Identification of components of the endoplasmic reticulum and Golgi complex by murine autoreactive monoclonal antibodies.
A number of autoreactive monoclonal antibodies have been produced by the fusion of spleen cells from unprimed BALB/c mice. The specificities of two of these monoclonal autoantibodies, MUI 38 and MUI 100, have been further examined. By indirect immunofluorescence, monoclonal antibody MUI 38 showed discrete perinuclear staining of acetone-fixed murine 3T3 fibroblasts, which was similar to that obtained with the Golgi vital stain, C6-NBD-ceramide, and with rhodamine-labelled wheat germ agglutinin. Furthermore, the staining pattern with antibody MUI 38 in cells treated with either monensin, taxol or nocodazol was altered in a manner consistent with the known effects of these drugs on Golgi morphology. In contrast, monoclonal antibody MUI 100 showed a diffuse cytoplasmic staining pattern, similar to FITC-Con A, indicative of reactivity with the endoplasmic reticulum. At high dilutions antibody MUI 100 showed only a perinuclear staining pattern, indicating that MUI 100 reacted with the Golgi as well as the endoplasmic reticulum. Both monoclonal antibodies are IgM kappa class and both showed reactivity with acetone-fixed fibroblasts from a number of species, indicating that the antigens are highly conserved. By immunoblotting with total membranes of murine 3T3 cells under either reducing or non-reducing conditions, monoclonal antibody MUI 100 reacted with a number of components with apparent molecular weights (MW) from 27,000 to 63,000. This reactivity was abolished when the 3T3 membranes were treated with sodium periodate, indicating antibody MUI 100 may be specific for carbohydrate. In addition, MUI 100, but not MUI 38, possessed rheumatoid factor activity, reacting with IgG from normal sera of a number of different species. Furthermore, monoclonal antibody MUI 100 was shown to be specific for the Fc domain of IgG. Absorption of MUI 100 antibody with normal rabbit IgG-Sepharose reduced the anti-endoplasmic reticulum reactivity, therefore both activities are attributable to the same antibody.
Kooy
J
J
Department of Pathology and Immunology, Monash Medical School, Monash University, Melbourne, Australia.
Underwood
J R
JR
Gleeson
P A
PA
D000818:Animals; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D001323:Autoantibodies; D001324:Autoantigens / Q000032:analysis*; D006224:Cricetinae; D004721:Endoplasmic Reticulum / Q000276:immunology*; D005455:Fluorescent Antibody Technique; D006056:Golgi Apparatus / Q000276:immunology*; D015151:Immunoblotting; D007075:Immunoglobulin M / Q000032:analysis; D051379:Mice; D008807:Mice, Inbred BALB C; D051381:Rats; D012217:Rheumatoid Factor / Q000032:analysis; D013045:Species Specificity
D000911:Antibodies, Monoclonal; D001323:Autoantibodies; D001324:Autoantigens; D007075:Immunoglobulin M; D012217:Rheumatoid Factor