Monoclonal antibodies against histone H5. Epitope mapping and binding to chromatin.
Monoclonal antibodies against chicken erythrocyte histone H5 were produced. Nine hybridomas of different clonal origin were selected, and the antibodies were purified by affinity chromatography. Typing of the antibodies indicated that all but one (IgM) belong to the IgG1 class and contain kappa light chains. Indirect immunoprecipitation, solid-phase radioimmunoassay, and competitive inhibition assays using various H5 fragments revealed that the antigen-binding sites were localized on the central region of H5 (GH5, residues 22-100). Results of immunoblots from gels containing different denaturing agents indicate that some of the antibodies recognize related continuous epitopes localized at the junction of the GH5 with the rest of the molecule. Competition experiments between pairs of the eight different IgGs suggest that they recognize at least seven distinct sites on GH5. The epitopes appear to represent different regions of GH5 although some of them overlap. In general, the antibodies recognize epitopes which are not too accessible to the environment in the native conformation of the histone. All of the antibodies examined, except one of them (5H10), react with nuclei and chromatin from the erythroid cells but not from other cell lines. The site recognized by 5H10 is likely to be one of the regions where GH5 interacts with the nucleosome. No cross-reactivity of the antibodies with other histones including H1, H2A, H2B, H3, H4, and rat liver histone H1(0) was observed.
M
M
Rózalski
L
L
Lafleur
A
A
Ruiz-Carrillo
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity; D001666:Binding Sites, Antibody; D001667:Binding, Competitive; D002467:Cell Nucleus / Q000276:immunology; D002645:Chickens; D002843:Chromatin / Q000276:immunology*; D003101:Collodion; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes / Q000276:immunology*; D004912:Erythrocytes / Q000032:analysis; D006657:Histones / Q000097:blood / Q000276:immunology*; D006825:Hybridomas / Q000276:immunology; D007163:Immunosorbent Techniques; D010446:Peptide Fragments / Q000276:immunology; D051381:Rats
D000911:Antibodies, Monoclonal; D002843:Chromatin; D000939:Epitopes; D006657:Histones; D010446:Peptide Fragments; D003101:Collodion