Detection of Corynebacterium equi-specific antibody in horses by enzyme-linked immunosorbent assay

An enzyme-linked immunosorbant assay was developed to measure naturally occurring Corynebacterium equi specific antibody in horse serum. Antibody against C equi was demonstrated in normal adults and was passively transferred to foals. Adult levels of specific antibody were reached by 5 to 6 months of age in healthy foals. Decreased early antibody levels were demonstrated in a limited number of foals with confirmed C equi infection.     

H5 antibody detection by blocking enzyme-linked immunosorbent assay using a monoclonal antibody

Many commercial enzyme-linked immunosorbent assays (ELISAs) are unable to differentiate antibody responses to different avian influenza virus (AIV) subtypes. Developing an ELISA for specifically detecting the H5 antibody is the purpose of this study. Four monoclonal antibodies (Mabs) were raised using A/duck/Yunlin/04 (H5N2). They were confirmed as being specific to H5. Two of these antibodies showed hemagglutination inhibition (HI) activity using the HI test. Using immunodot blot assays, three Mabs recognized both Eurasian and American H5, whereas the other Mab recognized only the tested Eurasian H5 virus. When testing denatured H5 antigen, one of the Mabs lost its antigen binding activity using Western blotting. For detecting the H5 humoral response in serum, one monoclonal antibody was purified and labeled with horseradish peroxidase to set up a blocking ELISA. Chicken sera that blocked H5 Mab binding by > 29% were considered H5 antibody positive. Inhibition percentages for sera from chickens infected with other AIV subtypes, H1 to H15, were < 29%. This blocking ELISA was used for 478 field chicken serum samples. The results showed that the sensitivity and specificity of this ELISA were 98.3% (232/236) and 95.9% (232/242), respectively. This blocking ELISA could be used specifically for detecting the H5 humoral responses in chickens.     

Detection of fish antibody against protein antigen of Aeromonas salmonicida by enzyme-linked immunosorbent assay using biotin-avidin system

Antibody against Aeromonas salmonicida was detected in sera from immunised or experimentally infected rainbow trout by enzyme-linked immunosorbent assay (ELISA) using the biotin-avidin system. The ELISA titre correlated well with the agglutinin titres of the sera, but the ELISA was found to be more sensitive than the agglutination test. When the rainbow trout serum was separated by column chromatography, antibody activity (determined by ELISA and agglutination test) was detected in the IgM fractions. Minimum cross reaction was observed in the ELISA system between antigen prepared from A salmonicida and antibodies against Vibrio species and other species of Aeromonas. The specificity of the ELISA was also confirmed by inhibition test. Immunisation of rainbow trout with a virulent strain of A salmonicida provided good protection, though no correlation was observed between the protection and the ELISA titres of sera.     

An enzyme-linked immunosorbent assay for the convenient serodiagnosis of contagious equine metritis in mares.

An enzyme-linked immunosorbent assay (ELISA) was developed for the serodiagnosis of contagious equine metritis (CEM), a sexually transmitted disease caused by Taylorella equigenitalis. Antigen preparation was simple, and antigens derived from both classical and atypical forms of T. equigenitalis enabled detection of antibody responses elicted in horses experimentally exposed to either form of the bacterium. Sera serially obtained from these horses from 0 to 63 days postexposure were tested by the traditional complement fixation test (CFT) for CEM and with the ELISA, using both antigens separately. There was close agreement between CFT and ELISA methodologies during the postexposure time period used to detect CEM serodiagnostically in regulatory animal health testing programs. Unlike the CFT, which requires an overnight incubation step, the ELISAs are more convenient and can be completed in 3 hours.     

Performance of an enzyme-linked immunosorbent assay for the diagnosis of Brucella ovis infection in rams

AIM: To describe the performance characteristics (sensitivity and specificity) of an enzyme-linked immunosorbent assay (ELISA) for the diagnosis of Brucella ovis infection in rams. METHODS: Sera from a negative (n = 2535) and a positive (n = 589) reference population were tested in an ELISA for anti-B. ovis antibodies and cut-off values calculated from the raw, log10-transformed and fitted data. Statistical methods were used to fit curves to the frequency distribution of the data and receiver-operated characteristics analysis used to optimise the cut-off values. RESULTS: Analysis of the frequency distribution of the positive ELISA values suggested a normal distribution of the data, whereas, in the case of the negative population, a Pearson type IV curve appeared to best fit the data. The cut-off values calculated as the mean plus 1.96 standard deviations (s.d.) from the raw, log-transformed and fitted ELISA data did not differ markedly. The differences were much greater at the mean plus 3.09 s.d. cut-off, with the cut-off value calculated from the log-transformed data giving a much better estimate of specificity. Optimisation (minimisation of classification error) of the cut-off calculated from the fitted curves suggested varying cut-off values, depending on the prevalence of B. ovis infection. Discussion: Calculation of cut-off values from curves that were fitted from the observed data give more accurate estimations of the performance characteristics of an assay than traditional calculations from observed values. They also allow the calculation of optimal cut-off values taking into account the prevalence of B. ovis infection and give additional information about the performance of the assay at cut-off values varied according to the epidemiological situation.     

Detection of antibody to Mycoplasma F38 in goat sera by an enzyme-linked immunosorbent assay

Summary An indirect enzyme-linked immunosorbent assay (ELISA) was developed to screen goat sera at a single dilution for antibody to mycoplasma F38. Antibody was detected in sera of six convalescent goats following experimental infection. Antibody was also detected in 34 sera three to four weeks after vaccination. No antibody was detected in 164 sera from goats without a history of vaccination or infection with contagious caprine pleuropneumonia. The ELISA was more sensitive than the complement fixation test in detecting antibody in vaccinated goats.

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Deteccion De Anticuerpos Contra Micoplasma F38 En Suero Caprino Mediante La Prueba Elisa

Resumen Se utilizó la prueba ELISA para detectar anticuerpos contra micoplasma F38. Se detectaron anticuerpos en el suero de seis cabras convalecientes, después de la infección experimental. Los anticuerpos también se detectaron en 34 sueros, tres a cuatro semanas después de la vacunación. Ciento sesenta y cuatro sueros de cabras sin historia de vacunación e infección con pleuroneumonía caprina, se encontraron libres de anticuerpos. La prueba enzimática ELISA fue más sensitiva que la prueba de fijación de complemento para detectar anticuerpos en cabras vacunadas.

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Detection De l'Anticorps Dirige Vers Le Mycoplasma F38 Dans Des Serums De Chevre Par Un Test Immuno-Enzymatique

Résumé Un test immuno-enzymatique indirect (ELISA) a été développé pour trier à une seule dilution les sérums de chèvre vis-à-vis de l'anticorps anti-mycoplasma F38. L'anticorps a été détecté dans les sérums de six chèvres convalescentes après infection expérimentale. L'anticorps a également été détecté dans 34 sérums trois à quatre semaines après vaccination. Les sérums de 164 chèvres sans commémoratifs de vaccination ou d'infection par la pleuropneumonie contagieuse caprine ne possédaient pas d'anticorps. L'ELISA est plus sensible que le test de fixation du complément pour détecter l'anticorps chez les chèvres vaccinées.

     

Detection of Salmonella dublin mammary gland infection in carrier cows, using an enzyme-linked immunosorbent assay for antibody in milk or serum

An ELISA has been developed for measurement of milk and serum IgG concentrations directed against Salmonella dublin. Four groups of cows were studied: group A--7 experimentally challenge-exposed cows (infected, recovered group); group B--6 normal uninfected randomly selected control cows; group C--7 naturally occurring S dublin carrier cows; and group D--6 normal uninfected S dublin negative cows from the same herd as group C. Group-A cows were inoculated orally, or inoculated orally and then IV, but none became a S dublin carrier. As expected, all 7 group-A cows responded with a marked increase in ELISA titer after oral exposure to virulent S dublin, starting with a mean serum titer of 17.7% and reaching a peak mean serum titer of 79.3% approximately 76 days after initial exposure. As determined by necropsy and organ culturing of the remaining cows, none of the group-A cows became carriers. The mean serum ELISA titer for group-B uninfected control cows was 14.1% (SD +/- 12.8%). The mean milk ELISA titer was -1.0% (SD +/- 5.5%). Colostrum and then milk gave false-positive results for up to 2 weeks after onset of lactation. Group-B cows were culture negative for S dublin in feces and milk during lactation, and when tissues were cultured after euthanasia. Milk and serum samples for ELISA, and milk and fecal samples for culturing were taken from all group-A and -B cows twice a week for 6 months. Statistical correlation (P less than 0.05) was found between serum and milk ELISA titers.(ABSTRACT TRUNCATED AT 250 WORDS)     

Detection of antibody in Aleutian disease of mink: comparison of enzyme-linked immunosorbent assay and counterimmunoelectrophoresis

Experiments were undertaken to investigate the potential of the enzyme-linked immunosorbent assay (ELISA) as a screening test for the diagnosis of the 2 known naturally occurring forms of Aleutian disease of mink. Anti-Aleutian disease virus (ADV) antibody activity was not detectable in the sera of mink with nonprogressive Aleutian disease despite the demonstration of antibody by counterimmunoelectrophoresis (CIEP) in the same sera. Anti-ADV antibody was detectable in 93% of sera from mink at various stages of experimentally induced progressive Aleutian disease. False-negative reactions occurred in sera which demonstrated high anti-ADV antibody titers by CIEP. As a consequence of the high prevalence of false-negative reactions, the ELISA was not considered to be an effective screening test. However, using CIEP as an indicator of ADV infection, the ELISA may be useful in differentiating mink with nonprogressive Aleutian disease from mink with progressive Aleutian disease.     

Sandwich enzyme-linked immunosorbent assay by using monoclonal antibody for detection of Clostridium perfringens enterotoxin

Sandwich enzyme-linked immunosorbent assay (ELISA) was developed for the quantitative estimation of Clostridium perfringens enterotoxin (CPE) with monoclonal and polyclonal antibodies as capturing and detecting antibodies, respectively. The dose-dependent relationship between absorbance at 405 nm and concentration of purified CPE was obtained over the range of 0.64-400 ng/ml. The sandwich ELISA was fond to detect crude CPE in culture and CPE in 10% fecal extracts. This method is convenient, rapid and sensitive for specific detection of CPE.     

A recombinant-based feline immunodeficiency virus antibody enzyme-linked immunosorbent assay.

We have developed an antibody detection enzyme-linked immunosorbent assay (ELISA) for the identification of animals infected by feline immunodeficiency virus (FIV). The ELISA solid-phase antigen consists of recombinant FIV gag proteins expressed in bacteria. The proteins are purified from bacterial lysates as insoluble inclusion bodies. In the case of bacterially expressed p24gag, it is shown that all of the linear, sequential epitopes presented by viral p24 during infection are retained. Purified preparations can be substituted for solid-phase whole virus in the IDEXX PetChektm immunoassay. The antibody ELISA duplicates the sensitivity and specificity of the whole virus based PetChek plate assay.     

Development and evaluation of an enzyme-linked immunosorbent assay for bovine antibody to Pasteurella haemolytica: constructing an enzyme-linked immunosorbent assay titer

A 2-stage strategy was developed and evaluated for estimating serum antibody titer by use of ELISA and a series of dilutions. In stage 1, the linear response region and least-square estimate of the assay line slope were established from 9-point dilution assays. Provided that the reading was within the linear response region, this information was used in the stage-2 estimation of titer from a single absorbance reading. Operationally, 2 fixed dilutions were selected, one suitably low and one suitably high, to provide at least one reading within the linear region. The procedure should save considerable time when a large number of assays are to be performed. Stage 1 required approximately twenty 9-point assays, but all subsequent assays required only 2 fixed dilutions.     

Detection of antibody against Mycoplasma mycoides subsp mycoides in cattle by an enzyme-linked immunosorbent assay.

The results of an enzyme-linked immunosorbent assay (ELISA) For the detection of antibody against Mycoplasma mycoides subsp mycoides are presented. Antibody was detected in the sera of cattle at least 19 months after recovery from an infection and at least 23 months after vaccination. Almost half the sera of some animals in an area of Nigera where contagious bovine pleuropneumonia is enzootic contained antibody. Antibody was rarely detected when the same sera were examined by other established serological tests, emphasising the sensitivity of the ELISA.     

Serodiagnosis of ovine paratuberculosis, using lipoarabinomannan in an enzyme-linked immunosorbent assay

The use of lipoarabinomannan (LAM; obtained from Mycobacterium paratuberculosis) in and ELISA (LAM-ELISA) to test 75 sheep sera from a paratuberculosis-infected flock resulted in an approximate threefold increase in sensitivity (from 23.5% to 70.6%), compared with the use of Annau's polysaccharide in a complement fixation test (P-CFT). Even after manipulation of the LAM-ELISA cut-off value to produce a specificity of 100% to match that of the P-CFT, the sensitivity still was approximately twofold greater than that of the P-CFT. Anti-bovine monoclonal antiglobulin-enzyme conjugates matched commercially available anti-ovine polyclonal antiglobulin-enzyme conjugates with respect to sensitivity and specificity. False-positive results were found to be less frequent after combining 2 serodiagnostic tests, LAM-ELISA and D antigenagar gel immunodiffusion, resulting in an increase in specificity from 88.1% to 95.2%. The repeatability of true seropositive and seronegative results was found to be 89.5% and 91.1%, respectively, for sera obtained less than or equal to 1 month prior to slaughter and 91.7% and 95.5%, respectively, for reanalysis of sera obtained at the time of slaughter.     

Detection of antibody to turkey coronavirus by antibody-capture enzyme-linked immunosorbent assay utilizing infectious bronchitis virus antigen

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.     

Detection of mink enteritis virus in mink feces, using enzyme-linked immunosorbent assay, hemagglutination, and electron microscopy

Twenty-five mink were inoculated with mink enteritis virus (MEV). Fecal specimens were collected daily and were simultaneously evaluated for MEV antigen by use of a direct enzyme-linked immunosorbent assay (ELISA), hemagglutination (HA), and electron microscopy. Results of the evaluations indicated that MEV was shed in the feces on postinoculation days 5 and 6. The virus was not detectable by ELISA or HA after postinoculation day 6, although viruses were found in reduced numbers by use of electron microscopy. The ELISA was specific for MEV, and the sensitivity of the ELISA for MEV was comparable with that of HA.     

An enzyme-linked immunosorbent assay using nuclear antigen for detection of feline herpesvirus 1 antibody.

To detect antibody against feline herpesvirus 1 (FHV-1) in the sera of cats, the sensitivity and specificity of an enzyme-linked immunosorbent assay (ELISA) using nuclear antigen was investigated. The standardized optical density readings (ODs) of the ELISA obtained by the 1-step serum dilution (1:80) method were compared with the serum neutralization test (SNT) results, with a correlation of 0.993, and with the hemagglutination inhibition (HI) test results, with a correlation of 0.851. The ODs for the ELISA titers were obtained using the serial serum dilution method and were compared with the SNT results, with a correlation of 0.933, and with the HI test results, with a correlation of 0.987. In the experimental infection of 4 specific-pathogen-free cats, the results of different serologic tests (SNT and HI) and the ELISA using the serial serum dilution method revealed rapid production of antibodies after inoculation, whereas the ELISA using the one-step serum dilution method indicated that titers increased more slowly. These results indicate that with the present ELISA using nuclear antigen, there are fewer demands on time and labor, making the method convenient for monitoring FHV-1 infection.     

Detection of IgM rheumatoid factor in canine serum using a standardized enzyme-linked immunosorbent assay.

An ELISA measuring IgM rheumatoid factor (RF) in dog serum is presented. Dog sera and a human IgM RF standard, calibrated against the international World Health Organisation (WHO) standard, are compared. It is concluded that the human IgM RF standard may be used as reference serum in the canine assay, which makes it possible to compare results from different veterinary laboratories.     

Single-dilution enzyme-linked immunosorbent assay for quantification of antigen-specific salmonid antibody.

An enzyme-linked immunosorbent assay (ELISA) was developed on the basis of testing a single dilution of serum to quantify the level of antibody to the p57 protein of Renibacterium salmoninarum in sockeye salmon (Oncorhynchus nerka). The levels of antibody were interpolated from a standard curve constructed by relating the optical densities (OD) produced by several dilutions of a high-titer rainbow trout (O. mykiss) antiserum to the p57 protein. The ELISA OD values produced by as many as 36 test sera on each microplate were compared with the standard curve to calculate the antigen-specific antibody activity. Repeated measurements of 36 samples on 3 microplates on each of 6 assay dates indicated that the mean intraassay coefficient of variation (CV) was 6.68% (range, 0-23%) and the mean interassay CV was 8.29% (range, 4-16%). The antibody levels determined for the serum sample from 24 sockeye salmon vaccinated with a recombinant p57 protein generally were correlated with the levels determined by endpoint titration (r2 = 0.936) and with results from another ELISA that was based on extrapolation of antibody levels from a standard curve (r2 = 0.956). The single-dilution antibody ELISA described here increases the number of samples that can be tested on each microplate compared with immunoassays based on analysis of several dilutions of each test serum. It includes controls for interassay standardization and can be used to test fish weighing <3 g.