title: Fine specificities of autoantibodies directed against the Ro, La, Sm, RNP, and Jo-1 proteins defined by two-dimensional gel electrophoresis and immunoblotting.
abstract: Although useful for specific purposes, immunofluorescence, precipitation in agarose gels, and the m.w. estimation of RNA or proteins immunoprecipitated from transformed cells often provide partial or ambiguous definition of autoantibody specificity. We have analyzed organ and cell extracts by one-and two-dimensional electrophoresis together with Western blotting to define the fine specificities of antibodies to the ribonucleoprotein (RNP) antigens Ro, La, Sm, RNP and Jo-1. One-dimensional analysis identified the Ro protein as a 57 kilodalton (kd) protein, although many anti-Ro sera also react with a 50 kd protein. La antisera react with 50 and 43 kd proteins. The 50 kd La protein readily breaks down into 43, 25, and smaller immunoreactive cleavage products. Partial proteolysis of Ro and La proteins in human spleen extracts produces similar immunoreactive products, providing evidence for a common structure. The major immunoreactive Sm antigens defined by human polyclonal antisera and a mouse monoclonal antiserum were doublets of 25/26 and 16/18 kd, whereas anti-RNP sera reacted with a protein of 68 kd. Most Sm-RNP antisera contained antibodies reactive with additional proteins, especially when whole cell extracts were used as a source of antigens. Two-dimensional analysis provided characteristic maps of the antigens. Ro and La were acidic, and La showed a unique set of acidic charge isomers at 50 and 43 kd. Anti-Sm antibodies reacted with discrete dots corresponding to both the acidic and basic regions of the first-dimension (charge) gels, whereas the RNP antigen showed a series of basic charge isomers of 68 kd. Many anti-Sm-RNP sera reacted with other closely spaced proteins of a similar charge and size to the Sm and RNP antigens, suggesting antibody cross-reactivity or reactivity with closely related functional proteins. Although Jo-1 had the same m.w. as the undegraded La antigen, the fingerprints were quite distinctive on two-dimensional electrophoresis. The results of this study indicate how the source and preparation of antigen extracts, as well as protein degradation, influence the m.w. determinations of soluble protein antigens. With these factors taken into account, two-dimensional fractionation with immunoblotting provides a highly discriminating, sensitive, and reproducible method of analysis of autoantibody specificity. This technique can be used to standardize reference antisera and to study protein antigens in normal and abnormal cell and tissue extracts, and could lead to new or more precise correlations with clinical disease.
authors.forename: K B
authors.initials: KB
authors.lastname: Elkon
authors.forename: P W
authors.initials: PW
authors.lastname: Jankowski
mesh_terms: D000818:Animals; D000974:Antibodies, Antinuclear / Q000032:analysis*; D000918:Antibody Specificity; D000937:Antigen-Antibody Reactions; D000941:Antigens / Q000276:immunology*; D001324:Autoantigens / Q000276:immunology*; D003101:Collodion; D004591:Electrophoresis, Polyacrylamide Gel; D005455:Fluorescent Antibody Technique; D006566:Herpesviridae Infections / Q000276:immunology; D006801:Humans; D010209:Paper; D011696:Purpura, Thrombocytopenic / Q000276:immunology; D020733:RNA, Small Cytoplasmic; D011817:Rabbits; D012261:Ribonucleoproteins; D055517:snRNP Core Proteins; D000097585:SS-B Antigen; D000099207:SS-A Antigen
chemical_list: D000974:Antibodies, Antinuclear; D000941:Antigens; D001324:Autoantigens; D003101:Collodion; D020733:RNA, Small Cytoplasmic; D012261:Ribonucleoproteins; D055517:snRNP Core Proteins; D000097585:SS-B Antigen; D000099207:SS-A Antigen; C116006:RO60 protein, human