes the development of an indirect immunoperoxidase assay (IIP) and an indirect enzyme-linked immunosorbent assay (ELISA) for detecting antibodies to chicken anemia virus (VAC). The IIP assay developed used CAV-infected MDCC-MSB1 cells for detecting antibody to CAV, whereas the ELISA utilized gradient-purified immunoadsorbed CAV as the target antigen. The IIP and ELISA were compared with the standard indirect immunofluorescent antibody (IFA) assay, which is more conventionally used to screen chicken serum for antibodies against CAV. Comparative test results of 185 field samples of chicken serum by these three methods were in agreement 84% of the time. Both IFA and IIP assays yielded fewer positive tests than did the ELISA. IFA and IIP assays were in agreement 93% of the time, as compared with 91% agreement of IIP and ELISA results, or 84% agreement for comparative IFA and ELISA results.     

Quantitative and Qualitative Study of Enzyme-linked Immunosorbent Assay to Detect IgG against Japanese Encephalitis Virus in Swine Sera

This study was designed to investigate the application of indirect enzyme-linked immunoassay (ELISA) in detecting IgG against Japanese encephalitis virus in swine sera and the qualitative nature of this test. The attenuated strain SA14-14-2 of Japanese encephalitis virus (JEV) was inoculated into 9-day-old chicken embryos and virus was harvested, purified and suspended in 0.9% saline as JEV antigen. The control antigen was prepared by the same method as for the antigen. In the ELISA, the optimal concentrations of antigen coated and dilution factor were selected using chi2 test. Ninety-two swine sera negative to haemagglutination inhibition (HI) were tested by this assay and the positive threshold was determined. The results of this study indicate that indirect ELISA has high specificity, sensitivity and reproducability. Simultaneous testing of 74 serum samples from nine pig farms was carried out to compare the existing HI test and the indirect ELISA. The coincidence rate of the two assays was 85.1% (63/74) and no significant difference was observed between them (p > 0.05). This ELISA test can detect 46 swine serum samples qualitatively and the titre of eight swine serum samples through endpoint dilution quantitatively within one 96-well plate.     

重组N蛋白抗原检测PRRSV抗体ELISA的研究Ⅳ.与IDEXX ELISA试剂盒及Western blot的比较

利用重组N蛋白抗原建立了检测猪繁殖与呼吸综合征病毒（PRRSV）抗体的ELISA方法并组装成试剂盒，将试制的3批试剂盒分别与IDEXX生产的试剂盒及Western—blot进行了符合率试验。试验结果表明，所研制的试剂盒与Western blot的符合率达97．67％以上。对460份临床血清分别用自制的试剂盒和IDEXX公司生产的试剂盒进行检测，其中有37份不相符合，用Western blot对这37份血清进行验证，有35份血清检测结果与自制的试刺盒检测结果一致。由此表明，自行研制的试剂盒，其特异性和敏感性均能满足目前临床上该疫病的流行病学分析或免疫抗体检测。     

副结核分枝杆菌MAP0862、MAP1345基因的串联表达及其在间接ELISA中的初步应用

为探索副结核特异性蛋白MAP0862与MAP1345在牛副结核病血清学诊断中的作用,将通过串联表达获得的融合蛋白MAP0862-1345纯化定量后包被酶标板,经过对反应条件的优化,初步建立了基于融合蛋白MAP0862-1345的间接ELISA诊断方法。使用建立的ELISA方法对牛副结核病阳性血清、牛结核病阳性血清、牛布病阳性血清、卡介苗免疫牛血清、健康牛血清检测的结果表明其具有较好的特异性;使用该方法与IDEXX副结核病抗体检测试剂盒共同对300份临床血清样本检测,总符合率为92.7%。