Enzyme linked immunosorbent assay for detection of antibodies againstBesnoitia besnoiti in cattle

The use of the ELISA method for the detection of antibodies to B. besnoiti in cattle is described and compared to the IFAT technique. One hundred and twenty-one sera were examined, of which 61 were sera of calves experimentally infected with B. besnoiti, 52 sera from field animals and eight were sera with high titres of antibodies to other parasites. The specificity of both assays correlates but ELISA seemed to be more sensitive. The ELISA technique provides a rapid and reliable method for the screening of B. besnoiti infection in cattle.     

Enzyme linked immunosorbent assay (ELISA) for the detection of antibodies to porcine cytomegalovirus.

The ELISA and indirect immunofluorescence test were compared on 56 porcine sera which were tested for antibodies to porcine cytomegalovirus. Viral antigens were prepared in cells of a pig fallopian tube line. The ELISA was found to be a sensitive reproducible and practical test to measure specific antibodies to this infection.     

Enzyme-linked immunosorbent assay for detecting antibodies to Brucella abortus in bovine milk and serum

An enzyme-linked immunosorbent assay (ELISA) method was evaluated for the detection of antibodies to Brucella abortus in cows milk. Milk samples from seropositive or -negative cows were sed to determine the distribution of absorbance values to classify milk as ELISA positive or ELISA negative. Brucella abortus was isolated from milk samples from 10 (45%) of the 22 cows whose milk and serum were ELISA positive. The ELISA was evaluated and determined to be an appropriate method for detecting antibodies to B abortus in bovine milk.     

An indirect enzyme linked immunosorbent assay for the detection of bovine antibodies to multiple Leptospira serovars

An indirect enzyme linked immunosorbent assay was developed for the detection of bovine antibodies to multiple pathogenic Leptospira serovars, including canicola, copenhageni (represents icterohaemorrhagiae), grippotyphosa, hardjobovis, pomona, and sejroe. The antigen utilized in this assay was a sonicated mixture of equal parts of killed whole cells of each of the 6 serovars named above. A mouse monoclonal antibody against bovine immunoglobulin (Ig)G₁ that was conjugated with horseradish peroxidase was used for detection of bound antibodies. This assay was evaluated with sera (n = 3107) that were microscopic agglutination test (MAT)-negative (at a 1:100 dilution) for each of the 6 serovars listed above and sera (n = 601) that were MAT-positive (at a 1:100 dilution) for 1, or any combination of the 6 listed serovars. In addition, sera from serial weekly bleedings of cows, which were individually experimentally infected with serovars hardjobovis, copenhageni, grippotyphosa, or canicola, were also tested in this assay.

At an optimal cut-off point determined by receiver operating characteristic (ROC) curve analysis, the relative sensitivity and specificity of the assay were 93.5% (95% confidence interval = 91.2% to 95.3%) and 94.7% (95% confidence interval = 93.9% to 95.5%), respectively. This assay was able to detect antibody in the sera of animals experimentally infected with serovar hardjobovis as early as 1 week postinoculation

     

Enzyme-linked immunosorbent assay to detect subgroup-specific antibodies to avian leukosis viruses

An enzyme-linked immunosorbent assay (ELISA) was developed to detect antibodies against avian leukosis viruses (ALV), using antigens extracted from Rous-associated virus-inoculated chicken embryo fibroblast (CEF) cells by Nonidet P-40 treatments. The antigens reacted strongly to sera of chickens immunized with antigenically homologous viruses, but weakly to those of chickens immunized with heterologous viruses. Antigens extracted from noninoculated CEF cells by the same procedures did not react to either of the immune sera. Normal control sera did not react to any of the antigens. Reactivities of immune sera were decreased markedly by the sera adsorbing with homologous Rous-associated virus-inoculated CEF cells, but not with heterologous CEF cells. The ELISA-specific optimal doses (the differences between the optimal doses with antigens from ALV-inoculated and noninoculated CEF cells) were correlated strongly with the virus-neutralization titers (r = 0.876, P less than 0.01). Examination of the antibody response from ALV-inoculated chickens revealed that ELISA detected antibodies at the same time or several weeks earlier than did the virus-neutralization test.     

Dot-enzyme linked immunosorbent assay as an alternative technique for the detection of bovine respiratory syncytial virus (BRSV) antibodies

A dot-ELISA test for the detection of anti-BRSV antibodies is described. The objective of this study was the standardisation of a test as a fast, inexpensive and effective alternative to detect anti-BRSV antibodies. Its sensitivity, specificity and usefulness were compared to a commercial ELISA-kit and to the standard serum neutralisation (SN) test. The standardisation of the technique was done using nitrocellulose disks soaked with a viral sample isolated in Brazil, BRSV-25-BR. The best results were obtained when the disks were sensitised with a purified antigen at a concentration of 0.7 microg/disk and the bovine serum was diluted 1: 200. The experiment used 423 samples of bovine serum collected in the main cattle breeding centres in Brazil. The standard SN, dot-ELISA technique and commercial ELISA kits scored 67.8%, 71.8% and 72.3% of the samples as positive, respectively. When compared to the SN test, the standardised dot-ELISA and the commercial ELISA tests presented relative sensitivities of 92.3% and 91.6% and relative specificities of 71.3% and 68.4% respectively. The results demonstrated that the dot-ELISA test is adequate for the objectives proposed by this study, being easy to use and economically viable. Thus, this test represents an alternative for BRSV serological diagnosis in the substitution of SN and commercial ELISA tests, recommendable for utilisation in laboratories with few resources.     

Development and evaluation of an enzyme-linked immunosorbent assay for detection of bovine antibodies to epizootic hemorrhagic disease of deer viruses.

An indirect enzyme-linked immunosorbent assay (I.ELISA) is described for detection of bovine serum antibody to epizootic hemorrhagic diseases of deer virus (EHDV). Serum samples, at a dilution of 1:200, were incubated with group-specific EHDV antigens, pre-adsorbed to microtiter plates. Bound antibodies were detected by a murine monoclonal antibody to bovine immunoglobulin (Ig)G1 (heavy-chain specific) conjugated with horseradish peroxidase. The performance of the I.ELISA in detecting antibodies to EHDV in sequential serum samples from calves experimentally infected with serotypes 1,2,3 and 4 was evaluated. The I.ELISA detected EHDV antibodies from 14 days postinfection when seroconversion by the standard agar gel immunodiffusion (AGID) test was also evident. The group-specific antibodies to EHDV increased exponentially during the first two to four weeks postinfection and remained relatively stable for about 12 months in some calves. Unlike observations with the AGID test, no reaction was seen in the I.ELISA between blue-tongue virus (BTV) antigen and sera from calves given a single dose of EHDV. The performance of the I.ELISA and AGID were compared using 3,135 AGID negative bovine field sera from herds in Ontario, Alberta and British Columbia and 130 AGID positive samples collected from cattle in 1987 and 1988 during and after outbreaks of EHD in the Okanagan Valley, British Columbia. The specificity and sensitivity of the assay relative to the AGID test were 99.3% and 91.5% respectively, with an overall agreement of 99.0% between the tests.(ABSTRACT TRUNCATED AT 250 WORDS)     

Simultaneous screening of bovine sera for antibodies to bluetongue and epizootic hemorrhagic disease of deer viruses by enzyme-linked immunosorbent assay.

An indirect enzyme-linked immunosorbent assay (I-ELISA) is described for simultaneous screening of bovine sera for detection of antibodies to bluetongue (BT) and epizootic hemorrhagic disease of deer (EHD) viruses (V). Optimal dilutions of BTV and EHDV antigens were combined and allowed to absorb on to the wells of microtiter plates. Appropriately diluted (1:100) bovine sera were allowed to incubate and the bound antibodies were detected by a murine monoclonal antibody (MAb) to bovine immunoglobulin (H-Chain) conjugated with horseradish peroxidase. The performance of the combined (C) I-ELISA in detecting antibodies to BTV and EHDV in sequential serum samples from calves experimentally inoculated with BTV, serotype 10, EHDV, serotype 1 (New Jersey) or EHDV serotype 2 (Alberta) was evaluated. Comparable antibody profiles were demonstrable by the CI-ELISA and separate I-ELISAs using either BTV or EHDV antigens. The results suggest that the CI-ELISA offers many advantages over the standard agar gel immunodiffusion (AGID) test and has potential application as a rapid, sensitive, inter-group-specific and inexpensive test for simultaneous screening of bovine sera for antibodies to BTV and/or EHDV.     

Development of an enzyme linked immunosorbent assay (ELISA) for the detection of bovine serum antibody to bovine viral diarrhoea virus

An enzyme linked immunosorbent assay (ELISA) was developed to detect antibody to bovine viral diarrhoea virus (BVDV) in bovine serum. The ELISA results were compared with those of the serum neutralisation test (SNT) using serums from 6 experimentally infected calves bled at intervals from 0 to 154 days postinfection and 886 field samples. The optical density (OD) produced by a single dilution of test serum was compared with a standard curve and the result expressed in ELISA units. Despite wide variation between absolute ELISA and SNT results, an agreement of 97% was obtained when reciprocal SNT titres greater than or equal to 8 and ELISA units greater than or equal to 10 were taken as indicative of a specific reaction. The ELISA was shown to be an efficient method of measuring antibody in bovine serum samples and would assist in any large scale screening of cattle herds for BVDV antibody.     

Evaluation of a covalent mix-enzyme linked immunosorbent assay for screening of Salmonella antibodies in pig serum

In this study, a commercial Salmonella covalent mix-enzyme linked immunosorbent assay (ELISA) for serological detection of Salmonella infection in swine was evaluated by comparing it with the conventional fecal culture method and inter-laboratory proficiency testing, using a panel of sera tested in 5 laboratories from Europe and North America. Comparison with culture results showed that 88.5% of 26 culture-positive animals were ELISA positive, as were 55% of 60 animals from 2 culture-positive pig herds. Of 90 animals from 2 high health farms with no clinical symptoms of salmonellosis, 98.9% tested negative. The interlaboratory comparison study found a kappa value of 0.9 between our laboratory (using an automated system) and the manufacturer laboratory (using the manual method). Comparison of ELISA results from all 5 participating laboratories showed very good to excellent agreement, between 85% and 97.5%. We found this assay to be useful for the screening of antibodies against Salmonella present in swine serum. It agrees well with bacterial cultures, is reproducible, sensitive, specific, repeatable, and suitable for automation.     

Enzyme-linked immunosorbent assay for antibodies to Campylobacter fetus in bovine vaginal mucus

An enzyme-linked immunosorbent assay (ELISA) was used to detect antibodies to Campylobacter fetus subspecies venerealis in bovine vaginal mucus. The results of testing 168 samples from experimentally infected, field cases and control cows showed that the ELISA was more sensitive than the vaginal mucus agglutination test and also detected antibodies in earlier stages of infection.     

Detection of antibodies against reticuloendotheliosis viruses by an enzyme-linked immunosorbent assay

A microplate indirect enzyme-linked immunosorbent assay (ELISA) for antibodies against reticuloendotheliosis virus (REV) was consistently more sensitive than indirect immunofluorescent-antibody tests. Limits of antibody detection were comparable to those obtained in virus neutralizations. Detection of REV-infected chickens long after infection and after immunofluorescent antibody has waned makes ELISA especially suitable for screening chicken flocks.     

An enzyme-linked immunosorbent assay for enzootic bovine leukosis virus antibodies

An enzyme-linked immunosorbent assay (ELISA) for detecting antibodies to enzootic bovine leukosis (EBL) virus is described and its sensitivity compared with that of the agar gel immunodiffusion test (AGIDT) using 198 sera collected in Great Britain. There was 95 per cent agreement between the ELISA and AGIDT, when sera with positive/negative ratio (P/N) values of 1 . 5 or greater were considered positive. A total of 259 out of 264 sera (98 per cent) collected in Northern Ireland had P/N values of less than 1 . 5, the remaining sera having P/N values of 1 . 5 and 1 . 6. As Northern Ireland is clinically and serologically free of EBL infection it is proposed that sera with P/N values of 1 . 5 and 1 . 6, which account for approximately 3.5 per cent of the total sera tested, are considered doubtful and should be tested by another serological test.     

Enzyme-linked immunosorbent assay for the detection of antibodies to bovine viral diarrhea virus in bovine sera

A specific and sensitive enzyme-linked immunosorbent assay (ELISA) was established for the detection of antibodies to bovine viral diarrhea virus (BVDV) in bovine sera. Polyethylene-glycol concentrated, equilibrium density gradient purified BVDV was used as test antigen at an optimal amount of 1 microgram/well, whereas the optimal concentration of conjugate was at 1/2000 dilution. The standardized test encountered no non-specific reaction with test sera at a starting dilution of 1/10. A total of 50 bovine serum samples was assayed for the presence of antibodies against BVDV by ELISA and serum neutralization test (SNT). A positive correlation between the 2 tests was found. However, ELISA could be as much as 500-fold more sensitive than SNT in detecting low levels of BVDV antibodies.     

An enzyme-linked immunosorbent assay for detection of antibodies against bovine pestivirus

A competitive enzyme-linked immunosorbent assay was developed and compared with the serum neutralisation test for bovine pestivirus using 508 cattle sera and serial serum samples from a goat hyperimmunized with five bovine pestivirus isolates. There was 96.7% agreement between the two tests. The relative sensitivity of the enzyme-linked immunosorbent assay compared to the serum neutralisation test was 95.2% and the relative specificity was 99.4%. The titres of individual animals in the assay did not show a close correlation with serum neutralisation test titres. This may be because the antibodies measured in the two tests are directed against different viral proteins. The enzyme-linked immunosorbent assay has the advantage of being quicker and cheaper than the serum neutralisation test. The configuration used in the ELISA means sera from all species can be tested for pestivirus antibody using the same set of reagents.     

Blocking enzyme-linked immunosorbent assay for detection of antibodies against bovine enterovirus

A blocking enzyme-linked immunosorbent assay (ELISA) was developed for detecting antibodies against bovine enterovirus (BEV) in bovine sera. In this ELISA, bovine serum samples were allowed to react with captured viral antigens (by specific chicken IgG), before the addition of specific mouse IgG for measuring non-occupied viral epitopes. The ELISA was slightly more sensitive and required a shorter time period than traditional serum neutralisation (SN). Among the 871 bovine serum samples tested so far, the titres produced by this assay had a significant correlation with those recorded by SN. The ELISA could be used as an alternative assay for SN in a large-scale BEV antibody investigation.     

牛尿中玉米赤霉醇残留酶联免疫检测方法的研究

在制备了玉米赤霉醇单克隆抗体的基础上 ,建立了牛尿中玉米赤霉醇残留的 EL ISA检测方法 ,确定了各种溶液的最适工作浓度 ,并对最低检测限、5 0 %抑制浓度和空白牛尿添加回收试验进行了研究。本方法的最低检测限为 0 .6 ng/ml,5 0 %抑制浓度为 3.0 ng/ml,以 10、2 1和 35 ng/ml浓度添加空白牛尿 ,回收率在 70 .0 %～ 116 .0 %之间 ,变异系数在 6 .0 %～ 15 .9%之间。此方法快速、灵敏、方便 ,满足了牛尿中玉米赤霉醇残留检测的要求     

Enzyme-linked immunosorbent assay for detecting antibodies to Aujeszky's disease virus in pigs

An indirect micro enzyme-linked immunosorbent assay (ELISA) for detecting antibodies to Aujeszky's disease virus in pigs is described. A control antigen prepared from infected cells was included for each serum tested. Of 243 sera from serologically positive farms, 175 (72 per cent) and 147 (60 per cent) were positive by the ELISA test and microtitre serum neutralisation test, respectively. Failure to include a control antigen for each serum would have resulted in 14 sera (6 per cent) being differently recorded. Results for sera from experimental and field infections indicated that seroconversion was more quickly detected by the ELISA test than the microtitre serum neutralisation test. In addition to greater sensitivity the ELISA test has other advantages over the serum neutralisation test. ELISA is a rapid, cheap test which is not dependent on a continuous supply of cell cultures and which can be readily automated.