Chicken IFN-gamma monoclonal antibodies and their application in enzyme-linked immunosorbent assay

Twelve mabs against native or recombinant chicken IFN-gamma were produced and characterized by virus neutralization, ELISA, and Western blot assays. No data were obtained to suggest that the form of the immunogen (native versus recombinant) influenced the antigenic specificity of the mabs produced. While only two antibodies inhibited the in vitro virus neutralizing activity of IFN-gamma, other evidence indicated that the specificity of these mabs was indeed directed against IFN-gamma. By Western blot analysis, all antibodies identified a 17-kDa IFN-gamma polypeptide. Using a direct binding ELISA incorporating these mabs, a high correlation with IFN-gamma detected by in vitro virus neutralization was observed. The IFN-gamma ELISA was also capable of measuring cytokine levels in the sera of chickens orally infected with Eimeria maxima. At 8 and 10 days post-primary infection, significantly higher (p<0. 001) levels of serum IFN-gamma were detected in E. maxima infected chickens compared to uninfected controls. These results indicate that a mab-based direct binding ELISA is suitable to measure chicken IFN-gamma in a variety of formats.     

Cloning, sequencing and expression of white rhinoceros (Ceratotherium simum) interferon-gamma (IFN-gamma) and the production of rhinoceros IFN-gamma specific antibodies

Bovine tuberculosis (BTB) is endemic in African buffalo (Syncerus caffer) in the Kruger National Park (KNP). In addition to buffalo, Mycobacterium bovis has been found in at least 14 other mammalian species in South Africa, including kudu (Tragelaphus strepsiceros), Chacma baboon (Papio ursinus) and lion (Panthera leo). This has raised concern about the spillover into other potentially susceptible species like rhinoceros, thus jeopardising breeding and relocation projects aiming at the conservation of biodiversity. Hence, procedures to screen for and diagnose BTB in black rhinoceros (Diceros bicornis) and white rhinoceros (Ceratotherium simum) need to be in place. The Interferon-gamma (IFN-gamma) assay is used as a routine diagnostic tool to determine infection of cattle and recently African buffalo, with M. bovis and other mycobacteria. The aim of the present work was to develop reagents to set up a rhinoceros IFN-gamma (RhIFN-gamma) assay. The white rhinoceros IFN-gamma gene was cloned, sequenced and expressed as a mature protein. Amino acid (aa) sequence analysis revealed that RhIFN-gamma shares a homology of 90% with equine IFN-gamma. Monoclonal antibodies, as well as polyclonal chicken antibodies (Yolk Immunoglobulin-IgY) with specificity for recombinant RhIFN-gamma were produced. Using the monoclonals as capture antibodies and the polyclonal IgY for detection, it was shown that recombinant as well as native white rhinoceros IFN-gamma was recognised. This preliminary IFN-gamma enzyme-linked immunosorbent assay (ELISA), has the potential to be developed into a diagnostic assay for M. bovis infection in rhinoceros.     

New ELISA test for detection of bovine leukemia virus infections in cattle, using bacterially synthesized p24

A new ELISA test is described for the detection of antibodies to bovine leukemia virus protein p24. This test employs a bacterially synthesized p24 antigen which represents a hybrid protein consisting of beta-galactosidase and about 70% of the mature viral p24. The antigen preparation was enriched from Escherichia coli cells to 95% purity and was used for the detection of antibodies in cattle. In a selected set of 100 positive field sera, 97 could be verified by the new test.     

Development and characterization of monoclonal antibodies to chicken interleukin-15

The chicken IL-15 gene was recently cloned and shown to encode a polypeptide with T cell growth factor activity similar to IL-2. To further characterize the chemical and biological properties of chicken IL-15, we generated a panel of monoclonal antibodies against bacterially expressed protein and characterized their binding specificities. All antibodies were reactive by ELISA with recombinant IL-15, but not IL-2, and identified a 15kDa recombinant chicken IL-15 by Western blot analysis. Two antibodies inhibited IL-15-induced proliferation of splenic lymphoblast cells. These monoclonal antibodies will be useful for further structural and immunological studies of chicken IL-15.     

Characterization of monoclonal antibodies to equine interleukin-10 and detection of T regulatory 1 cells in horses

Interleukin-10 (IL-10) terminates inflammatory immune responses and inhibits activation and effector functions of T-cells, monocytes, macrophages and dendritic cells. IL-10 has also been found to be a key cytokine expressed by subpopulations of regulatory T-cells. In this report, we describe the generation and characterization of three monoclonal antibodies (mAbs) to equine IL-10. The antibodies were found to be specific for equine IL-10 using different recombinant equine cytokine/IgG fusion proteins. Two of the anti-equine IL-10 mAbs were selected for ELISA to detect secreted IL-10 in supernatants of mitogen stimulated equine peripheral blood mononuclear cells (PBMC). The sensitivity of the ELISA for detecting secreted IL-10 was found to be around 200pg/ml. The production of intracellular IL-10 was measured in equine PBMC by flow cytometry. PBMC were stimulated with phorbol 12-myristate 13-acetate (PMA) and ionomycin in the presence of the secretion blocker Brefeldin A. All three anti-IL-10 mAbs detected a positive population in PMA stimulated lymphocytes which was absent in the medium controls. Around 80% of the IL-10(+) cells were CD4(+). Another 15% were CD8(+) cells. Double staining with IL-4 or interferon-gamma (IFN-gamma) indicated that PMA and ionomycin stimulation induced 80% IL-10(+)/IFN-gamma(+) lymphocytes, while only 5% IL-10(+)/IL-4(+) cells were observed. By calculation, at least 60% of the IL-10(+)/IFN-gamma(+) cells were CD4(+) lymphocytes. This expression profile corresponds to the recently described T regulatory 1 (T(R)1) cell phenotype. In summary, the new mAbs to equine IL-10 detected native equine IL-10 by ELISA and flow cytometry and can be used for further characterization of this important regulatory cytokine in horses.     

Antigen-induced production of interferon-gamma in samples of peripheral lymph nodes from sheep experimentally inoculated with Mycobacterium avium subsp. paratuberculosis.

The production of interferon-gamma (IFN-gamma) in response to Johnin purified protein derivate was measured in samples of the prescapular lymph node (PLN) from 10 sheep, aged 2 years, and nine sheep, aged 1 year that had been inoculated orally with Mycobacterium avium subsp. paratuberculosis within their first month of life. Ten non-inoculated sheep, aged 1 year, constituted the negative control group. The results obtained in the PLN IFN-gamma assay were compared with those derived from serological tests: a complement fixation test (CFT), agar gel diffusion test (AGID) and enzyme-linked immunosorbent assay (ELISA), as well as an IFN-gamma test on samples of blood. Among the 19 inoculated sheep, 16 gave positive reactions in the PLN IFN-gamma assay on samples incubated overnight, and 18 tested positive when the assay was applied to PLN samples incubated for 48h. In comparison, three, four and seven inoculated sheep gave positive reactions in the ELISA, CFT and in the blood IFN-gamma assay on samples incubated overnight, respectively. The AGID and IFN-gamma assay on blood samples incubated for 48h detected eight inoculated animals. Twelve inoculated sheep, that tested positive in the PLN IFN-gamma assay were clinically normal, gave negative results in an IS900-based polymerase chain reaction (PCR) assay on samples of ileum and ileocaecal lymph node and had no histological evidence of paratuberculosis, but tested positive on more than two occasions in sequential serological testing before necropsy. None of the 10 non-inoculated sheep tested positive in the AGID, CFT, ELISA, blood IFN-gamma assay on samples incubated overnight and for 48h or the PLN IFN-gamma assay on samples incubated overnight, but one gave a positive result in the PLN IFN-gamma assay on samples stimulated for 48h. It is likely that the positive reactions obtained by the PLN IFN-gamma assay in the 12 inoculated sheep that tested negative in the PCR assay and histopathological examination represents immunological evidence of latent infection or previous exposure to M. paratuberculosis rather than active infection.     

Interferon-gamma response of PBMC indicates productive pseudorabies virus (PRV) infection in swine

In Chinese Meishan/German Landrace cross-bred swine F2 generation interferon gamma (IFN-gamma) production by peripheral blood mononuclear cells (PBMC) was determined directly ex vivo at different time points after survival of a virulent pseudorabies virus (PRV) infection. This reactivity was compared with the reactivity of na?ve PBMC. Significant IFN-gamma production was determined in ELISA and ELISPOT only after in vitro PBMC re-stimulation with PRV and not with the closely related bovine herpesvirus BHV-1. The PRV-specific IFN-gamma secretion from re-stimulated PBMC showed high levels 6 days after infection, before the presence of serum antibodies, and it persisted at a high level over a 3 months period. The response of a group of eight piglets infected intranasally with PRV varied. Only two animals showed the expected typical fever response. PRV specific IFN-gamma production by PBMC clearly indicated that infection had occurred. Early significant IFN-gamma production by primed PBMC turned out to be a reliable and specific ex vivo marker for cellular response against productive PRV infection in swine before antibody formation.     

Antigen-specific IFN-gamma and IL-4 production in caprine herpesvirus infected goats

The analysis of cytokines secreted by antigen-specific lymphocytes is hampered in goats by the paucity of species-specific reagents yet it is crucial to study immune responses to infections. To overcome this limit, two commercial kits designed to measure soluble bovine IL-4 (by ELISA) and frequencies of bovine IFN-gamma secreting cells (by ELISPOT) were tested for cross-reactivity in goats. In addition, an ELISA specific to bovine/ovine IL-4 and employing two monoclonal antibodies, clones CC313 and CC314, was tested as well. Concanavalin A-stimulated caprine peripheral blood mononuclear cells (PBMCs) cultures were studied and they exhibited high levels of soluble IL-4 and high frequencies of IFN-gamma secreting cells. In addition, the two IL-4 ELISAs detected similar amounts of cytokine. To start defining the cytokine response triggered by caprine herpesvirus 1 (CpHV-1) infection, PBMC cultures were setup from goats naturally or experimentally infected with CpHV-1. High frequencies of IFN-gamma producing cells and low, when detectable, levels of soluble IL-4 were observed in CpHV-1-specific PBMC cultures from both groups of infected goats. Thus, the availability of cross-reactive research tools can expand cytokine studies in goats and can implement the research on immunity to other caprine infections.     

Monoclonal antibodies reactive with chicken interleukin-17

Chicken interleukin-17 (chIL-17) gene was previously characterized through cloning from a chicken intestinal expressed sequence tag (EST) cDNA library. To further investigate the biological properties of chIL-17, six monoclonal antibodies (mAbs) against a bacterially expressed chIL-17 recombinant protein were produced and their binding specificities characterized. Antibodies which were initially selected on the basis of their specific binding reactivity with recombinant chIL-17 in ELISA were further characterized by Western blot analysis. Monoclonal antibodies specific for chIL-17 identified 20 and 21kDa protein bands in the culture supernatant and cell lysate of CU205 cells. These mAbs also recognized specific bands for chIL-17 in the cell lysate from conconavalin A (Con A)-activated, but not from normal splenic lymphocytes. Furthermore, these mAbs detected a 16kDa protein in the lysate of CU205 cells treated with tunicamycin and stained an intracellular protein in CU205 cells in flow cytometric analysis. Together, these results indicate that these new mAbs are specific for chIL-17 and will be a useful tool for structural and immunological studies of IL-17 in poultry.     

A comparison of the interferon gamma assay with the absorbed ELISA for the diagnosis of Johne''s disease in cattle

Two interferon gamma (IFN-gamma) assays, the IFN-gamma enzyme immunoassay (EIA) and the IFN-gamma bioassay and an absorbed ELISA were used to screen 6 cattle herds for Johne's disease. Each herd had a history of Johne's disease but the majority of infected animals did not show clinical signs. The disease status of the cattle, which were removed from the herds, was confirmed by bacteriological culture of faeces or histopathological examination and culture of tissues collected at necropsy. The sensitivities of the IFN-gamma assays and the absorbed ELISA were determined using test results from infected animals. The sensitivity of the IFN-gamma EIA in detecting subclinical (71.8 to 93.3%) and clinical animals (100%) was not significantly different. However, the IFN-gamma bioassay and the absorbed ELISA were more sensitive in detecting cattle with advanced infections (80%) than those that were subclinically affected (16.7 to 33.3%).     

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.     

Evaluation of a dot ELISA kit for measuring immunoglobulin M antibodies to canine parvovirus and distemper virus

A dot ELISA for the detection of immunoglobulin M (IgM) antibodies to canine distemper virus (CDC) and canine parvovirus (CPV) was assessed. The titres of IgM antibodies to CDV and CPV in 100 dogs were measured by the Immunocomb ELISA kit and compared with the results derived from the immunofluorescence assay (IFA). There was a strong correlation between the results of the dot ELISA technique and the IFA (P < 0.001). The dot ELISA kit was also used to assess the changes in the levels of immunoglobulin G (IgG) and IgM antibodies to CPV and CDV in 10 puppies vaccinated with a polyvalent vaccine. High levels of IgM antibodies to CPV were first detected seven days after they were vaccinated, and after nine days all the pups had high titres of IgG antibodies to CPV. High levels of IgM antibodies to CDV were detected after nine days and the highest average titres were recorded after 12 days. IgG antibodies to CDV were present from nine days after vaccination.     

An evaluation of a modified interferon-gamma assay for the detection of paratuberculosis in dairy herds

Whole blood samples were obtained from multiple dairy herds in Pennsylvannia and in Wisconsin which were previously determined to be infected with Mycobacterium paratuberculosis (MpS) (Johne's disease) by fecal culture. Blood samples were shipped overnight to the National Animal Disease Center (NADC) in Ames, IA for processing and interferon-gamma (IFN-gamma) analysis. Blood samples were incubated alone (non-stimulated) or with concanavalin A (ConA), a T-cell mitogen used as a positive control in the assay, for 18h. In addition, samples were incubated with M. avium purified protein derivative (AvPPD), M. bovis purified protein derivative (BoPPD), or a whole cell sonicate of M. paratuberculosis for 18h to elicit antigen-specific IFN-gamma production. After incubation, plasma was harvested and analyzed for IFN-gamma by ELISA. Values for IFN-gamma for non-stimulated blood samples (background) were consistently low for animals in all herds evaluated. In contrast, ConA stimulation of blood samples evoked a significant secretion of IFN-gamma regardless of infection status or fecal culture results for individual cows, indicating that immune cells were still viable after overnight shipment and capable of responding to stimulation. Antigen-specific IFN-gamma results were positively correlated with infection status as determined by previous fecal shedding and/or current fecal shedding of M. paratuberculosis. Accuracy of the IFN-gamma assay for correctly predicting infection status of individual cows in the herds with low levels of infection ranged from 50 to 75% when used as a single test. Combined use of the IFN-gamma test and a commercial ELISA antibody test accurately predicted infection status of 73% of cows from a dairy herd with a high level of M. paratuberculosis infection and 90% from a well-characterized group of dairy cows at the NADC. These results indicate that the antigen-specific IFN-gamma assay is a very sensitive diagnostic tool for detection of subclinical paratuberculosis in cattle and may be useful on an individual animal basis to remove infected animals from the herd.     

Comparison of an interferon-gamma to a phospholipase D enzyme-linked immunosorbent assay for diagnosis of Corynebacterium pseudotuberculosis infection in experimentally infected goats

The optimal method of control of caseous lymphadenitis of goats caused by Corynebacterium pseudotuberculosis is eradication of infection by identification and removal of infected carrier animals. The objective of this study was to compare detection of C. pseudotuberculosis experimentally infected goats using a commercially available bovine interferon-gamma (IFN-gamma) whole blood enzyme-linked immunosorbent assay (ELISA) to serological response to a recombinant phospholipase D (PLD) ELISA. The tests were assessed repeatedly over 1 year in three infected and three non-infected goats. Using a IFN-gamma optical density cut-off at 0.10 as positive under the conditions used, the test accurately detected C. pseudotuberculosis experimentally infected goats over a 363 day period with a reliability of 89.2% and non-infected goats with a reliability of 97.1%. Using a cut-off value of the mean for negative samples plus two standard deviations, the PLD ELISA detected C. pseudotuberculosis experimentally infected goats over this period with a reliability of 81.0% and non-infected goats with a reliability of 97.0%. The PLD ELISA was however more predictive than the IFN-gamma ELISA of the presence of lesions observed at postmortem examination of infected goats.     

单抗阻断ELISA检测副鸡嗜血杆菌血清型特异性抗体的研究

本研究在副鸡嗜血杆菌Ａ、Ｃ型特异性单克隆抗体的基础上成功地建立了能区分Ａ、Ｃ血清型抗体的阻断ＥＬＩＳ诊断方法,并应用此法对９种常见病原体阳性血清及多份免疫鸡。     

Two newly developed Erns-based ELISAs allow the differentiation of Classical Swine Fever virus-infected from marker-vaccinated animals and the discrimination of pestivirus antibodies

New generations of Classical Swine Fever virus (CSFV) marker vaccines have recently been developed in order to make emergency vaccination in case of a CSF outbreak more feasible. However, the application of a marker vaccine is dependent on the availability of an accompanying discriminatory test allowing differentiation of infected from vaccinated animals (DIVA). CP7_E2alf, the most promising live marker vaccine candidate currently available, is a genetically modified Bovine Viral Diarrhea virus expressing the E2 glycoprotein of CSFV strain Alfort/187. The DIVA principle going along with CP7_E2alf is based on the detection of CSFV E^rns-specific antibodies that are raised in the host upon CSFV infection but not after vaccination with the marker vaccine. The aim of this study was to develop novel DIVA tests to be used in combination with CP7_E2alf. Two indirect ELISAs (one for screening, the other one for confirmation purposes) using bacterially expressed recombinant E^rns proteins were designed and evaluated. Both ELISAs detected CSFV-specific antibodies against a broad range of strains and genotypes, and as early as 10 days after infection. They were able to distinguish CSFV-infected pigs from pigs vaccinated with CP7_E2alf and allowed discrimination of antibodies against ruminant pestiviruses in both, sera from domestic pigs and wild boar. Sensitivity and specificity of the screening ELISA was ≥95%. Thus, the ELISAs represent promising DIVA diagnostic tools, as well as an alternative to traditional pestivirus antibody differentiation by serum neutralization test.     

Use of ELISPOT and ELISA to evaluate IFN-gamma, IL-10 and IL-4 responses in conventional pigs

ELISPOT and ELISA were standardised for pig interferon-gamma (IFN-gamma), interleukin-10 (IL-10) and interleukin-4 (IL-4) with the aim to study the evolution of the immune response in conventional pigs from birth to 6 months of age and also to compare results of both techniques. Five pigs were bled at 1, 6, 9, 12 and 22 weeks of age and peripheral blood mononuclear cells (PBMC) were stimulated with phytohemagglutinin. The frequencies of cytokine secreting cells (CSC) and the levels of secreted cytokines were compared. For IFN-gamma the mean of CSC increased with age (p<0.05) from an average of 486/10(6) PBMC at first week of age to 1256/10(6) PBMC at 22 weeks of age. No correlation was found between the number of IFN-gamma CSC and the cytokine levels obtained by ELISA. For IL-10, frequencies of CSC did not increase with age of pigs, having a low of 315/10(6) PBMC at first week of age and a high of 1485/10(6) PBMC at six weeks. Comparison of ELISA and ELISPOT results for IL-10 showed a certain degree of correlation (r=0.74; p<0.05). Spontaneous secretion was observed in unstimulated cultures. For IL-4, frequencies of CSC were low (50-70/10(6) PBMC). In this case, comparison of ELISA and ELISPOT could not be done because cytokine levels in culture supernatants were often below the detection limit of the IL-4 ELISA. All these values can serve as a reference for future studies and also, our observations suggest that ELISPOT and ELISA should be carefully interpreted and do not necessarily correlate.     

Use of a recombinant maedi-visna virus protein ELISA for the serologic diagnosis of lentivirus infections in small ruminants.

Highly purified recombinant gag and env proteins derived from Icelandic strain 1514 of maedi-visna virus were used in an indirect enzyme immunoassay (ELISA) to detect antibodies to small ruminant lentiviruses in sheep and goat sera. The recombinant protein-based ELISA performed very well relative to whole maedi-visna virus and whole caprine arthritis-encephalitis-virus-based ELISAs in its ability to detect anti-maedi visna virus and anti-caprine arthritis-encephalitis virus antibodies, despite the antigenic and genomic variability that is known to exist within and between these two small ruminant lentiviruses. The data suggest that these recombinant maedi-visna virus proteins can be reliably used in an ELISA for the routine serodiagnosis of lentiviral infections in sheep and goats.     

Clinical evaluation of a peptide-ELISA based upon N-terminal B-cell epitope of the VapA protein for diagnosis of Rhodococcus equi pneumonia in foals

A total of 227 field samples from naturally exposed foals aged between 3 weeks and 6 months were used in an evaluation of a peptide-based enzyme-linked immunosorbent assay (ELISA) for diagnosis of Rhodococcus equi infection. A biotinylated peptide derived from the virulence-associated protein A (VapA) of R. equi, a horse pathogen, was synthesized and designated as PN11-14. The peptide corresponds to the N-terminal B-cell epitope TSLNLQKDEPNGRASDTAGQ of the VapA protein. Based upon a serum immunoglobulin (Ig)G titre of 512 as a positive cut-off value for the R. equi infection, the ELISA provided the overall sensitivity of 47.62%, specificity of 69.67% and an accuracy of 59.47% with a positive predictive value of 57.47% for true R. equi pneumonia. The assay was improved by detecting VapA-specific IgGb antibodies against N-terminal B-cell epitope of the VapA protein rather than IgG antibodies. The VapA-IgGb ELISA showed the overall sensitivity of 70.47%, specificity of 72.13% and accuracy of 71.36% with a positive predictive value of 68.52%. Diagnosis of R. equi disease in 6-week-old foals showed that the VapA-IgGb ELISA provided an increasing trend (P=0.0572) in sensitivity of 82.4% in comparison with the VapA-IgG ELISA which showed the sensitivity of 58.8%. However, differences in specificity of both tests were statistically insignificant (P=0.357) as analysed by the McNemar test. These results indicated that detection of VapA-specific IgGb antibodies may be a better predictor of R. equi disease in foals.     

Prevalence of Neospora caninum infection in Australian (NSW) dairy cattle estimated by a newly validated ELISA for milk

AIM: To determine the performance characteristics of an Institut Pourquier (IP) enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies against Neospora caninum in bovine milk and subsequent determination of the prevalence of N. caninum infection in New South Wales (NSW) dairy cattle. METHODS: Matching serum and milk samples from 93 cattle were assayed in two commercially available ELISAs for the detection of anti-N. caninum antibodies. Serum test results of one ELISA (IDEXX) were used to determine the N. caninum infection status of the cattle. Optimised cut-off values for the IP ELISA using milk samples were determined by two-graph receiver operating characteristic (TG-ROC) analysis and then applied to a representative sample of 398 milk samples from dairy herds around NSW. RESULTS: When this ELISA was applied to a representative collection of 398 milk samples from dairy cattle across NSW it demonstrated a 21.1% prevalence of N. caninum infection in those cattle. From the TG-ROC analysis an IP ELISA protocol was derived which suggested a cut-off threshold that would allow milk testing with 97% sensitivity and specificity, respectively, relative to serum testing. CONCLUSIONS: The prevalence of N. caninum in NSW dairy cattle was higher than previously believed. When used on individual milk samples this ELISA demonstrated high sensitivity and specificity and so could be used to accurately identify N. caninum infection. TG-ROC analysis of the IP ELISA optimised the protocol and prescribed cut-off values enabling the ELISA to be used for the screening of N. caninum antibodies in the milk of dairy cattle.