Enzyme-linked immunosorbent assay for detection of bluetongue virus antibodies

The immune response to bluetongue virus in sheep and cattle was studied by applying a newly developed indirect enzyme-linked immunosorbent assay (ELISA). Purified virus obtained by sucrose gradient centrifugation was used at a concentration of 0.01 optical density units (formula: see text) to coat individual wells (200 microliter) of a microtitration plate. Dilution of antigen was performed in 0.05 M carbonate buffer, pH 9.6, and adsorption lasted for at least 16 hours at 4 C. Coated plates retained their activity for 10 weeks when stored at 4 C. Sera recovered from experimentally infected sheep and cattle were tested together with known negative sera. A good correlation between results was obtained with the modified complement-fixation test and the ELISA; however, the ELISA proved to be more sensitive. The group specificity of the ELISA was proven by testing various type-specific sheep and cattle immune sera. The ELISA has potential for the detection of group-specific antibodies to bluetongue virus infection.     

Enzyme-linked immunosorbent assay using solubilised antigen for detection of antibodies toAnaplasma marginale

An enzyme-linked immunosorbent assay (ELISA) was used to detect antibodies against Anaplasma marginale. A marginale bodies were separated from parasitised erythrocytes by a modified nitrogen decompression method, sonicated, then solubilised with Triton X-100 and used as the ELISA antigen. In this ELISA system the required amount of antigen protein was 16.2 ng for each well. In the course of experimental infections, of calves, significant antibody levels were detected by ELISA and the complement fixation test (CFT) at almost the same time. Antibodies against A. marginale were detectable for longer periods using the ELISA than using the CFT. Sera from calves infected with Babesia bigemina, B. bovis, B. ovata, Theileria sergenti and Eperythrozoon wenyoni gave no reaction; however, antisera against A. centrale did react with the A. marginale ELISA antigen.     

Enzyme-linked immunosorbent assay for measurement of allergen-specific IgE antibodies in canine serum

A micro-ELISA, using horseradish peroxidase-conjugated anti-canine IgE and polystyrene microtitration wells for detection of allergen-specific IgE in canine serum, was developed. Specificity of anti-canine IgE was confirmed by reversed cutaneous anaphylaxis evaluations, gel-precipitation reactions, immunoelectrophoresis, immunoaffinity chromatography, and heat inactivation. Individual allergen blanks were used to account for variable nonspecific binding among various allergens, and results were normalized using 4 reference sera. Coefficients of variation for intra-assay and interassay variability ranged from 0.77 to 5.66% and 3.15 to 9.83%, respectively. Results observed with wells coated with mixtures of various allergen extracts yielded results approximately equal to results (average) of wells containing individual components. Agreement between ELISA and skin test results ranged from 43 to 64%, depending on allergen used.     

Enzyme-linked immunosorbent assay for the detection of antibodies to infectious bronchitis virus

An enzyme-linked immunosorbent assay (ELISA) for antibodies to avian infectious bronchitis (IB) virus is described. The immune response of chickens following vaccination with IB virus was monitored using this test, and the titers were compared with those obtained by serum neutralization. The ELISA appears to be suitable for IB serology.     

Enzyme-linked immunosorbent assay for the detection of antibodies to Hypoderma bovis in cattle

An adaptation of the enzyme-linked immunosorbent assay has been shown to detect antibodies in the circulation of cattle infected with Hypoderma bovis. Strong positive reactions were obtained from all infected cattle, even those which harboured only one larva. A strong cross reaction was observed in sera taken from cattle infected with H lineatum, but not in cattle infected with Fasciola hepatica or Ostertagia ostertagi.     

Enzyme-linked immunosorbent assay for detection of antibodies against Mycobacterium paratuberculosis in goats

Using a heat and sonicated Mycobacterium paratuberculosis Cordoba antigen (COA1) and the commercial protoplasmic-antigen (PPA-3) as antigens, an ELISA for detecting goat antibodies was standardized. When 2 reference populations, 1 positive (17 goats) and the other negative (63 goats) to disease, were used, this test showed 87.5% sensitivity and 93.6% specificity for COA1, and 88.2 and 95.2%, respectively for PPA-3. Absorption with M phlei was performed; no significant differences were found for COA1, but a lower sensitivity was found with PPA-3. This test was not especially affected by cross-reactivity with other mycobacterial disease because when 9 goats with M bovis infection were included in the M paratuberculosis control group, the specificity was only slightly different for absorbed (94.4%) and nonabsorbed sera (91.7%) for COA1, and (93.1 and 94.4%, respectively) for PPA-3. This test was used to study the percentage of seropositive goats for M paratuberculosis in 3 herds with different prevalences. Among 251 goats in southern Spain (Huelva), 40% were found positive for COA1 and 41% for PPA-3. Among 242 goats studied in southern Spain (Córdoba), 10.0% were positive for COA1 and 13.0% for PPA-3. In the Canary Island population of 176 goats, 3% were positive for COA1 and 0.5% for PPA-3. According to the accuracies of both positive and negative predictions, our test could be applied to populations with high prevalence to prevent additions to the herd and to cull infected animals (with 40% prevalence, the positive and negative predictive values are 90%), and to prevent adding infected animals to populations with moderate or low prevalence.     

Enzyme-linked immunosorbent assay for the detection of bovine papillomavirus

An enzyme-linked immunosorbent assay has been developed to detect and quantitate bovine papillomavirus in partially purified and in purified viral preparations, using rabbit antiserum against group-specific papillomavirus structural antigens and alkaline phosphatase-labeled affinity purified goat antibody to rabbit immunoglobulin G. Viral detection correlated well with negative-stain electron microscopy of the various preparations and peroxidase-antiperoxidase staining of paraffin sections of the original fibropapillomas. The technique is rapidly done and will detect minute amounts of viral protein (1 ng/ml).     

Enzyme-linked immunosorbent assay for the detection of canine coronavirus and its antibody in dogs.

Two methods of enzyme-linked immunosorbent assay (ELISA) were developed for the diagnosis of canine coronavirus (CCV) infection in dogs. One ELISA, in which CCV-infected CRFK cell lysate is used as antigen, is for the detection and titration of antibody against CCV, and the other ELISA uses the double antibody sandwich method for the detection of CCV antigen. The first ELISA procedure demonstrated antibody responses in dogs inoculated with CCV, as did the virus neutralization test; the second ELISA detected specific CCV antigen in feces and organ homogenates of inoculated dogs.     

Enzyme-linked immunosorbent assay for the detection of antibodies to the sheep scab mite Psoroptes ovis

An enzyme-linked immunosorbent assay has been shown to detect antibodies in the circulation of sheep infected with Psoroptes ovis. Strong positive reactions were obtained from 19 sheep with four-month-old infections. No cross reaction was observed with sera obtained from sheep infected with either Fasciola hepatica, Nematodirus battus, Ostertagia circumcincta or Damalinia ovis.     

Enzyme-linked immunosorbent assay for the detection of porcine epidemic diarrhea coronavirus antibodies in swine sera

An enzyme-linked immunosorbent assay (ELISA) for detecting serum antibodies to the porcine epidemic diarrhea coronavirus (PEDV) was established by using cell culture-grown PEDV as antigen for coating. Ultracentrifugation through 20 and 45% (w/w) sucrose cushions proved to be the best antigen purification method. Examination of 1024 swine sera showed a high specificity and a greater sensitivity of the ELISA, when compared with indirect immunofluorescence. Reference sera with high antibody titers to PEDV originated from two pigs experimentally infected with PEDV. Three different antigen purification methods and the advantages of the ELISA compared with an immunofluorescence test are discussed.     

Enzyme-linked immunosorbent assay for canine serum ferritin, using monoclonal anti-canine ferritin immunoglobulin G

Immunoassay of serum ferritin is currently used to evaluate the clinical iron status of human beings, horses, cattle, and swine. Because ferritins are immunologically species specific, a separate assay must be developed for each species. We have developed an ELISA for serum ferritin in dogs, using a monoclonal anti-canine ferritin antibody. Ferritin standards were linear (r = 0.997) from 0 to 80 ng/ml. Recovery of ferritin from canine serum was 94%. Dilutions of pooled canine serum were linear from 0 to 50% (r = 0.994). Within-assay coefficient of variability was 5.5%, whereas assay-to-assay coefficient of variability ranged from 12.5 to 21%. This assay should provide a nonsurgical means of accurately estimating dogs' iron stores.     

Rapid detection of canine parvovirus in feces using monoclonal antibodies and enzyme-linked immunosorbent assay

Monoclonal antibodies were used to develop a double antibody enzyme-linked immunosorbent assay for the detection of canine parvovirus (CPV) antigen in fecal samples. The assay was specific for the hemagglutinating protein of CPV and detected as little as 1.5 ng of virus within a 15-minute incubation period. The use of monoclonal antibodies against 2 epitopes on the CPV antigen permitted the simultaneous addition of test sample and enzyme-conjugated antibody, thus considerably simplifying the manipulations required for the assay. Results were visually determined without special instrumentation. Clinical studies revealed greater than 95% correlation between enzyme-linked immunosorbent assay results and hemagglutination titers.     

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 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