Monoclonal antibody against kanamycin was prepared, and competitive direct ELISA and immunochromatographic assay were developed using the antibody to detect kanamycin in animal plasma and milk. The monoclonal antibody produced was identified to be IgG1, which has a kappa light chain. No cross-reactivity of the antibody was detected with other aminoglycosides, indicating that the monoclonal antibody was highly specific for kanamycin. Based on competitive direct ELISA, the detection limits of kanamycin were determined to be 1.1 ng/ml in PBS, 1.4 ng/ml in plasma, and 1.0 ng/ml in milk. The concentration of intramuscularly injected kanamycin was successfully monitored in rabbit plasma with competitive direct ELISA. Based on the colloidal gold-based immunochromatographic assay, the detection limits of kanamycin were estimated to be about 6-8 ng/ml in PBS, plasma, and milk. The immunochromatographic assay would be suitable for rapid and simple screening of kanamycin residues in veterinary medicine. Screened positives can be confirmed using a more sensitive laboratory method such as competitive direct ELISA. Therefore, the assays developed in this study could be used to complement each other as well as other laboratory findings. Moreover, instead of slaughtering the animals to obtain test samples, these methods could be applied to determine kanamycin concentration in the plasma of live animals.
Animals ; Anti-Bacterial Agents/*analysis ; Antibodies, Monoclonal ; Chromatography/methods/veterinary ; Enzyme-Linked Immunosorbent Assay/methods/*veterinary ; Kanamycin/*analysis ; Mice ; Milk/*chemistry ; Rabbits

An ELISA was established to measure bovine IgG directed against the recombinant antigenic determinant of Nc-p43, a major surface antigen of Neospora caninum. In a previous study, two thirds of the Cterminal of the molecule was expressed as a 6 x His tagged protein (Ncp43P) for ELISA using 2/3 of the N-terminal of SAG1 from Toxoplasma gondii as a control (TgSAG1A). Among 852 cattle sera collected from stock farms scattered nation-wide, 103 sera (12.1%) were found to react with Ncp43P positively, but no positive reaction was observed with TgSAG1A. This study shows that Ncp43P could be available as an efficient antigen for the diagnosis of neosporosis in cattle. Furthermore, it together with TgSAG1A, could be useful for the differential diagnosis of N. caninum and T. gondii infections in other mammals.
Animals ; Antibodies, Protozoan ; Cattle ; Cattle Diseases/diagnosis/epidemiology ; Coccidiosis/diagnosis/epidemiology/veterinary ; Enzyme-Linked Immunosorbent Assay/methods/*veterinary ; Immunoglobulin G/*analysis ; Neospora/*immunology ; Protozoan Proteins/*immunology ; Seroepidemiologic Studies

The Japanese encephalitis virus (JEV) is one of causative agents of reproductive failure in pregnant sows. An indirect enzyme-linked immunosorbent assay (I-ELISA) was examined for its potential use in the rapid monitoring of the JEV, and the results were compared with those from the hemagglutination inhibition (HI) and serum neutralization (SN) tests. The comparative analysis showed that the results of I-ELISA showed a significant correlation with the conventional HI (r = 0.867) and SN tests (r = 0.804), respectively. When the I-ELISA results were compared with the traditional diagnostic assays, the sensitivity of the I-ELISA was 94.3% with the HI test and 93.7% with the SN test, respectively. The specificity was found to be 81.4% and 80.0% with the HI and SN tests, respectively. To determine the applicability of I-ELISA in the field, the serum samples from 720 pigs were collected from 4 regions in Korea between July and August 2004. The results indicated that 21.7% of screened pigs were seropositive for the JEV. The seropositive rates of JEV in the 4 provinces were 12.6% in Gyeonggi, 45.0% in Gyeongnam, 16.7% in Jeonbuk, and 12.2% in Jeju. The I-ELISA methodology developed in this study was shown to have considerable sensitivity and specificity through a comparison with HI and the SN tests. Therefore, it might be one of convenient methods for screening a large number of samples in various fields.
Animals ; Antibodies, Viral/blood ; Antigens, Viral/immunology ; Encephalitis Virus, Japanese/immunology/*isolation&purification ; Encephalitis, Japanese/blood/immunology/*veterinary/virology ; Enzyme-Linked Immunosorbent Assay/methods/*veterinary ; Female ; Hemagglutination Inhibition Tests/veterinary ; Korea ; Neutralization Tests/veterinary ; Swine ; Swine Diseases/blood/immunology/*virology

Escherichia coli (E. coli) strains were collected from young diarrheic calves in farms and field. Strains that expressed the K99 (F5) antigen were identified by agglutination tests using reference antibodies to K99 antigen and electron microscopy. The K99 antigen from a selected field strain (SAR-14) was heat-extracted and fractionated on a Sepharose CL-4B column. Further purification was carried out by sodium deoxycholate treatment and/or ion-exchange chromatography. Monoclonal antibodies to purified K99 antigen were produced by the hybridoma technique, and a specific clone, NEK99-5.6.12, was selected for propagation in tissue culture. The antibodies, thus obtained, were affinity-purified, characterized and coated onto Giemsastained Cowan-I strain of Staphylococcus aureus (S. aureus). The antibody-coated S. aureus were used in a coagglutination test to detect K99+ E. coli isolated from feces of diarrheic calves. The specificity of the test was validated against reference monoclonal antibodies used in co-agglutination tests, as well as in ELISA. Specificity of the monoclonal antibodies was also tested against various Gram negative bacteria. The developed antibodies specifically detected purified K99 antigen in immunoblots, as well as K99+ E. coli in ELISA and co-agglutination tests. The co-agglutination test was specific and convenient for large-scale screening of K99+ E. coli isolates.
Agglutination Tests/methods/*veterinary ; Animals ; *Animals, Newborn ; Antibodies, Monoclonal/*immunology ; Antigens, Surface/immunology/isolation & purification ; Bacterial Toxins/immunology/isolation & purification ; Cattle ; Cattle Diseases/*immunology/*microbiology ; Chromatography, Gel/veterinary ; Chromatography, Ion Exchange/veterinary ; Chromatography, Liquid/veterinary ; Diarrhea/immunology/*veterinary ; Electrophoresis, Polyacrylamide Gel/veterinary ; Enzyme-Linked Immunosorbent Assay/veterinary ; Escherichia coli/*immunology ; Escherichia coli Infections/immunology/*veterinary ; Immunoblotting/veterinary ; Staphylococcus aureus

Interleukin-6 (IL-6) is introduced as a marker of disease. At present, a variety of method may be used to quantify expression of this protein. Antigen capture-ELISA is a sensitive and accurate quantification method previously used with ovine, rat, and human IL-6 proteins. However, it has never been reported to quantify porcine IL-6 protein using capture ELISA. In this study, we generated and characterized a set of IgY and mono-specific polyclonal antibodies to recombinant porcine IL-6 (rpIL-6), and combining these with a sensitive and specific capture-ELISA for a diagnostic purpose. cDNA encoding the mature protein coding region of porcine IL-6 was cloned and expressed with pQE-30UA expression vector. rpIL-6 was then expressed and purified by using Ni-NTA resin. Protein mass of 24 kDa was found with SDS-PAGE and the identity of the protein was confirmed by Western-blot. Production of polyclonal antibodies against rpIL-6 was performed using the purified rpIL-6 in mice and hens. An antigen capture-ELISA was developed with the antibodies after their extraction. To compare the IL-6 level in the different sanitary state of farms, pig sera were randomly collected and concentration of IL-6 in the sera was measured with the antigen capture-ELISA. The capture-ELISA with the optimal concentration of antibodies, in this study, was able to detect about 10 ng/ml of rpIL-6. IL-6 levels determined with the capture-ELISA in pig sera showed positive correlation with the sanitary states of the farms. These results suggested that the developed antigen capture-ELISA could be a good tool for the screening of microbial infection in pig farms.
Animals ; Biological Markers/blood ; Blotting, Western/veterinary ; Chickens ; Cloning, Molecular ; DNA, Complementary/genetics/isolation&purification ; Electrophoresis, Polyacrylamide Gel/veterinary ; Enzyme-Linked Immunosorbent Assay/methods/*veterinary ; Female ; Immunoglobulins/*blood ; Interleukin-6/*immunology ; Mice ; Mice, Inbred ICR ; Recombinant Proteins/immunology ; Swine/*immunology

The antigenicity of Photobacterium damselae (Ph. d.)subsp. piscicida, cultured in four different growth media[tryptone soya broth (TSB), glucose-rich medium (GRM),iron-depleted TSB (TSB+IR-), and iron-depleted GRM(GRM+IR-)] was compared by enzyme-linked immuno-sorbent assay (ELISA) and Western blot analysis usingsera obtained from sea bass (Dicentrarchus labrax) raisedagainst live or heat-killed Ph. d. subsp. piscicida. Theantigenic expression of Ph. d. subsp. piscicida was found todiffer depending on the culture medium used. A significantlyhigher antibody response was obtained with iron-depletedbacteria by ELISA compared with non-iron depletedbacteria obtained from the sera of sea bass raised againstlive Ph. d. subsp. piscicida. The sera from sea bass raisedagainst live bacteria showed a band at 22kDa in bacteriacultured in TSB+IR- or GRM+IR- when bacteria thathad been freshly isolated from fish were used for thescreening, while bands at 24 and 47kDa were observedwith bacteria cultured in TSB or GRM. When bacteriawere passaged several times on tryptic soya agar prior toculturing in the four different media, only bands at 24 and47kDa were recognized, regardless of the medium used toculture the bacteria. It would appear that the molecularweight of Ph. d. subsp. piscicida antigens change in thepresence of iron restriction, and sera from sea bassinfected with live bacteria are able to detect epitopes onthe antigens after this shift in molecular weight.
Animals ; Antibodies, Bacterial/blood ; Antigens, Bacterial/immunology/*metabolism ; Bass/blood/*immunology ; Blotting, Western/veterinary ; Cell Count/methods ; Culture Media ; Enzyme-Linked Immunosorbent Assay/veterinary ; Fish Diseases/immunology/*microbiology ; Molecular Weight ; Pasteurella Infections/immunology/microbiology/*veterinary ; Photobacterium/*immunology

There is an ongoing need for standardized, easily renewable immunoreagents for detecting African horsesickness virus (AHSV). Two phage displayed single-chain variable fragment (scFv) antibodies, selected from a semi-synthetic chicken antibody library, were used to develop double antibody sandwich enzyme-linked immunosorbent assays (DAS-ELISAs) to detect AHSV. In the DAS-ELISAs, the scFv previously selected with directly immobilized AHSV-3 functioned as a serotype-specific reagent that recognized only AHSV-3. In contrast, the one selected with AHSV-8 captured by IgG against AHSV-3 recognized all nine AHSV serotypes but not the Bryanston strain of equine encephalosis virus. Serving as evidence for its serogroup-specificity. These two scFvs can help to rapidly confirm the presence of AHSV while additional serotype-specific scFvs may simplify AHSV serotyping.
African horse sickness virus/*isolation & purification ; Animals ; Antibodies, Immobilized ; Antibodies, Viral/*immunology ; Cercopithecus aethiops ; Chickens ; Enzyme-Linked Immunosorbent Assay/methods/*veterinary ; Immunoglobulin G ; *Peptide Library ; Serologic Tests/methods/veterinary ; Serotyping ; Single-Chain Antibodies/*immunology ; Vero Cells

Apx IV, a forth RTX toxin indentified in Actionbacillus pleuropneumoniae recently, is expressed by all A. pleuropneumoniae regardless the serotypes and inducible only in vivo toxin, so it is the optimal to develop species-specific and differentiated diagnostic assay. Here the 2445bp DNA fragment of apxIVA gene of A. pleuroneumoniae was amplified and fused in-frame to the downstream of the T7 promoter and 6 His Tag of the prokaryotic expression vector pET-28b. The construct was transformed into E. coli BL21(DE3). After induction by 1.0 mol/L IPTG, a recombinant protein about 90 kD in size, designed as ApxIVAN, was detected, which was present as inclusion bodies and reacted specifically with swine antisera to the APP-serotype-1 by dot-blot. An indirect ELISA (ApxIVA-ELISA) was developed using purified recombinant ApxIVAN from the inclusion bodies as described previously, which had excellent specificity to A. pleuroneunoniae. Using the ApxIVA-ELISA, the ApxIV antibodies were not detected in the inactivated APP bacterins vaccinated pigs, but were detected in A. pleuropneumoniae serotype 1, 2 and 7 infected pigs and mice. These results suggested that ApxIVA-ELISA can be used not only to detect all serotypes of APP, but also to differentiate the naturally infected and inactivated vaccine immunized pigs.
Actinobacillus Infections ; diagnosis ; microbiology ; veterinary ; Actinobacillus pleuropneumoniae ; genetics ; immunology ; metabolism ; Bacterial Proteins ; genetics ; immunology ; metabolism ; Cloning, Molecular ; Enzyme-Linked Immunosorbent Assay ; methods ; veterinary ; Gene Expression ; Genes, Bacterial ; Recombinant Fusion Proteins ; genetics ; immunology ; metabolism

Infectious bursal disease (IBD) is an acute and highly contagious disease of young chickens caused by Birnavirus. Mortality of infected birds can be best prevented if injected with antibodies. The present study was an attempt to raise specific hyper-immune polyclonal antibodies against IBD virus in Pakistan. Commercial layers divided into four groups were injected with IBD vaccine subcutaneously according to four different treatment regimens. Eggs were collected daily and antibodies were purified from yolk with dextran sulphate. Titers of antibodies in serum and yolk were evaluated with enzyme linked immunosorbant assay and agar gel precipitation test. Antibody titers were significantly higher in yolk than serum. Eggs collected at 28 days post-vaccination had maximum antibody titers. Of treatment regimens, T3 was found to be most effective for hyperimmunization. Lyophilized antibodies stored at 4oC did not lose their activity till the end of experiment. IBD virus infected birds were injected with purified antibodies which induced 92% recovery as compared to control birds. The study implicates that the purified antibodies may be useful as a therapeutic agent to cure IBD infected birds.
Animals ; Antibodies, Viral/blood ; Birnaviridae Infections/immunology/*therapy/*veterinary/virology ; *Chickens ; Egg Yolk/immunology/virology ; Enzyme-Linked Immunosorbent Assay/veterinary ; Female ; Immunization/methods/*veterinary ; Immunoglobulins/*immunology ; Immunotherapy/methods/veterinary ; Infectious bursal disease virus/*immunology ; Poultry Diseases/immunology/*therapy/*virology ; Precipitin Tests/veterinary ; Viral Vaccines/*immunology/therapeutic use

Porcine circovirus type 2 (PCV2) is closely related to the postweaning multisystemic wasting syndrome (PMWS). In this study, the pig serum and tissue samples collected from different regions of Hangzhou District in Zhejiang Province of China between 2003 and 2005 were analyzed by enzyme-linked immunosorbent assay (ELISA) for PCV2 antibody and by polymerase chain reaction (PCR) for ORF2 gene. The results show that out of 1250 randomly collected serum samples, 500 sera (40%) were seropositive for PCV2. PCR results demonstrate that Hangzhou PCV2 with more than 50% Chinese PCV2 strains and French PCV2 formed Cluster A. Only one PCV2 from Hangzhou belonged to Cluster B with some other Chinese PCV2 and Netherlands's isolates. Cluster C consisted of PCV2 isolates from China, US, Canada, UK and Germany. The results indicate that the PCV2 infection was widespread in Hangzhou.
Animals ; Antigens ; chemistry ; China ; Circoviridae Infections ; genetics ; veterinary ; Enzyme-Linked Immunosorbent Assay ; methods ; Humans ; Open Reading Frames ; Phylogeny ; Polymerase Chain Reaction ; methods ; Sequence Analysis, DNA ; Swine ; Swine Diseases ; genetics