Comparison of methods for the analysis of outer membrane antigens of Neisseria meningitidis by western blotting.
The method of extraction of outer membrane proteins (OMPs), the conditions of electrophoretic transfer, and the conditions of antibody binding, were compared in Western blotting studies of Neisseria meningitidis outer membrane antigens. The OMP profiles obtained by SDS-PAGE of outer membrane vesicles extracted with lithium chloride/sodium acetate were compared with profiles obtained by Sarkosyl extraction; these profiles were further compared with the patterns obtained by 125I-labelling of surface-exposed proteins. Sarkosyl extracts gave profiles most closely resembling those of 125I-labelled whole-cells and gave the best resolution of the major proteins. After transfer in Tris-glycine-methanol buffer some proteins, including the major proteins, were not completely transferred and remained in the gel, with the class 2/3 and 5 proteins not effectively detected on nitrocellulose by amido black staining. There was weak antibody recognition of the class 1 and 4 proteins but good recognition of lipooligosaccharide (LOS) and H8 antigen. Empigen BB had no effect on renaturation of the class 1 protein. When 0.1% SDS was incorporated in the same buffer all of the proteins were removed from the gel, and although the major proteins bound to nitrocellulose other proteins did not. There was weak antibody recognition of the class 1 and 4 proteins, stronger reaction to the class 5 protein, but no recognition of the class 2 protein, LOS or H8 antigen, Empigen BB slightly enhanced antibody recognition of the class 1 protein. After transfer in Tris-glycine buffer, all the major proteins were transferred and bound to nitrocellulose and, other than the class 2 protein, were recognised by antibody, both in the presence or absence of Empigen BB, as were LOS and the H8 antigen. Differences existed in the patterns of antibody recognition between the lithium and the Sarkosyl extracts; additional proteins were recognised in the lithium extracts. The surface-labelling studies indicated, however, that some of these proteins were not surface-exposed. Some minor proteins appeared to be more highly immunogenic than the major proteins.
Microbial Pathogenicity Research Group, Clinical Research Centre, Harrow, Middlesex, U.K.
R L
RL
Davies
R A
RA
Wall
S P
SP
Borriello
D000085:Acetates; D019342:Acetic Acid; D000907:Antibodies, Bacterial / Q000032:analysis; D000942:Antigens, Bacterial / Q000032:analysis* / Q000302:isolation & purification; D000954:Antigens, Surface / Q000032:analysis; D001425:Bacterial Outer Membrane Proteins / Q000032:analysis* / Q000302:isolation & purification; D015153:Blotting, Western; D002021:Buffers; D002712:Chlorides; D003902:Detergents; D006801:Humans; D007457:Iodine Radioisotopes; D008094:Lithium; D018021:Lithium Chloride; D008589:Meningococcal Infections / Q000276:immunology*; D009345:Neisseria meningitidis / Q000276:immunology*; D009930:Organic Chemicals; D011487:Protein Conformation; D012521:Sarcosine / Q000031:analogs & derivatives
D000085:Acetates; D000907:Antibodies, Bacterial; D000942:Antigens, Bacterial; D000954:Antigens, Surface; D001425:Bacterial Outer Membrane Proteins; D002021:Buffers; D002712:Chlorides; D003902:Detergents; D007457:Iodine Radioisotopes; D009930:Organic Chemicals; C025231:sarkosyl; D008094:Lithium; D018021:Lithium Chloride; D019342:Acetic Acid; C041772:empigen BB; D012521:Sarcosine