Production and characterization of monoclonal antibodies specific for bovine gamma-interferon

Nine stable hybridoma cell lines were established which secreted specific monoclonal antibodies (MAbs) to bovine gamma-interferon (BoIFN-gamma). Specific binding of each of the MAbs to recombinant BoIFN-gamma (rBoIFN-gamma) was demonstrated in an indirect ELISA, whilst none of the MAbs bound to rBoIFN-alpha or rBoIFN-beta. In a Western blot the MAbs reacted with the 16 kDa and 32 kDa polypeptides present in rBoIFN-gamma preparations. Competitive ELISA's showed that four MAbs bound to one epitope on rBoIFN-gamma, and the other five MAbs bound to a separate epitope. Two MAbs, each recognising different epitopes, were shown to neutralise the anti-viral activity of natural BoIFN-gamma.     

Production and characterization of monoclonal antibodies to serotype specific antigens of Haemophilus pleuropneumoniae serotype 2

Mouse monoclonal antibodies were produced to Haemophilus pleuropneumoniae bacteria of the most common serotype 2. 11 hybridoma cultures were recovered that produced antibodies with moderate to strong reactivity to the antigen. 10 of these antibodies were specific to isolated capsular antigens from H. pleuropneumoniae of serotype 2, while one antibody reacted with capsular antigens from bacteria of all 8 serotypes. One hybridoma producing antibodies with a titre of 1:1000 was cloned and the antibody specificity studied further. The binding of this antibody (1F3) to whole bacteria, and capsular extracts isolated at different temperatures indicate that the antibody is specific for a thermostable polysaccharide antigen present in the cellular capsule of H. pleuropneumoniae of serotype 2.     

Production and characterisation of monoclonal antibodies specific for chicken interleukin-2.

Using genetic immunisation of mice, we produced antibodies against chicken interleukin-2 (ChIL-2), the first produced against a non-mammalian interleukin. After a final injection with a recombinant ChIL-2 protein, two stable hybridoma cell lines were established which secreted monoclonal antibodies (MAbs) against this cytokine. Specific binding of the two MAbs to recombinant ChIL-2 produced by Escherichia coli and COS-7 cells was demonstrated in an indirect ELISA, Western blotting and dot blots. Both of them were able to neutralise the biological activity of the ChIL-2, but neither allowed the detection of ChIL-2 by flow cytometry.     

Production and characterization of monoclonal antibodies to cervine herpesvirus-1.

Fourteen hybridoma cell lines secreting monoclonal antibodies (Mab) to cervine herpesvirus-1 (CerHV-1) produced following the fusion of NSO myeloma cells with splenocytes of BALB/c mice previously immunized with gradient purified CerHV-1 were selected using an indirect enzyme-linked immunosorbent assay (ELISA) employing CerHV-1 antigen and tested by the ELISA against four other ruminant alphaherpesviruses from cattle (bovine herpesvirus type 1.1 and 1.2) goat (caprine herpesvirus-2) and reindeer (rangiferine herpesvirus-1). Comparison of all five ruminant alphaherpesviruses with these Mabs confirmed their close antigenic relationships, with two Mabs reacting against all viruses. Ten Mabs which were able to differentiate between the viruses reacted with a 64 kDa polypeptide in a western blot. Four Mabs including two specific only for CerHV-1 with neutralizing activity against the virus used for immunization were directed against a 74 kDa viral protein.     

用DNA免疫研制鸡白细胞介素2单克隆抗体

构建鸡白细胞介素2(鸡IL-2)的真核重组质粒pcDNA-IL-2和原核重组质粒pET-IL-2,以重组质粒pcDNA-IL-2免疫BALB/c小鼠,取免疫小鼠的脾细胞和骨髓瘤细胞SP2/0进行融合,以重组质粒pET-IL-2在大肠杆菌BL21(DE_3)中诱导表达的蛋白作为筛选抗原,通过间接ELISA法对杂交瘤进行筛选,经亚克隆后共获得3株针对鸡IL-2的特异性单克隆抗体,并对这3株单克隆抗体的特性进行了鉴定。     

Production and characterization of monoclonal antibodies to bovine beta 2-microglobulin

In an attempt to isolate monoclonal antibodies specific for bovine lymphocytes, spleen cells from mice immunized with bovine lymphocytes were fused with the mouse myeloma cell line SP-2/0. The resulting hybridoma cell lines were tested for reactivity with bovine lymphocytes, polymorphonuclear neutrophils, RBC, gamma-globulin, kappa-casein, beta-casein, alpha-S1-casein, and beta 2-microglobulin (beta 2m) and with beta 2m from rabbits, goats, and human beings. None of the clones secreted anti-bovine lymphocyte-specific antibody. However, 4 secreted monoclonal antibodies to bovine beta 2m. They also reacted with beta 2m from rabbit, goat, and human being. One monoclonal antibody also was found to be reactive with bovine immunoglobulin. Monoclonal antibodies to beta 2m could serve as a tool to (1) explore the homology of the beta 2m molecule among various species, (2) examine the relationship of beta 2 m with the constant region of the immunoglobulin molecule, (3) quantitate bovine beta 2m in various body fluids and major histocompatibility antigens on cell surfaces, (4) help characterize those antigens in cattle, and (5) be used for tissue typing of those antigens.     

Production and characterisation of monoclonal antibodies specific for chicken interleukin-12

Using genetic immunisation of mice, we produced antibodies against chicken interleukin-12p40 (chIL-12p40), also known as IL-12β. After a final injection with a recombinant chIL-12p40 protein, several stable hybridoma cell lines were established which secreted monoclonal antibodies (mAbs) to this component of the heterodimeric IL-12 cytokine. Specific binding of three of the mAbs to COS-7 cell-derived recombinant chIL-12p40 and the chIL-12p70 heterodimer was demonstrated in an indirect ELISA, and in dot blots. Two of the mAbs were used to develop a capture ELISA, suitable for detecting both recombinant protein (chIL-12p40 and the heterodimeric p70 protein) and native chIL-12. The mAbs were further characterised to show utility in immunocytochemistry.     

抗Asia1型口蹄疫病毒单克隆抗体的制备及生物学特性鉴定

用纯化的Asia1型口蹄疫病毒免疫BALB/c小鼠,取免疫小鼠脾细胞与SP2/0骨髓瘤细胞进行融合,经间接ELISA和间接免疫荧光(IFA)筛选,有限稀释法克隆,获得了2株稳定分泌单克隆抗体的杂交瘤细胞株,分别命名为3H6、5G3,其细胞培养上清效价分别为1:64和1:128,小鼠腹水效价分别为1×10~(-4)和8×10~(-3);ELISA和IFA结果显示,2株单抗仅与Asial型口蹄疫病毒反应,不与O型口蹄疫病毒反应,表明它们均为抗Asial型口蹄疫病毒的型特异性单克隆抗体。westem blot结果显示,2株单克隆抗体均不与全病毒抗原反应,表明它们所针对的抗原表位均为构象表位。相加ELISA试验表明,两株单抗识别不同的抗原表位。经硫氰酸盐洗脱法测定,3H6和5G3的相对亲和力指数分别为1.0 mol/L和1.5 mol/L。这2株单抗的获得为建立口蹄疫病毒检测方法提供了强有力的工具。     

Characterization of monoclonal antibodies to bovine IgG1

Monoclonal antibodies were developed to bovine IgG1. In addition, production of monoclonal antibodies to bovine light chain is also reported. Monoclonal antibody specificities were initially determined by solid-phase enzyme immunoassay. The monoclonal antibovine IgG1 was shown by a specificity-independent isotyping solid-phase enzyme immunoassay to be mouse IgG1 with kappa light chains. Ascites derived monoclonal antibovine IgG1 antibodies were linked to cyanogen bromide-activated Sepharose and used for affinity isolation of bovine IgG1. The bovine IgG1 eluted from the affinity column was characterized using immuno-electrophoresis, acrylamide gel electrophoresis, isoelectric focusing and solid-phase enzyme immunoassay. Affinity chromatography using monoclonal antibodies provided both a verification of monoclonal antibody specificity and a rapid technique for the isolation of bovine IgG1. This technique may also be employed to remove IgG1 contaminants during purification of bovine IgA.     

Production and characterization of monoclonal antibodies to Ehrlichia risticii

Hybridomas producing monoclonal antibodies to Ehrlichia risticii were developed to provide a means of molecular investigation of the biochemical and immunopathologic characteristics of the organism. All of 6 stable monoclonal antibodies obtained were IgG isotypes. The ascitic fluid titers induced by the hybridomas ranged from 10(2) to 10(7). Competitive binding experiments conducted by ELISA and binding of labeled protein A to antigen-antibody complexes indicated competition among monoclonal antibodies. Two monoclonal antibodies (HybI and 14D4) were reactive in an indirect fluorescent antibody test; these antibodies also bound a maximum of labeled protein A, indicating recognition of epitopes on the surface of the ehrlichia. Protein specificity of monoclonal antibodies could not be demonstrated with western blot procedure. HybI monoclonal antibody, however, did precipitate the 28 kD protein from 125I-surface-labeled ehrlichiae and was shown to be specific to E risticii on the basis of nonreactivity with E sennetsu, using the indirect fluorescent antibody test. By use of the different monoclonal antibodies as probes, more definitive molecular studies now will be feasible.     

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.     

Comparative functional characterization of canine IgG subclasses

To date, very little is known about the functional characteristics of the four published canine IgG subclasses. It is not clear how each subclass engages the immune system via complement-dependent cytotoxicity (CDC) or antibody-dependent cell-mediated cytotoxicity (ADCC), or how long each antibody may last in serum. Such information is critical for understanding canine immunology and for the discovery of canine therapeutic monoclonal antibodies. Through both in vitro and ex vivo experiments to evaluate canine Fc's for effector function, complement binding, FcRn binding, and ADCC, we are now able to categorize canine subclasses by function. The subclasses share functional properties with the four human IgG subclasses and are reported herein with their function-based human analog. Canine Fc fusions, canine chimeras, and caninized antibodies were characterized. Canine subclasses A and D appear effector-function negative while subclasses B and C bind canine Fc gamma receptors and are positive for ADCC. All canine subclasses bind the neonatal Fc receptor except subclass C. By understanding canine IgGs in this way, we can apply what is known of human immunology toward translational and veterinary medicine. Thus, this body of work lays the foundation for evaluating canine IgG subclasses for therapeutic antibody development and builds upon the fundamental scholarship of canine immunology.     

Production and characterisation of two monoclonal antibodies recognising equine IgG Fc receptors

Despite the importance of IgG Fc receptors in the regulation of various immunological mechanisms, these receptors have not been well characterised in the domesticated animals including equines. This paper describes the production of two monoclonal antibodies (CVS 59 and CVS 61) that recognise equine IgG Fc receptors. Fusions were conducted using BALB/c mice hyperimmunised with equine peripheral blood mononuclear cells. Hybridoma supernatants were screened on the basis of their ability to inhibit the rosetting of equine antibody coated sheep erythrocytes with equine peripheral blood mononuclear cells. The mAbs were then characterised for cellular distribution of the antigen by flow cytometry. Using immunoprecipitation, it was shown that both under reducing and non-reducing conditions, CVS 59 and CVS 61 precipitated a 80 and 40 kD protein, respectively. From the functional study coupled with cellular distribution and molecular weight of the antigen recognised by these mAbs, it would appear that CVS 59 and CVS 61 recognise an epitope on equine equivalent of human and murine FcgammaRIII and FcgammaRII, respectively.     

Production and characterization of monoclonal antibodies against porcine interleukin-4

Interleukin 4 (IL-4) is an important regulatory cytokine produced by activated T lymphocytes and mast cells, and regulates the growth and differentiation of cells such as B and T lymphocytes. In the present study, recombinant thioredoxin (Trx)-porcine IL-4 (pIL-4) fusion protein was prepared by Escherichia coli (E. coli), and by using this protein as an immunogen, monoclonal antibodies (mAbs) against pIL-4 were produced to establish a basis for a research on immune responses in pigs. Six stable hybridoma cell lines were successfully established and specific binding of each mAb to recombinant pIL-4 produced by E. coli and insect cells infected with recombinant baculovirus was shown by enzyme-linked immunosorbent assay (ELISA) and/or immunoblot analysis. Isotype analyses of these mAbs revealed that the subclass of 5 out of 6 mAbs was IgG1 and the rest was IgG2b. Further, assessment of their epitopes by competition binding assay indicated that the mAbs obtained in this study bound to 4 different epitopes. The recombinant proteins and mAbs produced in this study will be useful tools for the assessment of porcine immune system.     

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.     

Production and characterization of monoclonal antibodies to bovid herpesvirus-4

Thirty-five hybridoma cell lines secreting monoclonal antibodies (Mabs) against bovid herpesvirus-4 (BHV-4) strain V. Test were produced. These hybrid cells resulted from the fusion of SP2/0 myeloma cells with splenocytes of BALB/c mice previously immunized with purified BHV-4. A modified indirect fluorescent antibody test (IFAT) was applied as a screening procedure and was compared with an indirect enzyme-linked immunosorbent assay. The selected Mabs were tested by the same IFAT against a panel of BHV-4 field isolates and against bovine herpesvirus-1, bovine herpesvirus-2 and alcelaphine herpesvirus-1 (AHV-1). Comparison of BHV-4 field isolates with Mabs confirmed their close antigenic relationships, but slight antigenic differences were observed between different isolates. One of the Mabs also reacted against AHV-1, indicating an antigenic relationship between BHV-4 and AHV-1. None of the Mabs reacting with BHV-4 possessed neutralizing activity against the strain used for immunization.     

Bovine monoclonal antibodies specific for bovine herpesvirus-1 glycoprotein gIII

Spleen cells from a calf immunized with bovine herpesvirus-1 (BHV-1) were fused with the nonsecreting murine cell line SP2/0. Several bovine-murine hybridomas secreting bovine immunoglobulins were stabilized. Of these, 9 hybridomas secreted bovine monoclonal antibodies that specifically bound to BHV-1 in a radioimmunoassay. Two of these monoclonal antibodies reacted specifically with BHV-1 in an indirect fluorescent antibody test and immunoprecipitated a BHV-1 glycoprotein with molecular mass of 97 kilodaltons.     

Production and characterization of monoclonal antibodies against Actinobacillus (Haemophilus) pleuropneumoniae serotype 2

Four hybridoma cell lines producing monoclonal antibodies (MAbs) against Actinobacillus (Haemophilus) pleuropneumoniae were established by fusion of mouse myeloma and spleen cells obtained from mice immunized with a serotype 2, strain SH-15. Enzyme-linked immunosorbent assay-inhibition tests with antigens obtained from 12 serotype strains of A. pleuropneumoniae and 9 other gram-negative bacteria showed that all the MAbs bound to only serotype 2 strains of A. pleuropneumoniae. The epitopes recognized by the MAbs were located on a carbohydrate moiety of lipopolysaccharide (LPS) of the organism, which was sensitive to periodate oxidation. In immunoblotting analyses of LPS obtained from A. pleuropneumoniae serotype 2, all the four MAbs reacted specifically with the characteristic ladder bands of LPS detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These results suggest that O-antigen side chains of the LPS are one of the antigenic determinants responsible for the serotype-specificity of A. pleuropneumoniae.     

Production and characterization of monoclonal antibodies specific for canine CD138 (syndecan‐1) for nuclear medicine preclinical trials on spontaneous tumours

We isolated 11 antibodies specific for canine CD138 (cCD138) to validate the interest of CD138 antigen targeting in dogs with spontaneous mammary carcinoma. The affinity of the monoclonal antibodies in the nanomolar range is suitable for immunohistochemistry and nuclear medicine applications. Four distinct epitopes were recognized on cCD138 by this panel of antibodies. CD138 expression in canine healthy tissues is comparable to that reported in humans. CD138 is frequently expressed in canine mammary carcinomas corresponding to the human triple negative breast cancer subtype, with cytoplasmic and membranous expression. In canine diffuse large B‐cell lymphoma, CD138 expression is associated with the ‘non‐germinal center’ phenotype corresponding to the most aggressive subtype in humans. This homology of CD138 expression between dogs and humans confirms the relevance of tumour‐bearing dogs as spontaneous models for nuclear medicine applications, especially for the evaluation of new tumour targeting strategies for diagnosis by phenotypic imaging and radio‐immunotherapy.