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.     

Evaluation of an enzyme-linked immunosorbent assay for the detection of transmissible gastroenteritis virus antibodies

An enzyme-linked immunosorbent assay (ELISA) using a detergent-solubilized antigen of purified virus was developed for detection of antibody against porcine transmissible gastroenteritis (TGE) virus in swine serum. The ELISA demonstrated antibody responses in pigs immunized intramuscularly with the attenuated TO-163 strain of TGE virus and in pigs orally infected with the virulent Shizuoka strain of the virus. The results of the ELISA were well correlated with those of the neutralization test. These results indicate the usefulness of the ELISA as a serological tool for TGE virus antibody.     

Enzyme-linked immunosorbent assay for detection of antibody to avian encephalomyelitis virus in chickens

An enzyme-linked immunosorbent assay (ELISA) for the detection of antibody to avian encephalomyelitis virus (AEV) has been developed for determining whether existing AEV control programs adequately protect breeder hens. A partially purified AEV antigen was bound to microcuvettes for reaction with specific primary antibody. A second antibody, rabbit anti-chicken immunoglobulin G (IgG) conjugated with horseradish peroxidase, was employed to react with bound primary IgG. The relative amount of bound primary IgG was detected using ortho-phenylenediamine as a substrate for enzymatic production of a chromogen by horseradish peroxidase. Intensity of absorbance of the chromogen at 490 nm was related to the bound primary antibody by the titration method. Negative antisera were surveyed to establish an appropriate positive/negative cutoff level at twice the mean absorbance of negative sera at a 1:100 dilution. The test reagents for the ELISA were optimized by reagent titrations utilizing known positive and negative antisera for discrimination. The optimized ELISA had a coefficient of variation of from 1.2 to 3.3 for within-assay titer and of 2.4 for between-assay mean titer. Even though the ELISA detected only specific IgG, it was as accurate as the virus-neutralization test for evaluating the immune status of hens to AEV. Moreover, the ELISA was more economical in the use of reagents, time, and personnel and was free from dependence on susceptible embryos. Since ELISAs can be standardized and measured with manual or automated instruments, the derived ELISA can be easily and economically used to evaluate the immune status of breeder hens in commercial poultry operations.     

Enzyme-linked immunosorbent assay for Brucella ovis specific antibody in ram sera

An enzyme-linked immunosorbent assay (ELISA) for the detection of Brucella ovis specific antibody in ram serum was compared with the currently employed complement fixation test (CFT). Rabbit anti-sheep IgG coupled to horseradish peroxidase was used as the antibody-enzyme conjugate and 2,2'-azino-di[3-ethylbenzthiazolin sulphonate (6)] as the substrate. The ratio of the optical density at 414 nm for positive and negative control sera (P/N ratio) was used to optimise the parameters of the test. Ram serum samples (16,527) were tested using ELISA and CFT (warm and cold) over a one year period. The ELISA was more sensitive and provided a more reliable measure of B ovis specific antibody than did the CFT. Implications of employing ELISA as the sole test in an eradication scheme are discussed.     

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 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.     

Leukocyte-aggregation assay for transmissible gastroenteritis of swine.

An in vitro leukocyte-aggregation assay was developed to detect the exposure of swine to transmissible gastroenteritis virus. Leukocytes in heparinized blood samples aggregated when mixed with test antigen prepared from transmissible gastroenteritis-infected swine testicle cell cultures. Twenty-two of 23 swine exposed 3 days or more were positive or suspects in the assay; 6 nonexposed swine were negative. Aggregation was shown as early as 3 days postexposure in 1 sow and persisted for as long as 14 months in another. Persistence of the assay was proved by repeated evaluations on 2 experimentally exposed swine.     

Enzyme-linked immunosorbent assay for detection of antibodies to Rift Valley fever virus in ovine and bovine sera

An enzyme-linked immunosorbent assay (ELISA) was developed for the rapid detection of antibodies to Rift Valley fever virus (RVFV) in ovine and bovine sera. Conditions to reduce nonspecific reactions were optimized. The ELISA results correlated with those of a plaque-reduction neutralization test, revealing 100% specificity and 90.7% sensitivity. In sera from sheep and cattle inoculated against RVFV, the hemagglutination-inhibition test in combination with the ELISA provided a better indication of response to killed RVFV vaccine than did either test alone.     

Enzyme-linked immunosorbent assay for the detection of antibodies to bovine virus diarrhea virus in sera from border disease virus-infected sheep.

An enzyme-linked immunosorbent assay (ELISA) was established for the rapid detection of specific antibodies against the causative agent of border disease in ovine sera. Polyethylene-glycol concentrated, equilibrium density gradient purified bovine virus diarrhea virus was used as test antigen. The optimal amount of antigen was 0.5 microgram/well, and the optimal concentration of conjugate was at 1/4,000 dilution. A total of 20 ovine serum samples, which had been collected from animals with or without border disease, were compared by ELISA and serum neutralization test for the detection of border disease-specific antibodies. ELISA was shown to be equally specific but less time-consuming and easier to perform than serum neutralization test. A positive correlation (r = 0.60) between the two tests was found.     

非洲猪瘟病毒抗体检测间接ELISA方法的建立

以基因工程表达的非洲猪瘟病毒VP73蛋白作为包被抗原,建立了间接ELISA方法,用以检测猪血清中抗非洲猪瘟VP73蛋白的抗体。该方法对非洲猪瘟标准阳性血清的检测灵敏度可以达到1∶2 560,与同类进口ELISA试剂盒相当。此方法只特异性检出非洲猪瘟阳性血清,而对猪传染性胸膜肺炎等5种猪传染病阳性血清的检测结果均为阴性,表明其具有良好的特异性。批内和批间重复性试验结果发现,检测同一份血清的变异系数小于10%,表明其重复性较好。包被好的酶标板37℃放置5d后,对同一份血清的检测敏感性无明显变化,初步表明其稳定性较好。利用建立的间接ELISA方法和进口ELISA试剂盒分别对150份血清样品进行非洲猪瘟血清抗体检测,结果表明本方法的特异性和敏感性分别为99.1%和94.3%,2种方法检测结果的符合率为98%。以上试验表明,本试验建立的间接ELISA方法具有良好的特异性和敏感性、较好的重复性和稳定性,可以满足临床检测的需求。     

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 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 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).