Quantitation of bovine immunoglobulin G2 antibodies binding Staphylococcus aureus, using a murine monoclonal antibody

A murine monoclonal antibody specific for bovine immunoglobulin (Ig) G2 was used to develop an enzyme-linked immunosorbent assay (ELISA) to measure serum antibodies against the bovine pathogen Staphylococcus aureus. Anion exchange chromatography was used to prepare IgG2 from serum taken from a mastitic cow infected with S aureus. Specific-IgG2 antibodies binding S aureus were purified with an affinity column, using a heat-killed, low protein A strain of S aureus (M-10). Purified antibodies did not contain IgG1 or IgM and were composed of greater than 95% heavy and light chains determined on the basis of polyacrylamide gel electrophoresis. Purified IgG2 anti-S aureus antibody was used as a reference in an ELISA that (i) could detect 5.0 ng of IgG2, (ii) was specific for IgG2 antibodies binding S aureus, (iii) was precise within and among different assays, (iv) yielded 112% recovery of the purified standard, and (v) when diluted in nonspecific IgG2, generated a curve that was parallel to the standard when a sample was serially diluted. A field study with cows having elevated California mastitis test scores showed evidence of infection with S aureus, as judged by a 59% increase (P less than 0.01) in IgG2 S aureus-specific antibodies and a 25% increase (P less than 0.05) in total IgG2 antibodies. There were no differences in IgG1 concentrations in plasma. Using indirect immunofluorescence, we also confirmed that bovine polymorphonuclear cells bound IgG2 preferentially over IgG1 or IgM. Measurement of antigen-specific IgG2 antibodies may therefore be useful as an index of specific antibody immunity to mastitis-causing organisms.     

Production and characterization of monoclonal antibodies against dog immunoglobulin isotypes

In this study, the effect of two oligodeoxynucleotide (ODN) sequences 5'GCT-AGA-CGT-TAG-CGT-3' (CpG-ODN) and 5'-GCT-AGA-GCT-TAG-GCT-3' (GpC-ODN) on the antigen-specific antibody and cellular immune response after intramuscular immunizations with OVA was analyzed in pigs. Pigs immunized with OVA supplemented with these ODNs showed a significantly enhanced primary antibody response in comparison with the control group which received OVA without ODN. This enhanced primary antibody response appeared ODN-sequence-independent as similar effects were seen in both ODN-groups. The OVA-specific antibody titers obtained after a single injection of antigen combined with either of both ODNs were as high as the titers in the control group after two injections. Furthermore, the ODN-supplemented animals showed significantly higher OVA-specific IgA antibodies in their saliva and nasal secretions at some time points after the first immunization. Proliferation assays showed that CpG- as well as GpC-ODN significantly enhanced the antigen-specific as well as the mitogen-induced proliferation in different lymphoid tissues. Furthermore, 48h after the third immunization the CpG-group showed a significantly decreased IL-6 mRNA expression in cells of the local draining lymph node but no significant difference in TGF-beta (Th3-like) and IL-10 (Th2-like). The ODN injected animals showed the tendency to have higher IFN-gamma (Th1-like) mRNA-expression in comparison with the control group. To our knowledge, these are the first in vivo studies in pigs, which demonstrate the appropriateness of CpG-ODN as immunostimulating adjuvants in vaccines for farm animals.     

Monoclonal antibodies against porcine immunoglobulin isotypes

Monoclonal antibodies (MCAs) against porcine immunoglobulin isotypes* G, G1, G2, M and A have been produced and characterized in detail. Epitope analysis using a competitive direct enzyme-linked immunosorbent assay (ELISA) indicated that the MCAs recognized 3 class-specific epitopes of IgG, 4 epitopes specific for IgG1, 3 epitopes specific for IgG2, 2 epitopes of IgM and 2 epitopes of IgA. Two MCAs against IgG2 were shown to react with an allotypic determinant (B2) and one MCA against IgM is probably allotype specific. The production of MCAs specific for IgG and for its subclasses G1 and G2 and, in addition, the one-step isolation of nearly pure IgG1 and IgG2 preparations by immunoaffinity chromatography using MCA 34.1.1a (anti-IgG2) confirmed the existence of at least two subclasses of IgG. Preliminary results further suggested the existence of a subpopulation of IgG1 which could be eluted selectively from Protein A-Sepharose columns at pH 5.0. MCA 34.17.2a appeared to react preferentially with this IgG1 subpopulation and could be used to isolate a similar IgG1 subpopulation by immuno-affinity chromatography. Several of the MCAs have been successfully applied for the detection of porcine immunoglobulin isotypes by a double antibody sandwich ELISA and for the (isotype-specific) detection of antibodies against various porcine viruses. The availability of a full set of MCAs against porcine immunoglobulin isotypes will stimulate and facilitate the further study of the porcine immune system.     

Generation and characterization of murine monoclonal antibodies specific for bovine immunoglobulin E

Monoclonal antibodies were produced against serum-derived bovine immunoglobulin E (IgE). Culture supernatants of hybridomas were initially screened by enzyme-linked immunosorbent assay (ELISA). Supernatant-derived antibodies were concentrated and further characterized using ELISA, reverse cutaneous anaphylaxis, immunohistochemical staining, and immunoblotting of IgE-containing samples separated by SDS-polyacrylamide gel electrophoresis (PAGE). Eight monoclonal antibodies showed specificity for bovine epsilon immunoglobulin heavy chain. Two antibodies (E2 and E32) reacted in immunoblots of SDS-PAGE of serum IgE under reducing conditions. Additionally, E2, E5, and E32 detected epsilon chain in serum separated by SDS-PAGE and then renatured. Antigen-specific IgE was detected in Western blots by E5 and E32. Immunoperoxidase staining of IgE-containing cells in mesenteric lymph node sections was detected with E5, E21 and E32. All eight antibodies produced positive reverse cutaneous anaphylaxis reactions in calf skin. All functioned well in ELISA as a plate-sensitizing reagent for quantitation of total IgE; E5 and E32 worked well as a primary antibody in antigen-specific IgE assays. These antibodies will be useful in research applications and in diagnostic assays.     

Quantitation of bovine immunoglobulins in culture fluids by use of sandwich radioimmunoassay with monoclonal antibodies

Bovine immunoglobulin isotype-specific murine monoclonal antibodies were used in sandwich radioimmunoassays to detect and quantitate bovine IgG1, IgG2, IgM, and IgA in culture fluids. The concentrations of bovine immunoglobulins in unknown samples were extrapolated from standard curves generated with bovine monoclonal immunoglobulins. The lowest detection limits for the bovine immunoglobulin isotypes ranged from 65 to 270 ng/ml.     

Characterization of bovine haemopoietic progenitor cells using monoclonal antibodies and fluorocytometry

Monoclonal antibodies against bovine leucocyte cell surface differentiation antigens were used in combination with a fluorescence activated cell sorter to enrich bovine haemopoietic progenitor cells present in bone marrow cell populations prior to in vitro culture. After two sequential centrifugations of the bone marrow cell suspension through Ficoll-Paque, the interface fraction was stained with a cocktail of monoclonal antibodies directed against mature monocytes/macrophages, granulocytes and lymphocytes. Using appropriate electronic window settings on a FACStar Plus, cells with a high 90 degrees light scattering property (granular cells), a low forward light scattering property (erythrocytes and reticulocytes) and cells positive for monoclonal antibodies specific for lineage-restricted leucocyte markers were removed and the negative cell fraction collected. These negatively-selected cells were stained with monoclonal antibodies specific for a pan-leucocyte or a MHC class II marker and the positive cell population was collected in a second sort and subsequently submitted to culture. All erythroid and granulocyte/macrophage colony forming cells expressed MHC class II antigens, as well as the pan-leucocyte antigen. These same progenitors did not bind any of a variety of monoclonal antibodies directed against lineage-specific antigens on lymphocytes, granulocytes or monocytes/macrophages, although they did bind monoclonal antibodies recognizing MHC class I antigens. Between 85% and 91% of the isolated cells seeded were capable of forming erythroid or granulocyte/macrophage colonies within 5 to 10 days, thus increasing the plating efficiency of these cell types in bone marrow populations by at least 60 fold.     

Diagnosis of bovine viral diarrhea virus infection using monoclonal antibodies

The monoclonal antibody (MAb) D89 against bovine viral diarrhea virus (BVDV) was used in conjunction with fluorescein-conjugated anti-mouse immunoglobulin in an indirect fluorescent antibody (IFA) procedure on frozen tissue sections and cell culture. During the 2-year study, BVDV was isolated from specimens submitted in 460 cases. The D89 Mab detected all but 2 BVDV isolates, both cytopathic. In 316 of the cases in which BVD virus was detected by IFA, specimens were inoculated on bovine turbinate cells and examined for BVDV antigens at 3-5, 10, and 20 days postinoculation. The BVDV was detected in 238/316 cases (75%) after 3-5 days incubation. The remainder were not detected until 10 or 20 days postinoculation. Virus isolation was enhanced in the early test if plates were centrifuged at the time of inoculation. Results suggest that D89 monoclonal antibody is a suitable diagnostic reagent for the detection of BVDV isolated from diagnostic specimens. The D89 MAb can be used for the detection of BVDV in both cell culture and tissues. Combination of D89 with another BVDV MAb (C17) did not improve the ability to detect BVDV in tissues compared to using D89 only, and the combined Mab's resulted in an increase in nonspecific fluorescence when used on tissues. Although pooling of different BVDV monoclonal antibodies may be necessary to detect all strains of BVDV in cell culture, pooling should be used with caution on tissues. Early detection of BVDV in cell culture by this IFA procedure permits faster confirmation of BVDV diagnosis when compared to the usual routine testing for noncytopathic BVDV at termination of first passage in cell culture.     

ELISA detection of bovine viral diarrhoea virus specific antibodies using recombinant antigen and monoclonal antibodies

A panel of monoclonal antibodies was prepared by immunization of BALB/c mice with Moredun (BD) virus strains. These antibodies were characterized by immunofluorescence and seroneutralization against BD, BVD and hog cholera (HC) virus strains, and radioimmunoprecipitation of BVD-infected cells extracts. The MAbs reacting with the majority of the Pestivirus strains recognize the 80 kDa antigen of the BVD cytophathic strains. The 80 kDa antigen of the BVD/Osloss virus strain has been cloned and expressed in E. coli as a fusion protein with beta-galactosidase. The fusion protein has been purified from inclusion bodies and used successfully as an antigen for ELISA detection of BVDV specific antibodies in bovine sera. A competitive ELISA using MAbs is more specific than a direct assay. These results compare well with the ones obtained with antigen extracted from BVDV-infected cells.     

Demonstration of antibodies against bovine leukemia virus (BLV) by blocking ELISA using bovine polyclonal anti-BLV immunoglobulin.

A blocking enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies against bovine leukemia virus (BLV) is described. The test is based on the biotin-streptavidin system using unlabelled polyclonal bovine IgG against BLV as catching antibody and biotinylated bovine anti-BLV IgG as detecting antibody. The sensitivity was found to be 50-100 times higher than the agar gel immunodiffusion test, with a specificity of practically 100%. The blocking ELISA proved to be suitable for detection of antibodies against BLV in serum and milk. In 34 paired milk/serum samples, the average ratio of BLV antibody titres was 1:26. So far, more than 700,000 sera have been screened by blocking ELISA for BLV antibodies in the course of the Danish surveillance programme for BLV infection.     

Characterization of surface markers of bovine gut mucosal leukocytes using monoclonal antibodies

The surface phenotypes of bovine intestinal leukocytes isolated from the intraepithelium (IEL), lamina propria (LPL) and Peyer's patches (PPL) of the small intestinal mucosa of normal adult cows were determined using monoclonal antibodies (mAb) specific to adult bovine peripheral blood leukocytes (PBL). Laser flow cytometric (LFC) analysis demonstrated that IEL contained significantly (P less than 0.1 to 0.02) fewer cells (26%) expressing the pan T cell phenotype in comparison to LPL (38%) and PPL (44%). Similarly, significantly (P less than 0.01 to 0.001) lower numbers of B cells were observed among IEL (10%) compared to LPL (28%) and PPL (33%). While approximately equal numbers of B7A1+ "null" cells (10%) and DH59B+ "Ia+ monocytes/granulocytes" (16.5%) were observed among the three intestinal cell populations, IEL contained significantly (P less than 0.1 to 0.05) lower numbers (19%) of T helper (Th) cells in comparison to LPL (44%) and PPL (38%). In contrast, lymphocytes with the T cytotoxic/suppressor (Tc/s) phenotype were significantly lower (P less than 0.01 to 0.001) among LPL (14.5%) compared to IEL (25%) and PPL (23%). While the numbers of cells expressing class I major histocompatibility complex (MHC) surface antigens (H58A+) were approximately equal among LPL (79%) and PPL (87%), a significant difference (P less than 0.02) was observed between IEL (71%) and PPL. Similarly, while approximately equal numbers of cells expressing the MHC class II surface phenotype were observed among LPL (42%) and PPL (46%), IEL contained significantly (P less than 0.01) fewer (31%) MHC class II cells in comparison to PPL. Enrichment for T cells by plastic adherence and Sephadex G-10/nylon wool fractionation revealed a significant (P less than 0.01) and proportional increase in T lymphocyte subsets expressing pan T, Th and Tc/s phenotypes among the three cell populations. Similarly, enrichment for B cells by the same techniques showed a significant (P less than 0.01) and proportional increase in cells expressing the panB cell phenotype among LPL and PPL. Marked differences in cell size distribution and cell surface density were observed when the three intestinal leukocyte populations were compared by LFC using monoclonal antibodies directed at various cell surface markers. Furthermore, considerable quantitative variations of each cell surface marker were observed among the individual animals tested. The results of this study indicate that bovine IEL, which contain a high percentage of cells (greater than 30%) with no known phenotype are significantly different from LPL and PPL which are phenotypically similar cell populations.(ABSTRACT TRUNCATED AT 400 WORDS)     

Quantitation and localization of immunoglobulin E containing cells in bovine lymphoid tissues

Although recent studies have begun to describe and quantify IgE responses in bovine serum and secretions, little is known about the distribution and quantity of IgE containing cells in cattle. In the present study, cells with cytoplasmic IgE were quantitated in bovine lymphoid tissues, using immunoperoxidase staining and evaluation by an image analysing computer (Quantimet). Frozen sections from retropharyngeal, bronchial and mesenteric lymph nodes, tonsil and spleen were stained from 11 calves, some of which had been exposed to antigen by aerosol or injection. Although individual variability was considerable, bronchial and mesenteric lymph nodes generally contained the greatest percentage of IgE containing cells, while retropharyngeal lymph node, tonsil, and spleen had less. Parenteral immunization with ovalbumin appeared to increase the splenic percentage, while aerosol exposure to ovalbumin was associated with a greater percentage of IgE containing cells in bronchial lymph nodes. Comparison of the present results with those reported for other species shows some similar trends in IgE localization.     

Preliminary serological characterization of bovine viral diarrhoea virus strains using monoclonal antibodies

Five monoclonal antibodies against the bovine viral diarrhoea (BVD) viral strain NADL were isolated and characterized by an indirect immunofluorescence assay. Extensive cross-reactions were detected when the antibodies were tested with 12 heterologous BVD and four hog cholera (HC) viral strains. One antibody reacted with all strains tested. Two antibodies were specific for cytopathogenic BVD viruses, but failed to react with HC virus. The other antibodies reacted to varying degrees with BVD and HC viral strains.     

Distribution of immunoglobulin isotypes and Salmonella antibodies in blood serum and meat juice of pigs

Using the close linear regression between the logarithm of the dilution degree of a sample and the logarithm of the extinction measured in an ELISA both the relative concentrations of immunoglobulines of the isotypes IgG, IgM and IgA and of the LPS antibodies against S. Typhimurium of the different isotypes in blood sera and meat juice of 15 slaughtered pigs were detected and compared. Furthermore the total concentration of antibodies against LPS of S. Typhimurium according to the "meat juice ELISA" were compared. Distribution of immunoglobulines between serum and meat juice revealed individual differences between the animals as well as between the different immunoglobulin-isotypes. Within the same isotype the ratio of the concentrations of anti-LPS Salmonella Typhimurium antibodies between serum and meat juice was significantly closer than relating the whole of immunoglobulines of the referred isotype. In order to detect pig herds with a high level of Salmonella exposure a comparison of the 1:30 diluted meat juice samples with the 1:400 diluted blood sera is justified, however, for detailed epidemiological or scientific studies there is a need to consider the existing differences between the immunoglobuline-isotypes as well as between the specificity of antibodies and of total immunoglobulines. While the concentration of Salmonella antibodies of the isotypes IgG1, IgG2 and IgA showed a clear and statistically significant correlation between both one below the other and with the total amount of Salmonella antibodies, this connection could not be established for the total amount of immunoglobulines of different isotypes and the IgM-antibodies.     

Quantitation of bovine immunoglobulin G2 by a competitive fluorescence immunoassay

Bovine immunoglobulin G2 (IgG2) was quantitated by fluoroimmunoassay (FIA) based on competition with fluorescein-conjugated IgG2 for Sepharose-bound anti-IgG2 antibodies. The optimal range was 0.1 to 1 microgram IgG2. The FIA was compared with the single radial immunodiffusion (SRD) test. Both methods gave comparable means and standard errors for IgG2 in serum. The FIA method was 30 times more sensitive.     

Investigation of bovine gut associated lymphoid tissue (GALT) using monoclonal antibodies against bovine lymphocytes

Gut associated lymphoid tissue of the small and large intestine of calves and cows has been compared morphologically and quantitatively using monoclonal antibodies to bovine lymphocytes. B cells were significantly decreased in the ileum of the cow compared to the calf. Significantly increased numbers of T cells were present in cell suspensions of all lymphoid areas of the cow compared to the calf. T lymphocyte subsets were quantified into cryostat sections of lymphoid tissues expressing BoT4, and BoT8 antigens demonstrated increased numbers in follicular and dome areas of the discrete Peyer's patches of the small and large intestine of the cow. BoT4+, BoT8+, and the non-BoT4/BoT8+ T cell subsets were increased in the mucosa of the cow as compared to the calf. Similarities in structure and lymphocyte composition of the discrete Peyer's patches of the small intestine, cecum and colon and isolated single follicles in the large intestine suggest similar functional properties.     

A competitive enzyme immunoassay for the detection of bovine antibodies to Brucella abortus using monoclonal antibodies

A competitive enzyme immunoassay (EIA) for the detection of circulating bovine antibodies to Brucella abortus has been developed using horseradish peroxidase conjugated monoclonal antibodies (MAb) raised against B. abortus cell surface antigens. Antibodies present in the serum of either vaccinated or infected cattle can apparently displace the conjugated MAb from the lipopolysaccharide antigen (LPS) in a quantitatively different manner allowing an assessment of immune status of the animal. The results from a panel of sera from animals with a known status of vaccination or infection indicated that the test was more selective in the detection and discrimination of infected from uninfected or immunized animals, than conventional complement fixation, agglutination or indirect enzyme immunoassay procedures.     

Antigenic analysis of feline and bovine Chlamydia psittaci with monoclonal antibodies.

Monoclonal antibodies were established for antigenic analysis of feline and bovine Chlamydia psittaci. The monoclonal antibodies recognized lipopolysaccharide (LPS), 56-64, 84 or 86 kDa antigens. At least 5 antibody-binding sites were detected on LPS with the monoclonal antibodies. The 56-64 kDa antigen was suggested to have both polypeptide and carbohydrate antibody binding sites. Immunoblotting analysis of cat and cattle sera indicated that the 56-64 kDa antigen is an important antigen in host immune response. The monoclonal antibodies are extremely useful tools to analyse the structure and function of chlamydial antigens.     

Production and characterization of monoclonal antibodies that recognize bovine Kit receptor.

Kit receptor is a transmembrane tyrosine kinase that is the receptor for stem cell factor (SCF). The extracellular domain of bovine Kit receptor (boKit) was produced by a baculovirus expression system. Six monoclonal antibody (MAb) clones designated as bK-1 to bK-6 were obtained upon immunization of mice with the recombinant protein. Immunoprecipitation and flow cytometric analysis indicated that all of the MAbs specifically bound to boKit expressed in COS-7 cells transfected with boKit cDNA. Four of the six MAbs neutralized the biological activity of recombinant bovine SCF, whereas the other two did not. The boKit-positive and boKit-negative cell fractions were sorted from cryopreserved bovine bone marrow cells by the use of MAb bK-1. Colony formation assays indicated that the cells which were able to grow in response to bovine SCF were enriched in the boKit-positive fraction. These MAbs would be valuable in studying possible boKit-positive cell species such as bovine hematopoietic cells, and in defining the biological role of Kit receptor in cattle.     

Differentiation of hog cholera and bovine virus diarrhoea viruses in pigs using monoclonal antibodies

Monoclonal antibodies against hog cholera and bovine viral diarrhoea viruses were assayed on organ tissue sections of experimentally infected animals. The animals had been infected simultaneously with both viruses. The antibodies were tested using an indirect immunofluorescence test and an indirect enzyme immunoassay with a biotin/streptavidin/peroxidase detection system. A polyclonal hyperimmune serum was used as a control in direct immunofluorescence tests. Both techniques based on monoclonal antibodies were more sensitive and more specific than the conventional test, the enzyme immunoassay being more sensitive than the immunofluorescence test. Small amounts of BVD viral antigen were demonstrable with monoclonal antibodies in most organ tissues.     

A functional and biochemical analysis of bovine class II MHC antigens using monoclonal antibodies

Monoclonal antibodies (MAbs) reacting with bovine (2) ovine (3), murine (1) or human (1) Class II MHC antigens were examined for reactivity with bovine peripheral blood leucocytes (PBL) and lymph node cells (LNC) by immunofluorescence, immunoprecipitation and the capacity to inhibit mixed lymphocyte responses (MLR), lectin- and antigen-induced blastogenesis. The 6 MAbs identified comparable percentages of Class II positive lymphocytes in PBL (40.8 to 54.2%) and LNC (6 to 11.5%) regardless of BoLA-A phenotype. Immunohistological staining of Class II MAb was localized principally to the lymphoid follicles in lymph nodes and to isolated epithelial reticular cells in the thymus. The anti-Class II MAb immunoprecipitated alpha- and beta- chains of 26-29K and 32-34K, respectively. These MAb inhibited proliferative responses in the MLR by between 25 and 74%, and diminished blastogenesis induced by specific antigens (purified protein derivative + PPD and ovalbumin) and B-lymphocyte mitogens (PPD, lipopolysaccharide and dextran sulphate) by between 45 and 75%, regardless of BoLA-A phenotype. In contrast, proliferation in response to concanavalin A and phytohaemagglutinin were unaffected by the anti- Class II MAb. Similarly these MAb did not affect lysis by cytotoxic T-lymphocytes, the activity of which was depressed by anti-Class I MAbs and monospecific alloantisera.