Systemic lupus erythematosus murine monoclonal DNA-binding antibodies recognize cytoplasmic and nuclear phosphorylated antigens that display cell cycle redistribution in HEp-2 cells.
The immunological basis for the production of autoantibodies characteristic of systemic lupus erythematosus (SLE) against a wide range of antigens remains obscure. The specificity of (NZB x NZW)F1 (BWF1) or MRL/Mp-lpr/lpr (MRL/lpr) mouse monoclonal antibodies (mAb) was examined by immunofluorescence, immunoblotting and immunoprecipitation techniques. Using non-synchronized HEp-2 cells as substrate, the murine mAb were classified by indirect immunofluorescence into five groups on the basis of their staining patterns of subcellular components in interphase and mitotic stages of the cell cycle. The nature of the antigens recognized by the murine lupus was assessed by immunoblotting experiments in total, cytoplasmic and nuclear cell extracts from HEp-2 cells. The six antibodies used recognized in total cell extracts a range of polypeptides with apparent molecular weights from 25,000 to 210,000. Three polypeptides of 130,000, 110,000 and 45,000 MW were recognized by all six antibodies in both nuclear and cytoplasmic extracts. Immunoprecipitation of total cellular extracts labelled with [35S]methionine showed almost the same pattern as obtained in the immunoblotting assay. The labelling in vivo of HEp-2 cells with [32P], followed by the immunoprecipitation of the [32P]cell lysate showed that these mAb recognized phosphorylated proteins. The progressive decrease in reactivity of these mAb following treatment with higher concentrations of alkaline phosphatase in both [32P]cell lysate or nitrocellulose membranes indicates that these mAb recognize phosphorylated epitopes.
Bronze-da-Rocha
E
E
Centro de Citologia Experimental da Universidade do Porto, Portugal.
Machado
C
C
Staines
N A
NA
Sunkel
C E
CE
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001323:Autoantibodies / Q000276:immunology*; D001324:Autoantigens / Q000737:chemistry / Q000276:immunology*; D002467:Cell Nucleus / Q000276:immunology; D002478:Cells, Cultured; D003429:Cross Reactions; D003593:Cytoplasm / Q000276:immunology; D004247:DNA / Q000378:metabolism*; D000939:Epitopes / Q000032:analysis; D005455:Fluorescent Antibody Technique; D006801:Humans; D008180:Lupus Erythematosus, Systemic / Q000276:immunology*; D051379:Mice; D008815:Mice, Inbred Strains; D008970:Molecular Weight; D010766:Phosphorylation
D000911:Antibodies, Monoclonal; D001323:Autoantibodies; D001324:Autoantigens; D000939:Epitopes; D004247:DNA