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 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.     

酶联免疫法测定鸡肉中尼卡巴嗪残留

为了建立鸡肉中尼卡巴嗪残留标志物4,4’-二硝基均二苯脲（DNC）残留酶联免疫（ELISA）检测方法。将N-琥珀酰-L-丙氨酰-L-丙氨酰-L-丙氨酸4-硝基苯胺（SAN）与载体蛋白偶联作为抗原免疫动物,获得抗DNC抗体,在此基础上建立了鸡肉中尼卡巴嗪残留的检测方法。人工抗原中SAN与载体蛋白的结合比约为12：1,血清效价1：2000倍,鸡肉中检测限为9．2μg／kg,添加回收率在49．4％～118％之间,批内、批间变异系数均小于20％。该方法在灵敏度、精密度和准确度方面均能满足兽药残留筛选检测要求。     

Enzyme-linked immunosorbent assay for the detection of Salmonella enteritidis infection in chickens

An ELISA was developed and tested for its ability to detect antibodies against Salmonella enteritidis in chickens. Various features of the ELISA were evaluated and optimized. The outer membrane protein antigens selected by use of the protein immunoblotting method made the assay specific and sensitive. The assay was evaluated in chickens experimentally infected with S enteritidis. Blood samples collected at weekly intervals after experimental infection with S enteritidis were analyzed by ELISA. Results of the ELISA were compared with those of conventional serum plate and microagglutination tests. The ELISA was more sensitive and specific in the detection of S enteritidis infection than the other 2 conventional tests.     

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 detection of transmissible gastroenteritis virus antibody in swine sera

An enzyme-linked immunosorbent assay (ELISA) was developed for detection and quantification of serum antibodies to transmissible gastroenteritis virus (TGEV) in swine. Sera from pigs inoculated with cell culture-origin TGEV or gut-origin TGEV were tested for anti-TGEV antibody by ELISA and by serum virus-neutralization test (NT). The ELISA detected antibody 3 days (av) sooner than did the NT when sera from pigs inoculated with cell culture-origin TGEV were tested and 1 day sooner than did the NT when sera from pigs inoculated with gut-origin TGEV were tested. The ELISA appeared to be more sensitive than the NT, since ELISA was more responsive to low-level antibody and ELISA titers exceeded NT titers.     

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 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 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 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.     

Comparative studies of Bacillus cereus strains isolated from various foods and food poisoning outbreaks.

Certain properties of 22 Bacillus cereus strains isolated from different foods and food poisoning episodes were investigated in order to evaluate possible différences between strains isolated from diarrhoeal and vomiting type food poisoning outbreaks.None of the strains isolated from vomiting type episodes produced acid from salicin and mannose, whereas 80 and 40 % of the strains from diarrhoeal type outbreaks were positive, respectively. No association between the antibiotic sensitivity pattern or the fatty acid composition and the source of a strain could be found, although some strains differed from the general pattern of B. cereus in some instances. No significant differences in the production of the skin factor between strains isolated from the two types of outbreaks were found either. The findings of this study support the observation that the food environment itself essentially affects the enterotoxin formation of B. cereus.     

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.     

Importance of newly detected staphylococcal enterotoxins for food safety

Staphylococcal enterotoxins (SEs) are emetic toxins causing food poisoning in humans, because of their biological activity and structural relatedness They have been classified as members of the pyrogenic exotoxin superantigen family Among them nine major antigenic types of emetic enterotoxins were recognized In recent years several newly detected SEs were also discriminated, but their occurrence and role in human and animal diseases are not fully understood Neverthless, evidences of their pathogenic role and broad distribution in staphylococcal strains cumulate Therefore their importance as potential risk factor for food safety becomes essential For this reason their properties, genetic determinants and supposed mechanisms of the pathogenic activity are discussed in respect of their potential hazard to human health.     

Enzyme-linked immunosorbent assay for diagnosis of equine infectious anemia

An enzyme-linked immunosorbent assay (ELISA) was elaborated for the detection of specific antibody to equine infectious anemia (EIA) antigen. Sera from horses experimentally infected with EIA virus were assayed by ELISA, complement fixation (CF) and immunodiffusion (ID) tests for antibody to EIA antigen. The ELISA technique was found to be much more sensitive than CF and ID tests. In addition, EIA specific antibody could be detected by ELISA at an earlier stage of infection than by CF or ID techniques. The applicability of the technique to diagnosis of EIA is discussed.     

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 detection of milk immunoglobulins to leukocidin toxin of Staphylococcus aureus

Leukocidin toxin from a bovine strain of Staphylococcus aureus was partially purified by ion exchange chromatography. An enzyme-linked immunosorbent assay was developed to quantitate antibodies specific for leukocidin in bovine milk. This was used to assay quarter samples from 88 cows in a S aureus-infected herd for antibody levels to the toxin. Milk samples from 65 cows with S aureus infections in at least one quarter produced a mean optical density of 1.054, whereas milk samples from 23 cows that were free of bacteria on cultural examination had a mean optical density of 0.584. There was a significant difference (P less than 0.001) in milk anti-leukocidin levels between these 2 groups. Evaluation of serum samples from 40 of these cows indicated that the milk anti-leukocidin concentrations were reflective of systemic anti-leukocidin values. The capability of 57 milk samples to neutralize the cytolytic effect of minimal amounts of leukocidin on bovine peripheral blood neutrophils was examined. Good correlation existed between the enzyme-linked immunosorbent assay antibody concentration and toxin-neutralizing capability of individual milk samples.     

Enzyme-linked immunosorbent assay methods for detection of feline leukemia virus and feline immunodeficiency virus.

Enzyme-linked immunosorbent assays have been widely used for diagnosis of FeLV and feline immunodeficiency virus (FIV) infections. Various ELISA kits for FeLV are available from several manufacturers. Although these tests are configured in a variety of formats, they are all direct antigen-detection systems for the viral core protein p27. On the other hand, ELISA for FIV exposure detects specific feline antibody to FIV. Basic immunoassay principles and the application of ELISA technology used in FeLV and FIV ELISA kits are described.