Preparation of a bovine monoclonal antibody to testosterone by interspecies fusion

This communication reports the first successful attempt to produce a hybridoma cell line secreting bovine immunoglobulin to a small hapten, starting with peripheral blood lymphocytes, rather than spleen or lymph node cells. A heteromyeloma line, sensitive to selective media, was made by fusing NS1/1-Ag4-1 mouse myeloma cells with bovine peripheral blood lymphocytes. This cell line was then fused with blood lymphocytes from a steer immunised with a testosterone immunogen. Cell cultures were screened using an ELISA specific for bovine antibodies to testosterone. Following repeated cloning, a cell line was established which secretes moderate levels of a specific, high affinity antibody to testosterone. This particular cell line has significant potential for veterinary application and the successful fusion demonstrates the possibilities of heteromyelomas for the development of non-murine monoclonal antibodies.     

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.     

Bovine monoclonal antibodies to the F5 (K99) pilus antigen of E. coli, produced by murine/bovine hybridomas

Lymph node cells from calves immunized with purified pilus antigen of K99+ enterotoxigenic E. coli (ETEC) were fused with mouse myeloma (NSO) cells, and with non-Ig producing mouse/calf hybridomas or with a bovine Ig-producing mouse/calf/calf secondary hybridoma. Lines secreting bovine monoclonal IgG1 specific for K99 pilus antigen in an ELISA were obtained in each case. The two lines derived from xenohybridoma fusion partners have been secreting anti-K99 bovine monoclonal antibody for over one year in continual passage. None of the antibodies cross-reacted with other pilus types including K88, CFAI, CFAII, 987P or CP; they all inhibited agglutination of horse RBC (which have a K99 receptor) in the presence of K99 antigen; they showed positive fluorescence in an indirect binding assay on K99+ ETEC and inhibited K99+ ETEC adhesion to piglet enterocytes. These antibodies have potential prophylactic and therapeutic use in control and treatment of diarrhoea.     

Protective activity to murine experimental brucellosis conferred by monoclonal antibody ISS/32 anti-B. abortus.

The protective properties of the monoclonal antibody ISS/32 anti-B. abortus were estimated by splenic infection with B. abortus 544. Five groups of Balb/c mice were used: two groups, previously vaccinated with a 45/20 antigen and a-LPS antigen, were challenged after 30 days intravenously by inoculation of 2.10(5) cells of B. abortus 544, one group was challenged with the same dose of B. abortus 544 preincubated with MAb-ISS/32 and another one with B. abortus 544 incubated with negative serum; the fifth group infected with B. abortus 544 only served as control. The results, expressed as an index of splenic infection, show significant protective properties of monoclonal antibody ISS/32. The infection index in the MAb-ISS/32 group of mice was a bit lower than in B. abortus 45/20 vaccine group.     

A canine lymphocyte surface antigen detectable by a monoclonal antibody (DT200).

A murine monoclonal antibody (DT200) was raised against a 210,000-dalton (210 K) lymphocyte surface protein (a member of the lymphocyte antigen known as T200) which was purified from a canine lymphoid tumor by preparative slab gel electrophoresis. In immunoblotting studies of electrophoretically separated plasma membranes from five cases of canine lymphoma, the antibody detected two antigenically intact peptides at 95 and 110 K which, based on previous polyclonal anti-210 K antiserum immunoblotting and peptide mapping studies, may represent the protease-resistant fragment of the canine T200 molecule. Since DT200 retains its reactivity in formalin-fixed, paraffin-embedded tissue sections, 13 dogs with malignant lymphoma and a panel of normal lymphoid and nonlymphoid tissues were studied using an indirect immunoperoxidase technique. The antigen was localized predominantly to the surface membrane of lymphoid cells. DT200 reacted strongly with all five histological subtypes of lymphoma tested while moderate reactivity was detected in normal B and T cell areas of lymph node, spleen and tonsil. Thymocytes and selected hemopoietic precursors were weakly reactive with DT200 while plasma cells, mature granulocytes, red cells and megakaryocytes were unstained. It was concluded that DT200 is a useful reagent for the diagnosis of malignant lymphoma particularly in extranodal sites and may prove valuable in the investigation of the structure and function of T200 in the dog.     

Production of monoclonal antibody against recombinant bovine sex-determining region Y (SRY) and their preferential binding to Y chromosome-bearing sperm

Separation of X and Y chromosome-bearing sperm is an appropriate method for the selection of desired sex of offspring to increase the profit in livestock industries. The purpose of this study was the production of a monoclonal antibody against recombinant bovine sex-determining region Y protein for separation Y sperm. The hybridoma cells from splenocytes of immunized female's balb/C mice and Sp2/0 cells were made. The binding affinity of our monoclonal antibody (mAbSRY2) was compared with mouse monoclonal SRY-15. The Western blot method indicated that mAbSRY2 successfully detected the rbSRY protein. The specificity and sensitivity of mAbSRY2 is comparable to SRY-15 commercially ones. The SRY gene in 100% of bull semen contains the Y chromosome that had the strongest binding affinity to mAbSRY2 was synthesized. In other words, the binding affinity of semen contains the X sperms near the negative control. In general, this immunological method can help to separate X from Y sperms. However, the mAbSRY2 is bind to Y-bearing sexed sperm, but in the future; the sexed sperms need to apply in farms.     

Production, characterization, and applications of a murine monoclonal antibody to dog erythrocyte antigen 1.1.

A murine IgM monoclonal antibody, which recognizes dog erythrocyte antigen (DEA) 1.1, has been produced. The antibody correctly identified canine RBC possessing DEA 1.1 in a panel of RBC typed by an independent laboratory. Reactivity of the monoclonal antibody was compared with canine anti-DEA 1.1 antiserum with 163 RBC samples from 145 dogs. Results of agglutination tests with the 2 reagents were in agreement for all samples. A card agglutination test that uses the monoclonal antibody with blood is described. A monoclonal antibody-based test should facilitate blood typing for DEA 1.1 in clinical practice.     

Characterization of a novel activation antigen on porcine lymphocytes recognized by monoclonal antibody 5A6/8

In this report, we describe the characterization of a novel activation antigen on porcine lymphocytes recognized by mAb 5A6/8. This antigen was detected on B and T cells 24 h after treatment with various stimuli. It was also found on alveolar macrophages, and at low levels on untreated monocytes. MAb 5A6/8 precipitated two bands of 45 and 50 kDa under non-reducing conditions, and of 22 and 28 kDa under reducing conditions. The cellular distribution, expression kinetics and/or molecular size of the 5A6/8 antigen differ from those of other known lymphocyte activation antigens. MAb 5A6/8 was able to inhibit lymphocyte proliferative responses driven by different stimuli, suggesting a role for this molecule in the events that lead to lymphocyte activation.     

A neutralizing monoclonal antibody to bovine rotavirus VP8 neutralizes rotavirus infection without inhibiting virus attachment to MA-104 cells.

VP8*, the N-terminal cleavage product of rotavirus VP4, contains the virus neutralizing epitopes in the hemagglutination domain. To map the neutralizing epitope, we developed monoclonal antibodies specific for VP4 of bovine rotavirus C486 (BRV). A neutralizing escape mutant was generated by one of these monoclonal antibodies (2E8) and a point mutation (Glu-->Asp) was found at aa 116 of VP8*. To investigate the effect of this mutation on the cellular binding and hemagglutination activities, the VP8* genes of the escape mutant and wild type (WT) virus were expressed in E. coli and their functional activities were compared. Both the escape mutant and WT virus VP8* showed hemagglutination and MA-104 cell binding activities. However, hemagglutination activity of the WT virus VP8* was inhibited by 2E8, but that of the escape mutant VP8* was not. These data indicate that the neutralizing epitope is located in the HA domain but is not critical for rotavirus attachment to MA-104 cells. To understand virus neutralization, radiolabelled BRV was incubated with 2E8 and the distribution of radioactivity in a CsCI density gradient was analysed as was the morphology of the virions in peak fractions. Interaction of 2E8 with rotavirus led to virus morphological changes with a concomitant shift in buoyant density. These data suggest that aa 116 influences the binding of 2E8 which in turn may alter virus integrity.     

FACS analysis of bovine leukemia virus (BLV)-infected cell lines with monoclonal antibodies (mAbs) to B cells and to monocytes/macrophages.

The eighteen monoclonal antibodies (mAbs) to B cells and the fourteen mAbs to accessory cells submitted to the workshop were analysed by FACS on three established, bovine leukemia virus (BLV)-infected bovine cell lines. Several mAbs of previously defined specificity were run in parallel. This analysis allowed us to gain further insight on the precise phenotype of those peculiar cells and to cluster the submitted mAbs according to their staining patterns. The BLV-infected cell lines seemed to belong to the B cell type though some of them lack detectable surface immunoglobulins. Moreover, all lines express the CD5 T cell marker and several myeloid markers.     

呋喃妥因代谢物AHD单抗的制备及鉴定

将呋喃妥因代谢物1-氨基-乙内酰胺脲(AHD)的衍生物1-氨基乙内酰脲-4-羧苯基肟(CPAHD)分别与牛血清蛋白(BSA)和Jeffamine修饰的卵清蛋白(OVA)偶联,经紫外光谱扫描鉴定,成功合成了免疫抗原CPAHD-BSA和包被抗原CPAHD-Jeffamine-OVA。应用杂交瘤技术将免疫鼠脾细胞与骨髓瘤细胞SP2/0融合,获得了稳定分泌抗4-NPAHD单克隆抗体的杂交瘤细胞株,命名为B10E7。使用本细胞株制备的McAb对4-NPAHD的半数抑制质量浓度(IC50)为3.65 ng/mL,与同类抗生素及其代谢物未发现交叉反应,显示高度的灵敏性和特异性。本抗体的研制为建立间接竞争酶联免疫方法奠定了基础。     

A new epitope on sheep CD45R molecule detected by a monoclonal antibody

This paper describes the production and characterization of a monoclonal antibody (mAb), Co-46D5, which recognizes a new epitope on the isoform of the homologous sheep leukocyte common antigen (LCA) or CD45. This nmAb was submitted to the 3rd workshop on ruminant leukocyte antigens and was assigned to a cluster reactive with B- and T-cells subsets. Co-46D recognizes a 220 kDa molecule on peripheral blood mononuclear cells (PBMC) and spleen cells but not on thymocytes. Flow cytometry (FCM) analysis shows that Co-46D5 reacted with 30% of PBMC and 50% of spleen cells and more than 95% of cells freshly isolated from lymphoid follicles of the ileal Peyer's patches (IPP) of young lambs. By immunohistochemistry, the antigen was detected mainly on B-cell areas of lymph nodes and spleen. It was also found on a subpopulation of medullar thymocytes. Based on these results, we assume that Co-46D5 recognizes a new epitope on the largest isoform of the sheep CD45 receptor, probably on the homologous to the human CD45RA isoform.     

Malignant fibrous histiocytoma (MFH) cells and macrophages/histiocytes have a common antigen recognized by a monoclonal antibody risen against a rat MFH-derived cloned cell line

A monoclonal antibody (B9) was generated by using a rat malignant fibrous histiocytoma (MFH)-derived cloned cell line (MT-8) as the immunogen. Immunohistochemically, B9 reacted specifically with a cytoplasmic antigen of MT-8 cells. Furthermore, B9 immunolabeled another MFH-derived cloned cells (MT-9) and histiocytic sarcoma cells, as well as macrophages/histiocytes in normal and diseased tissues of rats. These findings suggest the presence of a common antigen recognized by B9 between MFH cells and macrophages/histiocytes. This suggests that MFH cells may express histiocytic nature.     

Fasciola hepatica: studies on vaccination of rats and mice with a surface antigen prepared from fluke homogenate by means of a monoclonal antibody

T1 tegumental antigen was isolated from a homogenate of eight- to 10-week-old Fasciola hepatica using a T1-specific monoclonal antibody bound to sepharose in an antibody-affinity column. Rats and mice were vaccinated with T1 antigen in Freund's complete adjuvant, and control groups received equivalent amounts of non-T1 antigen (eluted from the antibody-affinity column) or ovalbumin. On completion of the immunisation programme, serum samples were collected for ELISA and IFA testing. The animals were challenged by oral infection with F hepatica metacercariae or, for several vaccinated rats, by intraperitoneal transplantation of live adult flukes. At autopsy, worm-burden and liver damage was assessed for each animal and the condition of transplanted flukes was examined. Comparison of test and control groups of animals showed that neither T1 nor non-T1 antigens provided significant protection against challenge, although specific antibody responses against the appropriate sensitising antigen were engendered. Flukes transplanted to the peritoneal cavity of immunised rats survived without damage, although they became encased in hollow fibrous capsules of host origin. The results lend support to the pre-existing concept that glycocalyx turnover by discharge of T1 secretory bodies at the apical surface of migrating flukes provides an efficient means of protection for the parasite against host immunity.     

Partial characterization of the antigen recognized by a monoclonal antibody to Ascaris suum ovary extracts

A monoclonal antibody produced against ovary extracts from the worm Ascaris suum showed immunoreactivity against granules in the rachis and oocytes, the inner layer of the eggshell and the middle layer of some egg, but not against either ovary wall or uterus wall. Furthermore, the same antigens were detected on the body surface of migrated larva in guinea pig lung, whereas none were detected in adult male worm or adult female worm, except for the female reproductive organs. The ovary extracts were passed through an affinity column and the eluted fractions analyzed by SDS-PAGE, Western blotting and native-PAGE. Western blotting after SDS-PAGE detected chemiluminescence primarily as three bands of about 70, 78 and 90 kDa. However, Western blotting after native-PAGE of the partially purified ovary extracts demonstrated only one band at a position of about 230 kDa. LC-nanoESI-MS/MS analysis of protein band gel slices from silver-stained SDS-PAGE revealed one peptide sequence "ILVGLIGTNR", that matched only the hypothetical protein F14D2.8 of Caenorhabditis elegans (gi/7499081).