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1.
用酶标记抗传染性法氏囊病毒（ＩＢＤＶ）单克隆抗体，建立夹心阻断ＥＬＩＳＡ，检测ＩＢＤＶ鸡血清抗体。用Ｄ７８细胞毒免疫２４日龄的雏鸡，每周采血一次，共４次，用夹心阻断ＥＬＩＳＡ、微量细胞中和试验（ＶＮ）、琼脂扩散试验（ＮＧＰ）检测鸡血清抗体，结果表明：夹心阻断ＥＬＩＳＡ同ＶＮ之间具有较高的相关性（ｒ＝０．８１２６），与ＡＧＰ的相关性较低（ｒ＝０Ｘ．７５７５）。  相似文献   

2.
采用鸡传染性法氏囊病（ＩＢＤ）病毒（ＩＢＤＶ－２５１２）研制了检测ＩＢＯＶ抗体的ＥＬＩＳＡ试剂盒。抗原最佳包被浓度为５．１２μｇ／ｍｌ，被检血清最佳稀释度为１：２００，酶标结合物最佳工作浓度１：１０００．本试剂盒与美国ＫＰＩＩＢＤ－ＥＬＩＳＡ试剂盒比较，结果敏感性、特异性、重复性均达到美国同类产品水平，且易于操作．在４℃可稳定保存６个月、对北京地区４个非免疫鸡群的１０３只鸡测得其阳性率为５４％，对４５只ＳＰＦ鸡的阳性率为０％。  相似文献   

3.
针对鸡新城疫病毒（ＮＤＶ）弱毒株Ｆ蛋白前体（Ｆ０）Ｆ２片段的特异结构,人工合成特异性多肽,将多肽与牛血清白蛋白（ＢＳＡ）化学偶联制和轩成全抗原后免疫小鼠,制备抗多肽血清。经ＥＬＩＳＡ检测,该抗体与ＭＤＶ弱毒株呈阳性反应,而与鸡痘病毒（ＦＰＶ）,鸡传染性腔上囊病病毒（ＩＢＤＶ）,ＭＤＶ强毒Ｆ４８Ｅ８株和四平析呈阴性反应,试验结果证明该抗体可以用于鉴别ＮＤＶ弱毒株。  相似文献   

4.
建立了一种以单一血清稀释度定量检测鸡血清中ＮＤＶ抗体的间接ＥＬＩＳＡ方法，经试验得出回归方程ＬｎＥＴ＝５．８７８＋０．５３７Ｐ／Ｎ．该方法特异性强，能被ＮＤＶ单抗所特异性阻断，比ＨＩ方法敏感。通过对ＳＰＦ鸡等进行的不同疫苗免疫前，后抗体动态测定表明，ＥＴ值与ＨＩ值之间呈一定的相关性，强毒攻击试验表明，ＥＴ值＞４０００时鸡群能得以保护，装配成的商品试剂盒保存期长达６个月。  相似文献   

5.
以抗鸡新城疫病毒（ＮＤＶ）单抗夹心ＥＬＩＳＡ试验为基础，在６０００，建立了ＰＥＧ－ＥＬＩＳＡ法，使用此法检测临诊样品，与单抗夹心ＥＬＩＳＡ相比，时间缩短７０分钟且提高了ＯＤ４９０值，易于识别。结果表明，ＰＥＧ－ＥＬＩＳＡＩ法具有实际应用价值。ＰＥＧ能加强抗原－抗体反应的速率和强度，这种效应在固相夹心ＥＬＩＳＡ第二步表现尤其明显。ＰＥＧ的这种非特异性效应对于检测其它抗原或抗体的ＥＬＩＳＡ试验可能具有  相似文献   

6.
作者建立了两种抗原捕捉酶联免疫吸附试验方法（多抗和单抗ＡＣ－ＥＬＩＳＡ），用于滴定用不同宿主系统增殖的传染性氏囊毒病，这些宿主系统包括ＢＧＭ－７０传代细胞系。鸡胚成纤维细胞和鸡法氏囊，两种检测方法都有较高的特异性，但是多克隆ＡＣ－ＥＬＩＳＡ比单克ＡＣ－ＥＬＩＳＡ更加敏感（Ｐ〈０．０５），结果还表明，滴定ＩＢＤＶ抗原的常规方法（细胞培养物和鸡胚）比多抗ＡＣ－ＥＬＩＳＡ更敏感。  相似文献   

7.
单抗介导的斑点ELISA检测鸡传染性支气管炎病毒的研究   总被引：1，自引：0，他引：1  
用群特异性的单克隆抗体作第一抗体,用酶标兔抗体ＩＢＶＩｇＧ作第二抗体,建立夹心法Ｄｏｔ－ＥＬＩＳＡ程序检测鸡传染性支气管炎病毒抗原。试验表明,本程序检测ＩＢＶ抗原高度敏感性和特异性。最低抗原检出量为０．５μｇ,约１００个气管环半数感染量（１００ＴＯＣＩＤ５０）,阳性检出率为９６％。  相似文献   

8.
用ＩＢＤ－ＥＬＩＳＡ快速诊断盒对ＩＢＤＶ阳性法氏囊囊毒及ＩＢＤ阳性出血清，ＩＢＤＶ阴性法氏囊及ＩＢＤ阴性血清和ＩＢ，ＩＬＴ，ＥＳＤ－７６，ＲＥＯ，ＮＤ，ＭＤ等６种鸡传染病的抗原及阳性血清检测，只有ＩＢＤＶ阳性法氏囊囊毒及阳性血清呈阳性反应，证明快速诊断盒对ＩＢＤＶ法氏囊囊毒抗原及其抗体的检测是特异的，与其它６种传染病的抗原和抗体无效叉反应。与ＡＧＰ对比试验结果表明，检测ＩＢＤＶ阳性法氏囊囊毒时，快  相似文献   

9.
建立了检测鸡痘病毒抗体的ＰＰＡ－ＥＬＩＳＡ。用该法检测７５份鸡血清样品，鸡痘病毒抗体检出率（２１．３３％）高于琼脂扩散试验（１０．６７％）。经血清抗体吸收和重复性试验，证明ＰＰＡ－ＥＬＩＳＡ法具有特异性强和重复性好特点。此外，对４种封闭液的封板结果表明：１０％犊牛血清ＰＢＳ／Ｔ效果最好，次之为０．２５％白明胶ＰＢＳ／Ｔ、０．５％牛血清白蛋白ＰＢＳ／Ｔ、０．１％牛血清白蛋白ＰＢＳ／Ｔ。  相似文献   

10.
用PPA—ELISA检测鸡痘病毒抗体的研究   总被引：7，自引：0，他引：7  
建立了检测鸡痘病毒抗体的ＰＰＡ－ＥＬＩＳＡ，用该法检测７５份鸡血清样品，鸡痘病毒抗体检出率（２１．３３％）高于琼凝扩散试验（１０．６７％），经血清抗体吸收和重复性试验，证明ＰＰＡ－ＥＬＩＳＡ法具有特异性强和重复性好特点，此外，对４种封闭液的封板结果表明，１０％犊牛血清ＰＢＳ／Ｔ效果最好，次之为０．２５％白明胶ＰＢＳ／Ｔ，０．５％牛血清白蛋白ＰＢＳ／Ｔ，０．１％牛血清白蛋白ＰＢＳ／Ｔ。  相似文献   

11.
A recombinant antigen-based single serum dilution enzyme-linked immunosorbent assay (ELISA) was developed to measure the specific antibody activity in sera of dogs with leptospirosis. The recombinant antigen developed and used in the assay was specific for the pathogenic serovars of Leptospira. A linear relationship was found to exist between the predicted antibody titres at a single working dilution of 1:1000 and the corresponding observed serum titres as determined by the standard serial-dilution method. Regression analysis was used to determine a standard curve from which an equation can be derived that allows demonstration of the mentioned correlation. The equation was then used to convert the corrected absorbance readings of the single working dilution directly into the predicted ELISA antibody titres. The assay was proved to be sensitive, specific and accurate as compared to the standard microscopic agglutination test (MAT).  相似文献   

12.
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.  相似文献   

13.
Serial dilution and single dilution enzyme linked immunosorbent assays (ELISA) were standardised and their sensitivity and specificity were compared for serodiagnosis of Babesia equi infection. The antibody titres of 24 donkey sera of known identity were determined separately by serial dilution ELISA using three different B. equi antigens namely whole merozoite (WM), cell membrane (CM) and high speed supernatant (HSS). The ratios of the optical density (OD) of known positive and known negative sera at different serum dilutions were calculated and termed as the positive/negative (P/N) ratio. The coefficients of correlation (r) were calculated between the P/N ratios at different dilutions of sera and the log10 antibody titres of the same sera were ascertained by serial dilution ELISA. The highest value of 'r' was obtained at a serum dilution of 1:200. From log10 antibody titre of sera (y) and their P/N ratio at a dilution of 1:200 (x), regression equations (y = a + bx) were calculated separately for the three antigens. Test sera were diluted to 1:200, their OD were read in duplicate wells and were converted to the P/N ratio. Antibody titres were predicted from the P/N ratio using a regression equation separately for the three antigens. Titres obtained by both ELISAs were not significantly different from each other, thus confirming that single dilution ELISA could be successfully used to replace conventional serial dilution ELISA. The sensitivity, specificity and predictive value of single dilution ELISA was validated statistically using 42 B. equi disease-positive sera and 106 B. equi disease-negative sera. The WM antigen was found to be the most sensitive with a higher predictive value for negative test sera as compared to the CM or HSS antigens. Sera positive for other equine infections including Babesia caballi showed no cross-reaction with the three B. equi antigens in ELISA, thus the test was immunologically specific. Antibody titres of 109 unknown field donkey/horse sera obtained by serial and single dilution ELISA using the WM antigen did not show any significant difference. Since the single dilution ELISA was found to be more economical, convenient, sensitive, specific than the serial dilution ELISA and has a high predictive value, it is suitable for use in sero-epidemiological studies on B. equi infections in the field.  相似文献   

14.
An enzyme-linked immunosorbent assay (ELISA) was developed to measure specific antibody activity in sera of chickens exposed to Newcastle disease virus (NDV). A near-linear relationship existed between the log of the corrected absorbance of antisera at a single working dilution and the corresponding observed serum titers as determined by a standard serial-dilution method. Regression analysis was used to construct a standard curve and extract an equation from this relationship. The equation was used to convert corrected absorbance readings of the single working dilution directly into predicted ELISA antibody activity titers. In a comparative study, a correlation (P less than 0.01) was found between ELISA and hemagglutination-inhibition (HI) antibody titers to NDV. ELISA titers were as much as 160 times greater than the HI titers. ELISA was also able to detect much lower levels of antibody activity than the HI test.  相似文献   

15.
猪非典型瘟病毒(APPV)是新近发现的仔猪先天震颤的病原体,其血清学检测方法亟待建立.E^rns蛋白是APPV诱导机体产生中和抗体的重要保守性抗原,是血清学检测的特异性靶标之一.本研究制备了猪非典型瘟病毒原核表达的Erns蛋白,以其作为包被抗原,建立了间接ELISA检测方法.结果表明,最佳抗原包被浓度为8μg/mL,最佳血清稀释度为1:10,阴阳性临界值D450=0.318,灵敏度达到1:64,特异性好,与猪瘟、猪伪狂犬、猪繁殖与呼吸障碍综合征、猪流感及圆环病毒病的阳性血清均不发生交叉反应.该检测方法重复性好,批内变异系数最大值为7.84%,批间变异系数最大值为8.57%.利用建立的间接ELISA方法对50份猪临床血清样本进行了检测,其阳性率为10%.基于Erns蛋白间接ELISA检测方法的建立为APPV的流行病学调查和临床诊断提供了有效的工具.  相似文献   

16.
An antibody-capture enzyme-linked immunosorbent assay (ELISA) for detection of antibody to turkey coronavirus (TCV) utilizing infectious bronchitis virus (IBV) antigen was developed. Anti-TCV hyperimmune turkey serum and normal turkey serum were used as positive or negative control serum for optimization of the ELISA system. Goat anti-turkey immunoglobulin G (light plus heavy chains) conjugated with horseradish peroxidase was used as detector antibody. The performance of the ELISA system was evaluated with 45 normal turkey sera and 325 turkey sera from the field and the cutoff point was determined. Serum samples of turkeys experimentally infected with TCV collected sequentially from 1 to 63 days postinfection were applied to the established antibody-capture ELISA using IBV antigens. The optimum conditions for differentiation between anti-TCV hyperimmune serum and normal turkey serum were serum dilution at 1:40 and conjugate dilution at 1:1600. Of the 325 sera from the field, 175 were positive for TCV by immunofluorescent antibody (IFA) assay. The sensitivity and specificity of the ELISA relative to IFA test were 93.1% and 96.7%, respectively, based on the results of serum samples from the field turkey flocks using the optimum cutoff point of 0.18 as determined by the logistic regression method. The ELISA values of all 45 normal turkey sera were completely separated from that of IFA-positive sera. The ELISA results of serum samples collected from turkeys experimentally infected with TCV were comparable to that of the IFA assay. Reactivity of anti-rotavirus, anti-reovirus, anti-adenovirus, or anti-enterovirus antibodies with the IBV antigens coated in the commercially available ELISA plates coated with IBV antigens could be utilized for detection of antibodies to TCV in antibody-capture ELISA.  相似文献   

17.
A fast, sensitive and reliable flow cytometry-based (FACS = fluorescence activated cell sorting) immunofluorescence inhibition assay (FACS-IFI) for the detection of virus-specific antibodies in sera is described. The method was evaluated using sera from cattle experimentally infected with bovine viral diarrhea virus (BVDV). Virus-infected cells, which were fixed and permeabilized, were incubated with diluted sera from immunized or control animals. Monoclonal antibodies (mabs) against different viral proteins were added, and detected with ALEXA488-conjugated goat-antimouse antibodies. The fluorescence signals were detected by flow cytometry and determined as mean channel values. Results were expressed as percent fluorescence inhibition compared to standardized negative sera. The FACS-IFI test with sera from experimentally infected animals was highly sensitive and specific. Comparison of the FACS-IFI results with a commercially available blocking ELISA, an indirect ELISA and the standard serum neutralization test showed a strong correlation. Furthermore, the detection of protein-specific antibodies was possible using the FACS-IFI test.  相似文献   

18.
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   

19.
A blocking enzyme-linked immunosorbent assay (ELISA) has been adapted to detect specific antibodies in bovine sera to respiratory syncytial virus using a horseradish peroxidase-labeled monoclonal antibody to the fusion protein of the virus. This assay plus an indirect blocking ELISA and indirect ELISA were used to detect antibodies to the bovine respiratory syncytial virus (BRSV) in 159 field-origin bovine sera. Results of these assays were compared with serum antibody titers measured by the serum neutralization (SN) test. Over a 56-day period, the mean neutralization titers and the mean delta absorbance values for the blocking ELISA, on the same sera, showed similar declines. However, the calculated correlation coefficients between mean SN titer and mean absorbance value for the blocking ELISA of the individual sera ranged from -0.2 to -0.5 depending on the source of sera. Similar values were obtained whether using crude or purified viral antigen in the assays. Corresponding calculated correlation coefficients were generally higher for the indirect blocking ELISA or indirect ELISA than for the blocking ELISA. The blocking ELISA was between 70 and 64% as sensitive as the serum neutralization test with a specificity of 100 or 90% using the crude and purified viral antigen, respectively. The indirect blocking ELISA and indirect ELISA had similar calculated sensitivities and specificities. The blocking ELISA was faster to run than either of the other ELISA's or the neutralization test. Further, nonspecific background absorbance was obviated because the blocking ELISA detects antibodies to 1 specific viral protein, the fusion protein. These studies suggest that the blocking ELISA should be useful as a serological test for BRSV antibodies.  相似文献   

20.
Infectious bursal disease virus (IBDV) causes a contagious immunosuppressive disease in chickens. The aim of the present study is to develop an enzyme-linked immunosorbent assay (ELISA) using the expressed VP2 or VP3 protein of IBDV as the coating antigen for detecting antibodies to IBDV. Experimental results were compared with virus neutralization assay and a commercial-available ELISA. These assays were used to examine the sera from farm chickens and chickens vaccinated experimentally. The VP3-based ELISA had a higher correlation coefficient (R(2)) of 0.812 with a commercial ELISA kit at a serum dilution of 1:500 than that of VP2-based ELISA (R(2)) of 0.671. The relative sensitivity between virus neutralization and VP2-ELISA and VP3-ELISA was 96% (251/262) and 100% (262/262), respectively, and that between virus neutralization and a commercial ELISA was 99% (257/261). Additionally, compared with virus neutralization assay, the reference technique for diagnosing IBDV, VP3-based ELISA had an agreement value of 99%, superior to that of VP2-based ELISA (95%) or the commercial kit (89%). These results revealed that the capability of either VP2-ELISA or VP3-ELISA in detecting the field chicken sera was comparable to the commercial one, which is generally used to replace the virus neutralization assay. However, the preparation of VP3 is derived from an Escherichia coli expression system with a high yield and purification efficiency by Ni(2+)-NTA gels, which is more favorable to the insect cell-derived particles formed by VP2. Therefore, VP3-ELISA could be developed as an efficient and low cost diagnostic method for IBDV infection in field chickens.  相似文献   

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