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Antibodies Directed Against Monomorphic and Evolutionary Conserved Self Epitopes may be Generated in ‘Knock‐Out’ Mice. Development of Monoclonal Antibodies Directed Against Monomorphic MHC Class I Determinants
Author(s) -
CLAESSON M. H.,
ENDEL B.,
ULRIK J.,
PEDERSEN L. Ø.,
SKOV S.,
BUDS S.
Publication year - 1994
Publication title -
scandinavian journal of immunology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.934
H-Index - 88
eISSN - 1365-3083
pISSN - 0300-9475
DOI - 10.1111/j.1365-3083.1994.tb03459.x
Subject(s) - monoclonal antibody , epitope , microbiology and biotechnology , biology , antibody , peripheral blood mononuclear cell , major histocompatibility complex , mhc class i , beta 2 microglobulin , antigen , t cell , b cell , cell culture , immunology , immune system , biochemistry , in vitro , genetics
Beta‐2 microglobulin (β 2 m) gene ‘knock‐out’ mice (C1D) were primed wilh purified H‐2K b and H‐2D b molecules and spleen cells from immunized mice were used to generate monoclonal antibody secreting B‐cell hybridomas. Approximately 0.2% of the Ig‐secreting primary microcultures contained H‐2 b binding antibodies. Three stable anti‐MHC class I (MHC‐I) antibody secreting hybridoma clones were established and subcloned. All three MoAbs precipitated radiolabelled H‐2 molecules as analysed by SDS PAGE, and all three MoAbs stained H‐2 b , H‐2 d , as well as H‐2 k cells by FACS analysis. The MoAbs stained to two β 2 m loss mutant cell lines, C4.4‐25‐ and R1E, suggesting that some MHC‐I heavy chain is exported to the cell surface even in the absence of endogenous β 2 m. Staining of murine cell lines kept under serum‐free culture conditions was strongly influenced by the addition of bovine or human serum as a source of exogenous β 2 m suggesting that xenogeneic β 2 m affects the conformation of class I molecules. Furthermore, all three MoAbs strongly stained the peptide transporter deficient cell line, RMA‐S, when cultured at 26°C, however, staining was reduced five‐fold when RMA‐S cells were cultured at 37°C. In total, these observations suggest that the MoAbs recognize conformational, presumably β 2 m and peptide dependent, self epitopes on MHC‐class I. One of the three MoAbs stained rat blood mononuclear blood cells (BMC), all three MoAbs stained hamster BMC, whereas two of the MoAbs stained human cells. These data suggest that the MoAbs recognize determinants which are conserved between species. All three antibodies strongly inhibited the development of CTLs generated in an allogeneic one‐way MLC, provided that the MoAbs were present during the first 24 h of culture. It is concluded that MoAbs reacting with monomorphic self epilopes may be generated using animals deleted of the gene of interest. The implications may be far reaching since such MoAbs potentially identify evolutionary conserved and physiologically important epitopes.