An enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies to bovine viral diarrhoea virus (BVDV) in cattle sera

A microtitre ELISA has been established for the quantitation of antibodies to bovine viral diarrhoea virus (BVDV). Single dilutions of sera were assayed and units of antibody were calculated from a standard curve. In order to detect the maximum number of responding animals both IgG1 and IgG2 antibody should be assayed, although detection of IgG1 alone was nearly as effective. The ELISA was as sensitive as the virus neutralization test for detection of antibody; comparison of an ELISA that detected IgG1 plus IgG2 antibody to BVDV with the virus neutralization test gave a correlation coefficient (r) of 0.89 (P less than 0.001 for 95 compared sera). Although similar amounts of IgG1 and IgG2 antibodies were present in sera from both experimentally- and naturally-infected cattle, antibody to BVDV in colostrum and in the sera from young calves was predominantly IgG1. The number of adult cows with antibody was 40 out of 41 while 36 of 44 calves reared in a beef unit were found to have produced antibody by the time they were 31.5 weeks old, an indication of the high prevalence of BVDV in the cattle population.     

An indirect enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies to maedi-visna virus

An indirect enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies to maedi-visna virus (mvv) in sheep is described, in which microtitre plates are with a partly purified preparation of mvv. The antibodies bound are detected by a horseradish peroxidase conjugate.The results obtained with ELISA on a total of 493 serum samples from several commercial flocks were compared to those of a routine agar gel precipitation test (AGPT) and a complement fixation test (CFT).All samples which scored positive in AGPT, CFT or both (20.8%) were also found positive by ELISA. In addition, with ELISA a further 11.5% of the samples were positive. Serum samples from maedi-free flocks, from sheep suffering from sheep pulmonary adenomatosis and from lambs immunized against other viruses were all negative by ELISA. The assay has been used routinely for some years and proved to be specific, sensitive and suited for screening of large numbers of serum samples.     

Rapid detection of viral-specific antibodies by enzyme-linked immunosorbent assay (ELISA)

The development of three separate rapid ELISAs for detecting antibodies in host serum to three different viruses is described. These include: 1. A direct antigen assay using enzyme labelled anti-canine Ig for detecting antibodies to canine parvovirus, 2. A competitive ELISA using a feline infectious peritonitis virus-specific monoclonal antibody labelled with enzyme, and 3. A competitive ELISA using an equine infectious anemia virus-specific monoclonal antibody and enzyme labelled antigen, p. 26. The utility and benefits of each of the three approaches is emphasized.     

An enzyme-linked immunosorbent assay (ELISA) for detection of antibodies against Toxocara vitulorum in water buffaloes

Toxocara vitulorum, a parasite of the small intestine of cattle and water buffaloes, is mainly acquired by calves via the colostrum/milk from infected cows. To understand the development of immune responses in calves, antibody levels to a soluble extract antigen (Ex) from T. vitulorum infective larvae were measured by an indirect ELISA with sera of 15 buffalo calves, which were sampled every 15 days for the first 180 days after birth and 9 buffalo cows during the perinatal period. From all serum samples examined during the first 180 days, antibody level was lowest and highest in calves at 1 day of age before and after suckling colostrum, respectively, suggesting that the origin of antibodies was the colostrum. Immediately after birth, antibody levels in suckled calves remained at high levels until day 15, began to decrease to lower levels between 15 and 30 days and remained relatively stable until 120 days. By comparing the immune responses of these animals with their parasitological status it was considered possible to determine if passively acquired or actively produced antibodies provided protection against the infection. High numbers of T. vitulorum eggs in the feces between 30 and 60 days indicated that passively acquired antibodies did not provide protection against the infection, at least during these first days, and the maximum fecal egg counts during 30-45 days were coincident with decreased antibody levels. Between 60 and 120 days, when serum antibodies were detected at reduced, but stable levels, adult nematodes were expelled from the intestines and no more T. vitulorum eggs were found, suggesting development of acquired resistance. However, the potential and functional protective role of the antibodies against T. vitulorum infection and the process of self-cure requires further investigation.     

An enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies to Anaplasma centrale and Anaplasma marginale

An enzyme-linked immunoassay (ELISA) was applied to detect antibodies to A. centrale and A. marginale using homologous and heterologous antigens. The assay was compared with the indirect fluorescent antibody (IFA) test, and although a similar degree of sensitivity was obtained, the ELISA test had several advantages. Partially purified Anaplasma initial bodies used for antigen preparations contained negligible amounts of residual erythrocytic material, and did not interfere with the specificity of the ELISA. The antigenic similarity between A. marginale and A. centrale was further substantiated by cross-reactivity obtained with heterologous antigens in both ELISA and IFA tests, and antibodies produced during natural infection with A. marginale were indistinguishable in both tests from those produced following vaccination with A. centrale.     

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.     

An enzyme-linked immunosorbent assay for the detection of vesicular stomatitis virus antibodies

A liquid-phase enzyme-linked immunosorbent assay (ELISA) was developed for the detection of vesicular stomatitis virus (VSV) types New Jersey (VSV-NJ) and Indiana subtype Indiana I (VSV-IND₁) antibodies in the sera of naturally and experimentally infected cattle, horses and swine. Four different VSV preparations were compared for use as antigen in the ELISA: virus used in neutralization tests, complement-fixation antigen, immunodiffusion ager gel antigen and viral glycoprotein. Comparative antibody titration results of virus neutralization (VN) and ELISA showed no statistically significant difference between serum titers obtained with the four antigens to VSV-NJ (P=0.21) and VSV-IND₁ (P=0.14). The viral glycoprotein antigen was incorporated in the ELISA system because it is non-infectious and induces neutralizing antibodies. The reliability and variability of the ELISA was determined by testing 516 bovine, equine and swine sera which originated from areas free of vesicular stomatitis, and by testing 186 sera from areas where outbreaks occur. ELISA and VN results were correlated (P < 0.001 for both serotypes), and no statiscafically difference was found between replications of the tests. The ELISA allows the testing of a larger number of sera in a shorter time than is possible with the VN test and it can be used in diagnostic laboratories in VSV-free areas for the support of epidemiological surveillance programs.     

Latest developments in the enzyme-linked immunosorbent assay (ELISA)

Enzyme-linked immunosorbent assays (ELISAs) for the serologic detection of both antigen and antibody in monitoring programs of commercial poultry flocks have begun to be recognized as an improvement over more conventional diagnostic procedures. The feasibility of employing double-antibody sandwich assays for the detection of virus without prior isolation of virus has been demonstrated and shows promise as the method of choice for the detection of lymphoid leukosis virus shedding. The versatility of indirect ELISA for the measurement of antibody induced by a wide variety of potential pathogens using a single basic overlapping ELISA system has also been demonstrated. It shows potential as a likely candidate to replace some of the more costly and time-consuming or less sensitive conventional serologic methods that do not overlap. Although some aspects of the two major types of immunoassays currently used in poultry health may need some modifications or improvements before delivery for routine use, it is likely that the use of computer-assisted ELISA will gain increased acceptance and use as the preferred way to efficiently and accurately monitor the health of poultry flocks on a broad scale.     

An enzyme-linked immunosorbent assay (ELISA) for infectious bursal disease virus.

The application of the indirect enzyme-linked immunosorbent assay (ELISA) for detection of infectious bursal disease virus antibodies in chicken serum was investigated. The test procedure involved the coating of concentrated infectious bursal disease virus antigen onto polystyrene tubes, followed by the addition of chicken anti-infectious bursal disease virus serum and horseradish peroxidase labeled rabbit anti-chicken globulin. As an indicator substrate, 5-aminosalicylic acid, with the oxidant H2O2 was added. The reaction was stopped by 3M NaOH and the colour intensity of the reaction mixtures read in a spectrophotometer at 449 nm. The ELISA test was found to be a precise, sensitive and reproducible means of measuring infectious bursal disease virus antibodies in chicken and turkey sera.     

An enzyme-linked immunosorbent assay (ELISA) for detection of Marek's disease virus-specific antibodies and its application in an experimental vaccine trial

An enzyme-linked immunosorbent assay (ELISA) for the detection of Marek's disease virus (MDV)-specific antibodies was developed. Chicken embryo cells (CEC) or chicken kidney cells (CKC) were infected with MDV vaccine strain CVI988/Rispens, and infected-cell lysates were prepared at day 5 post-infection by freeze-thawing. Uninfected-cell lysates served as negative controls. Sera were used at a 1 : 100 dilution and were added in parallel to wells containing the infected and uninfected cell lysates. The optical densities at 492 nm (OD(492 nm)) were measured after detection of bound chicken antibodies with anti-chicken IgG peroxidase conjugate and colour reactions using o-phenylenediamine (OPD) as a substrate. The best results concerning the signal-to-noise ratio were obtained by using CKC cells rather than CEC for antigen preparation. The OD(492 nm) of plasma or serum samples with infected CKC was <0.02 when samples of unvaccinated and unchallenged maternal antibody-negative white leghorn chickens were tested. Sera and plasma samples of positive control birds exhibited OD(492 nm) of <0.01 when tested with uninfected CKC. The assay was used to monitor a trial that compared experimental BAC DNA vaccines and a commercial vaccine. Sustained seroconversion and antibody titers that were constantly rising until day 84 after vaccination (71 days after challenge) was observed only when chickens did not develop Marek's disease. In contrast, chickens developing the disease mounted marginal and short-lived antibody titers only. We conclude that the developed ELISA may be a valuable tool for the evaluation of the efficacy of MDV vaccination under experimental but possibly also under field conditions.     

Detection of IgM antibodies against canine distemper virus in dog and mink sera employing enzyme-linked immunosorbent assay (ELISA)

An enzyme-linked immunosorbent assay (ELISA) for the detection of IgM antibodies against canine distemper virus (CDV) in canine and mink serum is described. The diagnostic potential of this technique was evaluated by analyzing sera from natural or experimental infections in dog and mink and negative control sera. These results were compared with results obtained in the developed CDV IgG ELISA and in the virus neutralization test. The IgM test, which requires only a single serum specimen, is a useful method for diagnosing current or recent CDV infections in dog and mink.     

An enzyme-linked immunosorbent assay for the detection of antibodies to Marek's disease virus

A reproducible enzyme-linked immunosorbent assay (ELISA) using Marek's disease virus (MDV)-infected cells for the detection of antibodies to MDV is described. The optimum number of MDV-infected chicken embryo fibroblasts (CEF) was 5 X 10(4)/well, and test sera were positive at 1:400 dilutions. Compared with a purified virus preparation, MDV-infected CEF produced high specific and low nonspecific reactivities. Wells coated with whole cells could be stored at 4 C or -20 C for at least 3 months without loss of reactivity. With antibody-negative sera, the cutoff absorbency was 0.20 units. The ELISA was 20-to-40-fold more sensitive than indirect immunofluorescence. Homologous combinations of antisera in wells coated with CEF infected with different MDV serotypes were more reactive at higher dilutions than were heterologous combinations. The procedure described is specific and suitable for large-scale screening of both chicken and monoclonal antibodies against MDV.     

An indirect enzyme-linked immunosorbent assay (ELISA) for measuring antibodies in chickens infected with infectious bursal disease virus

An immuno-enzyme assay for measuring infectious bursal disease antibodies in chickens is described. The test is performed rapidly after coating plates overnight with partially purified antigen prepared in cell culture. Coated plates can be stored for at last 4 months. The chromatographically purified rabbit anti-chicken immunoglobulin-G, conjugated to horseradish peroxidase, was used optimally at a dilution of 1:3,000. It could be stored for at least 10 months without a reduction in titer. The test is safe, highly reproducible, specific, and sensitive. Results can be read visually or by spectrophotometry. Antibodies could be detected as early as 4 days postinfection. Serum titers rose rapidly to high levels, ranging from 1:1,600 to 1:25,600 by one week postinfection. High titers persisted for up to one year. The results of this assay compare favorably with results obtained with the agar-gel precipitin and virus-neutralization tests.