Characterization of murine monoclonal antibodies against 60-kD Ro/SS-A and La/SS-B autoantigens.
Small cytoplasmic ribonucleoproteins (scRNPs) are important autoantigens in patients with systemic lupus erythematosus and Sjögren's syndrome. MoAbs against these proteins were made by immunization of BALB/c mice with purified human recombinant 60-kD Ro/SS-A or 50-kD La/SS-B proteins. Five stable hybridoma cell lines were obtained, of which four secreted anti-Ro/SS-A antibodies (clones 1D8, 1D11, 2G10 and 6G8) and one produced anti-La/SS-B antibodies (clone 7F6). The MoAbs were further characterized using four different immunoassays: immunofluorescence, immunoblotting, RNA precipitation combined with Northern blotting, and recombinant protein precipitation. All four MoAbs against Ro/SS-A recognized the native protein and one of them (2G10) recognized also intact scRNP particles. Interestingly, hY3-RNA was reproducibly not efficiently precipitated by MoAb 2G10. Epitope mapping using deletion mutants of the 60-kD Ro/SS-A antigen showed that MoAb 1D8 recognized the C-terminal part of this protein, while 1D11 and 2G10 recognized distinct epitopes in the region between the RNP motif and the putative zinc finger domain. The epitopes recognized by these MoAbs are highly conserved among species, and the epitope recognized by MoAb 2G10 may be identical to an autoepitope recognized by sera of patients. This is the first report describing the isolation and characterization of MoAbs of the IgG class against the 60-kD Ro/SS-A and La/SS-B autoantigens obtained by immunization with purified human recombinant proteins. These MoAbs can be of great use in studying the cellular processes in which scRNPs are involved, and may help to determine why these scRNPs become autoantigenic in autoimmune diseases.
Veldhoven
C H
CH
Department of Autoimmune Diseases, Netherlands Red Cross Blood Transfusion Service, Amsterdam.
Pruijn
G J
GJ
Meilof
J F
JF
Thijssen
J P
JP
Van der Kemp
A W
AW
Van Venrooij
W J
WJ
Smeenk
R J
RJ
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology* / Q000378:metabolism; D001324:Autoantigens / Q000276:immunology* / Q000378:metabolism; D018604:Epitope Mapping / Q000592:standards; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D011485:Protein Binding / Q000276:immunology; D020733:RNA, Small Cytoplasmic; D011994:Recombinant Proteins / Q000276:immunology; D012261:Ribonucleoproteins / Q000276:immunology* / Q000378:metabolism; D013045:Species Specificity; D000097585:SS-B Antigen; D000099207:SS-A Antigen
D000911:Antibodies, Monoclonal; D001324:Autoantigens; D020733:RNA, Small Cytoplasmic; D011994:Recombinant Proteins; D012261:Ribonucleoproteins; D000097585:SS-B Antigen; D000099207:SS-A Antigen; C116006:RO60 protein, human