A bovine monoclonal antibody detecting a class I BoLA antigen.

The bovine IgG1 monoclonal antibody (mAb) ILA70 was made by immunizing a calf with peripheral blood mononuclear cells (PBM) from a BoLA-w10 homozygous heifer and subsequently fusing lymphocytes from the local lymph-node with the heterohybridoma 53B3. Family and population studies, antibody binding inhibition and immunoprecipitation of the target antigen all indicate that ILA70 detects a polymorphic epitope on a bovine class I major histocompatibility complex (MHC) molecule. The antibody is complement fixing and so may be used in a standard cytotoxicity assay. Ascitic fluid with antibody activity many times greater than that of the tissue-culture supernatant has been prepared in nude mice. The antibody-producing heterohybridoma has been subcloned three times and appears to be stable. Such heterohybridomas may prove to be a valuable source of particularly discriminating and informative mAbs for the serological analysis of the products of the bovine MHC.     

A polymorphic monoclonal anti-BOLA class I antibody

Cytotoxic monoclonal antibody IVA 44 was generated after the intraperitoneal immunization with peripheral blood mononuclear (PBM) cells and the boost by the intrasplenic inoculation of skin graft. The detected membrane antigen isolated by immunoprecipitation appears to be composed of two subunits characteristic for the MHC class I molecules. The antibody IVA 44 exhibited a different reactivity: it recognized the BoLA A14 (A8) specificity in animals typed in the Fifth BoLA workshop, while it reacted with all A8 positive animals including subtypes A14 and A15 in Czech and Slovak cattle. It is concluded that mAb IVA 44 might detect the broad subtype of A8 covering A14 and certain A15 split(s). The diverse A15 reactivity of this mAb in the workshop and our population could be explained by the different occurrence of A15 splits in both populations.     

A new monoclonal antibody detecting ochratoxin A at the picogram level

Á. GYÖNGYÖSI-HORVÁTH, I. BARNA-VETRÓ AND L. SOLTI. 1996. A monoclonal antibody against ochratoxin A was produced after immunization of Balb/c mice with ochratoxin A-BSA. This antibody was of the IgGl heavy chain subclass with a k type light chain. The 50% inhibition value was 0.45 ng ml^-1 in a direct competitive ELISA and the detection limit was 42 pg ml^-1. This antibody is very specific, cross-reacting only with ochratoxin B (9.3%).     

T cell recognition of nonpolymorphic determinants on H-2 class I molecules

Recognition of polymorphic determinants on class I or class II MHC Ag is required for T lymphocyte responses. Using cell-size artificial membranes (pseudocytes) bearing H-2 class I Ag it is demonstrated that T cells can, in addition, recognize nonpolymorphic determinants on class I proteins. Pseudocytes bearing class I alloantigen stimulate in vitro generation of secondary allogeneic CTL responses. At a suboptimal alloantigen surface density, incorporation of class I molecules identical to those of the responder cells (self-H-2) or from third-party cells resulted in dramatically enhanced responses, whereas incorporation of class II proteins had no effect. The receptor that mediates recognition of conserved class I determinants has not been identified, but results of antibody blocking studies are consistent with the Lyt-2/3 complex of CTL having this role. Thus, class I proteins on Ag-bearing cells can have two distinct roles in T cell activation, one involving recognition of polymorphic determinants by the Ag-specific receptor and the other involving recognition of conserved determinants.     

Signal transduction through class I MHC by a monoclonal antibody that detects multiple murine and human class I molecules.

We have generated a hamster anti-mouse class I reactive mAb that is capable of activating T cells in the presence of the cofactor PMA, as assayed by both IFN-gamma production and cellular proliferation. This mAb detects an epitope present on the majority of murine class I molecules, with the known exceptions of H-2Kk and H-2Kq, and is therefore not beta 2-microglobulin-specific. It also recognizes multiple human class I molecules. The epitope recognized by this antibody maps to the class I alpha 1 domain. The activation properties of this mAb are not mediated exclusively through the glycosylphosphatidylinositol-linked Qa-2 molecule, as the antibody activates spleen cells from Qa-2 negative strains. Although class I molecules are not usually considered as activation Ag, these data demonstrate their potential for involvement in signal transduction.     

A monoclonal antibody detecting unusual Thy-1 determinants

20-10-5S is a monoclonal antibody produced by the fusion of C3H anti-C3H.SW splenocytes with the SP2/0 cell line. The antibody appears to react with Thy-1 determinants by several criteria including cytotoxicity patterns, functional assays, genetic analyses, and competitive binding experiments. However, the antibody and the determinants it detects are unusual in that: 1) 20-10-5S is autoreactive; 2) the antibody shows allospecificity for Thy-1.2 vs Thy-1.1 antigens only on peripheral lymphocytes and not on thymocytes; and 3) the antibody reacts only with determinants on murine T cells and not with antigens on brain tissue or on rat thymocytes. It therefore seems that 20-10-5S reacts with murine T cell-specific Thy-1 determinants that are lost or modified during maturation of the cells on which they are expressed.     

Influence of major histocompatibility complex H-2LD class I molecules on spleen colony-forming units

To test whether the major histocompatibility complex class I genes are involved in the regulation of hemopoiesis, the stem cell activities of BALB/c-H-2dm2 (Dm2) mice, which are defective in the expression of H-2L antigens, have been compared with those of the wild-type, BALB/c-Kh, in in vivo and in vitro stem cell assays. In spleen colony-forming unit assays, Dm2 as hosts consistently supported a smaller number of colonies than did BALB/c-Kh. However, both Dm2 and BALB/c-Kh supported a comparable number of colonies in in vitro granulocyte-macrophage colony-forming unit and erythroid colony-forming unit assays. These observations together suggest that the mutation in Dm2 has not affected the hemopoietic potential of the stem cells but may probably affect the hemopoietic microenvironment for the development of the stem cells.     

Serological analysis of H-2 and Ia molecules with monoclonal antibodies

Five H-2 and seven Ia monoclonal antibodies were tested against a panel of 43 independentH-2 haplotypes (11 of laboratory-mouse and 32 of wild-mouse origin), 33 recombinantH-2 haplotypes, and up to 74 wild mice. All the antibodies gave negative reactions in the PVP hemagglutination tests; all, however, gave positive reaction with some members of the panel in the dye-exclusion cytotoxic test. Four of the antibodies (H-2.m2,Ia.m2, Ia.m5 and Ia.m7) reacted identically to conventional antibodies detecting determinants H-2.2., and Ia-1.2, Ia-1.15, and Ia-5.7, respectively (this statement does not apply to wild mice in which minor differences in reactivity patterns of the corresponding antibodies were found: the reproducibility of these differences, however, could not be checked by absorption). Five other antibodies (H-2.m5, H-2.m3, H-2.m4, Ia.ml, and Ia.m6) had very similar though not identical reactivity patterns to conventional antibodies detecting determinants H-2.5, H-2.11, H-2.25, Ia-1.2, and Ia-1.19, respectively. The last three monoclonal antibodies (H-2.ml, Ia.m3, and Ia.m4) had a reactivity pattern that did not match those of any known conventional antibodies. The near identity or great similarity of many monoclonal and conventional antibodies indicates that the cleanest of the conventional antisera are truly monospecific, and gives credence to the H-2 serology as defined by conventional antibodies. The serological analysis of monoclonal antibodies supports the true existence of private and public determinants, and reveals that the H-2 and Ia determinants are complex, even when the antibody is simple.     

Separation of spleen colony-forming units and prothymocytes by use of a monoclonal antibody detecting an H-2K determinant

The density of H-2K antigens was determined on both the mouse hemopoietic stem cell, using an assay for spleen colony-forming units (CFU-S), and the prothymocyte, using a thymus repopulation assay. This was done by light-activated cell sorting of bone marrow cells labeled first with a biotinylated antibody against H-2Kk and then with avidin-fluorescein isothiocyanate. Almost all CFU-S were found to be present among the 4% bone marrow cells with high forward light scatter (FLS), low perpendicular light scatter (PLS), and bright immunofluorescence. Thymus regeneration by this brightly fluorescent fraction was delayed 3 days compared to thymus regeneration by unsorted cells, although the same number of CFU-S was present in each cell suspension. This delay indicates that differentiation from CFU-S to prothymocytes takes 3 days. The fraction of cells in the FLS/PLS window with dull anti-H-2Kk fluorescence contained few CFU-S and gave rise to a transient thymus regeneration. These findings indicate that the prothymocyte carries fewer H-2K antigens than does the CFU-S. The H-2K antigen is a marker with which CFU-S and prothymocytes can be separated. Therefore, during early T-cell differentiation, the number of H-2K molecules on the cell surface decreases (CFU-S----prothymocyte----cortical thymocyte). During maturation of T cells, a reexpression of H-2K molecules occurs, since lymph node cells and spleen cells were shown to be brightly positive for H-2K antigen.     

A new H-2.1-PositiveD region allele,D dx,controlling two molecules,H-2Ddx and H-2Ldx

The inbred strains GRS/A and LIS/A carry the haplotypeH-2 ^dx , which had earlier been shown to have theK ^d ,I ^f ,S ^f , andG ^f alleles and a previously unknownD region allele,D ^dx . We show here that theD ^dx allele determines a new private specificity, H-2.63, is H-2.28 negative, and determines at least one public specificity of the H-2.1 family. It is thus a second example (afterD ^k ) of a H-2.1-positive H-2.28-negativeD region allele. Capping experiments show that the D^dx product comprises two molecules: H-2D^dx bearing the private specificity H-2.63, and H-2L^dx, which is H-2.63-negative but reacts with sera against the H-2.1 family of specificities. SDS gel electrophoresis of detergent-solubilized immunoprecipitated D^dx products shows that the H-2L^dx antigen has a molecular weight of approximately 45,000 daltons and is associated with a smaller polypeptide (mol. wt. 12,000).     

Family organization of mouse H-2 class I genes

The H-2 complex of the mouse contains numerous class I genes of unknown function. These genes are here classified into families according to homology in the exons encoding the variable domains. There are one major and at least five minor families, whose members are partly clustered and partly interspersed on the mouse chromosome. DNA sequences show that not only Tla and Qa-2 but also other minor-family genes have intact coding domains. These may be expressible genes of novel types.     

Localization of H-2Kk in developing mouse teeth using monoclonal antibody

The in situ distribution of H-2 antigens during mouse tooth morphogenesis was investigated using monoclonal antibodies to H-2Kk and indirect immunofluorescent techniques. H-2 antigens were detected in the basement membrane region of fetal molars; they were absent from both the epithelial and dental mesenchyme. H-2 antigens were not found in newborn and 4-day-old mouse molars.