Cell surface display of rat invariant gamma chain: detection by monoclonal antibodies directed against a C-terminal gamma chain segment.
A series of 14 monoclonal antibodies (mAb) directed against the C-terminal part of the rat invariant gamma chain (amino acid 142-216) was generated using distinct fusion proteins that contain this gamma segment for immunization and hybridoma screening. Additional fusion protein were prepared carrying discrete regions of the gamma chain. Employing these reagents confirmed that the obtained mAb do indeed recognize the C-terminal portion of the invariant chain, as demonstrated by Western blot analysis. All mAb established recognize epitopes present on the native gamma chain, as revealed by immunoprecipitation analysis using nonionic detergent extracts of metabolically labeled Lewis rat splenocytes combined with two-dimensional gel electrophoresis. However, while the majority of the gamma chain-specific mAb precipitated gamma chain-containing polypeptide chain complexes in which immature, sialic acid-deficient and mature, terminally sialylated forms of the gamma chain were predominantly represented, a fraction of the antibodies preferentially precipitated the immature gamma forms. Cell surface binding of these two groups of mAb correlated with the immunoprecipitation data in that the former group of antibodies did bind to intact Lewis rat spleen cells, while essentially no binding was observed with the antibodies of the latter group. Double-fluorescence staining with the class II-specific fluorescein isothiocyanate-conjugated mAb OX3 and OX6, respectively, as well as a representative gamma chain-specific mAb visualized with phycoerythrin-coupled secondary antibody shows coexpression of class II determinants and the invariant chain at the cell surface.
Fisch
A
A
Institut für Immunologie, Universität Mainz, FRG.
Reske
K
K
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000944:Antigens, Differentiation, B-Lymphocyte; D000954:Antigens, Surface / Q000096:biosynthesis*; D015153:Blotting, Western; D003001:Cloning, Molecular; D015180:Electrophoresis, Gel, Two-Dimensional; D004926:Escherichia coli; D005434:Flow Cytometry; D005822:Genetic Vectors; D000949:Histocompatibility Antigens Class II / Q000096:biosynthesis* / Q000276:immunology; D006825:Hybridomas; D051379:Mice; D008807:Mice, Inbred BALB C; D010957:Plasmids; D051381:Rats; D011917:Rats, Inbred Lew; D011993:Recombinant Fusion Proteins; D013154:Spleen / Q000276:immunology
D000911:Antibodies, Monoclonal; D000944:Antigens, Differentiation, B-Lymphocyte; D000954:Antigens, Surface; D000949:Histocompatibility Antigens Class II; D011993:Recombinant Fusion Proteins; C045084:invariant chain