Demonstration of membrane association and surface location of Mycoplasma pneumoniae antigens using monoclonal antibodies.
We examined 10 monoclonal antibodies (mAbs) directed against Mycoplasma pneumoniae proteins of 200, 170, 67, 46 and 42 kDa, and one mAb directed against a glycolipid component. The membrane association of the antigens reacting with our mAbs was investigated, in particular by phase-fractionation involving use of the detergent Triton X-114. The 170 kDa protein was shown to be membrane-associated, and surface exposure of this antigen was demonstrated by its disappearance from SDS-PAGE patterns after treatment of intact mycoplasmas with proteolytic enzymes. Cross-reactions with protein antigens of Mycoplasma genitalium were also shown. A mAb directed against a component of a lipid extract, prepared by the method used for preparation of the antigen used in the complement fixation (CF) test for serological diagnosis of M. pneumoniae infection, reacted with one major and a few minor bands in thin-layer chromatography (TLC) of the crude extract. The glycolipid character of this major antigen was demonstrated by treatment of the extract with sodium periodate, and by development of the TLC with orcinol/ferric chloride. These reactive bands were the same as those detected by the use of polyclonal mouse antiserum and a human convalescent serum, a result showing that the CF antigen contains a glycolipid moiety reacting with our mAb. The surface exposure of this antigen was demonstrated by binding of mAbs to intact cells.
Hirschberg
L
L
Department of Bacteriology, Karolinska Institute, Stockholm, Sweden.
Holme
T
T
Hydén
N
N
D000911:Antibodies, Monoclonal / Q000276:immunology; D000942:Antigens, Bacterial / Q000032:analysis*; D000954:Antigens, Surface / Q000032:analysis*; D001798:Blood Proteins / Q000276:immunology; D040501:Calgranulin A; D003429:Cross Reactions; D004591:Electrophoresis, Polyacrylamide Gel; D006017:Glycolipids / Q000032:analysis; D009177:Mycoplasma pneumoniae / Q000276:immunology*
D000911:Antibodies, Monoclonal; D000942:Antigens, Bacterial; D000954:Antigens, Surface; D001798:Blood Proteins; D040501:Calgranulin A; D006017:Glycolipids; C026422:cystic fibrosis serum factor