Use of immunoblotting to characterize the mitochondrial antigens recognized by anti-mitochondrial autoantibodies.
Sera with anti-mitochondrial autoantibodies detected by indirect immunofluorescence and/or enzyme-linked immunosorbent assay (ELISA) were examined by immunoblotting against pig heart mitochondria. Seven types of reactions were defined, according to the pattern of the labelled bands. Type I sera reacted with 12 bands located within four zones. The most intensively labelled bands were located at 70, 67, 58, 63 and 43 kDa. Other types gave decreasing band numbers. When beef heart mitochondria were used, sera belonging to each of the above types had a profile of labelled bands which sometimes differed from those obtained with pig heart mitochondria. When the chloroform extracted F1-ATPase from beef heart mitochondria was used to prepare the immunoblots, primary biliary cirrhosis (PBC) sera with anti-mitochondria antibodies reacted with all the bands although zone A bands were less labelled. Rat liver mitochondria gave seven bands with type I sera among which the 57 and 35 kDa bands were specific for rat liver mitochondria, as shown by absorption tests. Sera of PBC patients were also tested in immunoblotting against rat liver subcellular fractions including mitoplasts, submitochondrial particles, inner membrane, outer membrane, matrix proteins and inter-membrane proteins. Antigenic bands of A and B zones were localized in the inner membrane and/or in the matrix proteins and the 35 kDa band in inter-membrane proteins. The outer membrane gave no reaction. The most frequent anti-mitochondrial autoantibody types in PBC were type II, then I, whilst for chronic active hepatitis type III was the most common. Type V was only seen in a patient suffering from a typical PBC. Some sera from patients with syphilis, collagenous colitis or progressive systemic sclerosis labelled one or two bands distinct from those labelled by the PBC sera. Sera from patients with drug-induced hepatitis with endoplasmic reticulum antibodies and with systemic lupus erythematosus were generally found negative by immunoblotting.
al-Hussami
O
O
Laboratoire d'Immunologie, UFR Alexis Carrel, Lyon, France.
Godinot
C
C
Monier
J M
JM
Trepo
C
C
Monier
J C
JC
D000818:Animals; D000937:Antigen-Antibody Reactions; D001323:Autoantibodies / Q000145:classification; D001324:Autoantigens / Q000032:analysis* / Q000276:immunology; D001327:Autoimmune Diseases / Q000145:classification / Q000276:immunology; D002417:Cattle; D006801:Humans; D007106:Immune Sera / Q000145:classification; D015151:Immunoblotting / Q000379:methods; D051379:Mice; D008928:Mitochondria / Q000032:analysis* / Q000276:immunology; D008929:Mitochondria, Heart / Q000032:analysis; D008930:Mitochondria, Liver / Q000032:analysis; D011817:Rabbits; D051381:Rats; D013552:Swine
D001323:Autoantibodies; D001324:Autoantigens; D007106:Immune Sera