A bovine monoclonal antibody to oestrone/oestradiol prepared by a (murine x bovine) xbovine interspecies fusion

A heterohybridoma was produced by the fusion of sensitized peripheral blood lymphocytes (PBLs) with a previously derived heteromyeloma, generated by the fusion of bovine PBLs with murine myeloma cells. The sensitized bovine PBLs were collected from a steer immunized with an oestradiol-ovalbumin conjugate. The cell lines resulting from the fusion were screened for the production of bovine antibodies to oestradiol. A stable heterohybridoma was isolated which secreted a bovine IgG1 to oestrone/oestradiol. The use of sensitized PBLs together with heteromyeloma fusion partners has proved to be a reliable and simple way of producing monoclonal antibodies against specific haptens.     

The bovine lymphoid system: II. Derivation and partial characterlzation of monoclonal antibodies against bovine peripheral blood lymphocytes

We have raised monoclonal antibodies to produce reagents specific for bovine lymphocyte subpopulations. Spleen cells from mice immunized with bovine peripheral blood lymphocytes were fused with X63-Ag8 myeloma cells and eleven myeloma-hybrids which secreted antibody specific for bovine lymphocytes were doubly cloned. Five of the hybrids secreted antibodies which bound to the majority of bovine lymphocytes. Two of these antibodies were specific for polymorphic antigens. One antibody bound to B lymphocytes and serum IgM molecules. The remaining five bound to subpopulations of lymphocytes. Four monoclonal antibodies bound only to bovine cells while six also bound to lymphocytes from other bovidae. None bound to human lymphocytes. We discuss the difficulty of correlating the specificities of monoclonal antibodies to functional lymphocyte subpopulations in outbred animals where few other defined markers are available.     

The bovine p150/95 molecule (CD11c/CD18) functions in primary cell-cell interaction.

The monoclonal antibody (MAb), C5B6, recognizes the CD11c/CD18 molecule on the surface of bovine peripheral blood monocytes. C5B6 was reactive with 69-83% monocytes, all granulocytes, and less than 5% of lymphocytes from cattle. Of the lymphocyte series, the antibody had specificity for large lymphocytes and two lines expressing T cell markers, but was not reactive with small lymphocytes, thymocytes, a tumor cell line of B-cell lineage, an interleukin 2 (IL2)-dependent T cell line, fibroblasts, or human, sheep, goat or pig peripheral blood mononuclear cells. No dual fluorescence was seen using C5B6 and antibodies to bovine IgM, CD2, CD4 or CD8. Immunoprecipitation of 125I labeled peripheral blood mononuclear cells with C5B6 antibody defined two bands: 150,000 and 95,000 Da. Antibody to the beta chain (CD18) of the leukocyte adhesion receptor family precipitates the 95 kDa beta subunit and the three associated alpha subunits (180, 165 and 150). The bands obtained using MAb C5B6 correlated with the p150/95 bands observed using an antibody that precipitated the alpha and beta chains of the leukocyte adhesion receptor family. Functionally, the primary but not the secondary proliferative response to alloantigens was inhibited by C5B6 MAb. No effect was seen using C5B6 MAb in cytotoxicity assays or in the secondary proliferative response to Brucella abortus or bovine herpes virus type 1.     

Preparation of monoclonal antibodies against cell surface antigens in cattle

The BALB/c mice were immunized three times with bovine lymphocytes and thrombocytes in vivo. Besides, dissociated spleens of the mice were immunized with bovine lymphocytes in vitro. Hybridomas producing monoclonal antibodies were formed as a result of the fusion of the spleen cells with the murine myeloma cells. On the whole, four fusions were performed after immunization in vivo and three fusions after immunization in vitro, and 70 stable hybridoma clones were obtained. Five monoclonal antibodies exhibited an identical specific reaction only with bovine thrombocytes, two antibodies reacted only with a certain limited population of bovine spleen cells. The remaining monoclonal antibodies exhibited no tissue specificity and were bound to the lymphocytes of peripheral blood, lymph nodes, thymus, and spleen, to thrombocytes, liver cells, and spermatozoa, but never to erythrocytes. As for the amount of the obtained hybridomas and specific antibodies, no significant difference was observed in the effectiveness of in vivo and in vitro immunization.     

Two monoclonal antibodies (CC17, CC29) recognizing an antigen (Bo5) on bovine T lymphocytes, analogous to human CD5

Two new monoclonal antibodies (CC17 and CC29) raised against bovine thymocytes are described. The antibodies, both of which were IgG1, recognize a molecule of approximately 67,000 molecular weight on bovine T cells. They react T cells in peripheral blood, the lymph node paracortex and the periateriolar lymphoid sheath in the spleen. Both the cortex and medulla of the thymus are stained but the medulla reacts more intensely. They do not stain B cells in peripheral blood, the ileal Peyer's patch, the cortex or the primary follicles in lymph nodes. No activity was found on cells outside the lymphoid system, i.e. monocytes, alveolar macrophages or endothelial and epithelial tissue. The antigen recognized is considered to be the bovine homologue of CD5 (T1) in humans and Lyt1 in mice. The mAbs appear to be particularly useful for detecting cells in the peripheral blood of young calves which are of the T cell lineage but do not express BoT2 or the mature pan T cell antigen recognized by mAb IL-A27 and may thus allow identification of a population of bovine lymphocytes previously described as null cells.     

Expression of Kit, the receptor for stem cell factor, in bovine peripheral blood

The expression of Kit, the receptor for stem cell factor (SCF), on bovine peripheral blood cells (PBCs) was examined by using monoclonal antibodies against the bovine Kit protein. Flow cytometric analysis showed that approximately 1.5% of PBCs expressed Kit. In cytospin preparations, the morphology of most Kit+ PBCs was similar to that of large lymphocytes. Subsets of Kit+ PBCs coexpressed CD3, IgM, and/or CD11b but not CD14 or G1. SCF did not induce the proliferation of Kit+ PBCs in vitro. These results indicate that Kit is expressed on subsets of lymphocytes in bovine peripheral blood, but the ligand of Kit, SCF, does not directly induce the proliferation of this cell population.     

Preparation and characterization of a monoclonal antibody against bovine CD5 lymphocyte surface antigen.

The M1 monoclonal antibody (mAb) was proved to recognize 51-70% of Bovine peripheral blood lymphocytes (PBL). The M1+ cells were SIg-. In spleen and lymph nodes, the M1 positive lymphocytes were located within the T cell areas. All the lymphoid follicles remained negative. In the thymus, 10% of thymocytes were M1+, most of them were located in the medulla. The M1 mAb did not inhibit spontaneous rosette formation by sheep erythrocytes and bovine lymphocytes. On the other hand, biochemical analysis of membrane antigen with bovine thymic tumor cell line LB203 gave a molecular weight of 75 kDa. Despite a slight difference in biochemical results (75 vs 67-69 kDa). Our data permit us to consider M1 mAb as a possible homologous of human anti-CD5 mAb. Finally, M1 cross-reacted with sheep peripheral blood T lymphocytes.     

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.     

Negative enrichment of bovine T lymphocytes with monoclonal antibodies and magnetic microspheres

A highly enriched population of bovine T lymphocytes was produced from peripheral blood leukocytes following the depletion of monoclonal antibody-labelled B lymphocytes and monocytes with magnetic microspheres. This negative-enrichment protocol was simple, rapid, and specific. Also, it had a high recovery efficiency and was consistently reproducible. The enriched T lymphocytes proliferated in response to recombinant bovine interleukin 2 and, following the addition of monocytes, to concanavalin A. This methodology made it possible to determine the proliferative responses of peripheral blood lymphocytes utilizing a constant number of T lymphocytes within each assay. In this way, the in vitro T lymphocyte responses were determined independent of changes in the number of responder cells within peripheral blood.     

Identification of bovine lymphocyte subpopulations by a combined bacterial adherence and fluorescent antibody technique

It is known that certain strains of bacteria bind selectively to subpopulations of human peripheral blood lymphocytes. We have developed a technique which used the specificity of bacterial binding concurrently with fluorescent antibody staining methods to identify 5 B-cell and 5 T-cell subpopulations of bovine lymphocytes. In addition, greater than 95% of the peripheral blood lymphocytes could be positively identified as being either T-cells or B cells. Using ethidium bromide-stained bacteria and lymphocytes in combination with fluorescent antibody staining to detect surface immunoglobulins or T-cell antigens, the method provided a simple yet highly specific technique for the enumeration of both B and T cells in 1 preparation of peripheral blood lymphocytes. The use of bacterial rosetting with fluorescent antibody staining was found to be easier and more reliable than the methods currently used to identify bovine B- and T-lymphocyte subpopulations.     

Preparation and characterization of monoclonal antibodies against bovine B lymphocyte surface antigens

Two monoclonal antibodies (MoAbs; BLMo-4 and BLMo-10) were prepared by immunizing with a cell line established from peripheral blood mononuclear cells (PBMC) of enzootic bovine leukosis (EBL) cattle. The specificities of these MoAbs were assayed using bovine PBMC. BLMo-4 reacted with all surface immunoglobulin-positive cells (SIg+ cells; B lymphocytes) and also recognized monocytes, but did not react with T lymphocytes. BLMo-10 recognized a majority, although not all, B lymphocytes, but did not react with either T lymphocytes or monocytes. The antigens recognized by BLMo-4 and BLMo-10 were not Ig, Fc or C3 receptors on the surface of B lymphocytes. The reactivity of the MoAbs with mononuclear cells from the lymphoid organs of adult cattle was studied. BLMo-4 and BLMo-10 did not react with any bone marrow cells. BLMo-10 reacted with 7.4% of thymocytes, and stained the medulla of the thymus in the immunoperoxidase assay. In the case of PBMC, spleen and lymph node cells, the percentage of cells positive for BLMo-4 was slightly higher than that of SIg+ cells, but BLMo-10 showed a slightly lower value.     

Differentiation antigens on bovine mononuclear phagocytes identified by monoclonal antibodies

Five monoclonal antibodies (MAb) produced against cell surface antigens on bovine mononuclear phagocytes (MPh) were characterized. None of the MAb recognized erythrocytes, thrombocytes, B lymphocytes or resting or activated T lymphocytes. Two MAb (IL-A22 and IL-A24) reacted with the majority of monocytes and granulocytes in peripheral blood, with 20-40% bone marrow cells comprising myelo-monocytic cells, and with a proportion of mature macrophages. Reactivity of the remaining three MAb was restricted to MPh: one of these (IL-A25) was apparently specific for pulmonary macrophages, whereas the molecules recognized by the other two (IL-A23 and CH16A) were expressed on subpopulations of blood monocytes and tissue macrophages. None of the MAb inhibited adherence of MPh to plasma-coated gelating surfaces or Fc-mediated rosette formation. One of the MAb, IL-A24, which reacts with MPh and granulocytes, inhibited antigen-specific proliferative response or peripheral blood mononuclear leukocytes (PBM) to the soluble antigen, keyhole limpet hemocyanin (KLH) but did not inhibit responses to concanavalin A or allogeneic leukocytes. This MAb was shown to react with two polypeptides of approximately 75 kD and 110 kD on the surface of peripheral blood monocytes.     

Investigation of cross-reactivity between commercially available antibodies directed against human, mouse, and rat lymphocyte surface antigens and surface markers on canine cells

Using automated flow cytometry, 23 commercially available antibodies (all but one of them monoclonal) raised against surface antigens of specific populations of human, rat, and mouse lymphocytes were tested for cross-reactivity to peripheral blood lymphocytes from five clinically healthy adult dogs. Of all the antibodies tested, only the polyclonal anti-asialo GM1 directed against mouse NK cells, and the monoclonal antibodies anti-HLA-DR directed against the human class II antigen and anti-B1, a human pan B cell marker, consistently labeled subpopulations of canine lymphocytes.     

Surface markers on bovine fetal lymphocytes and immunoglobulin synthesis in a congenital infection related to epizootic bovine abortion

Immunological parameters were studied among 23 late-term bovine fetuses. Epizootic bovine abortion (EBA) disease was induced in fetuses by feeding Ornithodoros coriaceus ticks on pregnant heifers. A spirochaete-like microorganism was detected in the blood of diseased fetuses and in inapparent natural infections in some abattoir-collected fetuses. Fetuses were classified according to stages of disease: EBA diseased (n = 10), EBA infected (n = 7) and normal (n = 6). Using flow cytometry, the presence of surface immunoglobulins (sIg) and peanut agglutinin (PNA) receptors were used to detect B and T lymphocytes, respectively. In peripheral blood of normal fetuses, most lymphocytes were identified as T or B cells, whereas about 20 per cent of lymphocytes in EBA diseased fetuses did not reveal the sIg or PNA receptor markers (null cells). Size and shape analyses by flow cytometry detected a population of enlarged lymphocytes in the EBA diseased fetuses. The numbers of cells bearing determinants reactive with monoclonal antibodies specific for bovine T cells (B26A and B29A) and B cells (TH21A) were considerably less than those expressing the PNA receptor and sIg. These results suggested that the monoclonal antibodies were binding to differentiation antigens which were not consistently expressed on the fetal cells. Radio-immunodiffusion was used to measure bovine IgM, IgG1 and IgG2 in fetal serum. The quantities of immunoglobulins were markedly increased in animals infected with the spirochaete-like organism (groups 1 and 2) and were assumed to result from fetal antibody synthesis.     

Establishment of an IL-2-dependent bovine T cell line and an assay of lymphocyte response inhibition in leukemic bovine serum.

The mechanism of immunosuppression induced by leukemic bovine serum was investigated with respect to lymphokine reactions using an interleukin 2 (IL-2)-dependent bovine T cell line generated from bovine peripheral blood lymphocytes (PBLs). The suppression of concanavalin A (con A)-induced PBL blastogenesis was observed at a high rate in leukemic cattle sera. The growth of IL-2-dependent bovine T cells and IL-2 production from con A-induced bovine PBLs were also inhibited by these sera, and particularly, the latter was correlated significantly to the degree of lymphocyte blastogenesis by the mitogen. Therefore, the lesser sensitivity of lymphocytes to IL-2 and the reduced IL-2 production by activated lymphocytes seem to play a role in suppressing the lymphocyte reaction.     

Changes in the peripheral leukocyte phenotype of calves in clinical cases of bronchopneumonia complicated with chlamydial co-infectious agent

This paper reports the alterations in peripheral blood leukocyte phenotype in respiratory diseased calves affected with chlamydial and non-chlamydial co-infectious agent. The etiological contribution of chlamydial infectious agent in examined clinical cases of enzootic bronchopneumoniae syndrome was confirmed in affected calves serologically both by complement fixation test (CF) and enzyme immunoassay (EIA). Changes in leukocyte subpopulations in the blood of the calves were detected both with routine haematological methods and by FCM using specific monoclonal antibodies directed against CD14, CD45, CD2, CD4, CD8 and WC4 (a specific surface marker for bovine B-lymphocytes). The results obtained by flow cytometry analysis indicate that polymorfonuclear neutrophils (PMNLs) and T lymphocytes, especially CD8-positive cells, may play a significant role in cellular immune response against Chlamydophila psittaci (Chl. psittaci) co-infection in calves suffering from enzootic bronchopneumonia syndrome. A repercussion of this was a significant increase of the cell numbers in peripheral blood of the infected animals. Effective recruitment from a reserve marginal pool of these cells into blood vessels and activation of bone marrow proliferation are probably the reason for their high circulating number.     

Characterisation of antibodies to bovine Toll-like receptor (TLR)-2 and cross-reactivity with ovine TLR2

Host recognition of conserved pathogen-associated molecular patterns (PAMPs) and their interactions with pattern-recognition receptors, including the Toll-like receptors (TLR) is essential for innate immune response induction. The TLR1 family (TLR1, 2, 6 and 10) is involved in the recognition of gram-positive and gram-negative bacteria and heterodimers of TLR1 or TLR6 with TLR2 are crucial for the identification of several PAMPs. Studies on cell surface expression of TLR in ruminants are hampered by the lack of specific antibodies and no convincingly cross-reactive anti-human antibodies have been described so far. We describe herein four antibodies which recognise bovine TLR2. Differences in TLR2 expression were evident on bovine antigen presenting cells with high level expression on peripheral blood monocytes and monocyte-derived macrophages. Lower levels of expression were evident on dendritic cell populations derived in vitro and ex vivo, and on alveolar macrophages. One of the antibodies recognised TLR2 expression on ovine peripheral blood monocytes. The identification of antibodies specific for bovine and ovine TLR2 will facilitate studies of the role of this important PRR in the initiation of immune responses to important pathogens.     

Erythrocyte rosettes--a marker for bovine T cells.

Many species of erythrocytes were investigated for their ability to form spontaneous rosette with bovine peripheral blood leukocytes and fetal thymocytes. Only sheep and chicken red blood cells gave rosettes. Using conditions shown optimum for the demonstration of human rosette forming cells, only low numbers of bovine rosettes were demonstrable. By changing culture conditions to include 100% fetal calf serum, neuraminidase treated erythrocytes and/or lymphocytes and optimizing the incubation times and temperature, up to 38% of peripheral blood leukocytes and 52% of thymocytes formed rosettes. A thymic origin of rosetting cells was ascribed to T cells for the following reasons: 1) thymocytes gave higher numbers than did peripheral blood leukocytes, 2) rosette forming cell numbers were increased in peripheral blood leukocyte subpopulations enriched in T cells by nylon column separation and 3) only very few rosette forming cells had surface immunoglobulin, a marker of B lymphocytes. The reasons why all T cells were not detected by the technique were discussed.     

Development and functional characterization of monoclonal antibodies recognizing chicken lymphocytes with natural killer cell activity.

We have developed two monoclonal antibodies which detect cell surface antigens present on chicken lymphocytes mediating natural killer (NK) cell activity against the avian tumor cell target. The monoclonal antibodies, K-14 and K-108, stained 17 and 6% of splenic lymphocytes, and 11 and 14% of peripheral blood lymphocytes (PBL), respectively, and fewer than 5% of thymic and bursal lymphocytes. Neither of these monoclonal antibodies stained adherent macrophages or the MC29-virus transformed monocytic cell line. Both monoclonal antibodies significantly inhibited NK cell activity in a standard 4 h 51Cr-release cytotoxicity assay using the LSCC-RP9 tumor cell line as target cells at an effector to target ratio of 50:1. Pretreatment of splenocytes with either monoclonal antibody in the presence of rabbit complement (C) resulted in a significant reduction in NK cell activity. However, the monoclonal antibody K-1 which detects normal chicken macrophages did not interfere with NK cell activity. The monoclonal antibody K-108 significantly blocked Fc receptor-mediated rosette formation of sheep red blood cells coated with IgG antibodies (EA) by 56% while the monoclonal antibody K-14 did not show a significant blocking. These results indicate that the monoclonal antibodies K-108 and K-14 identify different epitopes present on the surface of chicken splenic lymphocytes which mediate spontaneous NK cytotoxicity.     

Identification of two independent MHC class II antigens in a bovine lymphoblastoid cell line

Bovine major histocompatibility complex (MHC) class II antigens were investigated using monoclonal antibodies (MoAbs) with known MHC class II specificities in other species. Thirty-four MoAbs were tested for reactivity with bovine peripheral blood mononuclear (PBM) cells and the bovine lymphoblastoid cell line, BL3, by flow cytometry. Twenty-seven of 31 MoAbs tested, reacted with BL3 cells, and 22 of 25 MoAbs tested with PBM cells were reactive. MoAbs that reacted with BL3 cells were used to immunoprecipitate class II molecules from BL3 lysate labeled with [35S]methionine. Using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography, many MoAbs were found to immunoprecipitate a single band of approximately 31,000 relative mass (Mr). MoAbs yielding successful immunoprecipitations and with known antigen specificity in other species were then used in sequential immunoprecipitations and two dimensional (2-D) non-equilibrium pH gradient electrophoresis (NEPHGE). The HLA-DR specific MoAb H4 and the predominantly HLA-DQ specific MoAb CC11.23 were used to identify the presence of two independent antigens in BL3 cell lysate. These class II molecules consist of alpha and beta chains.