Purification and analysis with monoclonal antibodies of P2, the major outer membrane protein of nontypable Haemophilus influenzae.
The protein P2 comprises a large proportion of the outer membrane of nontypable Haemophilus influenzae and functions as a porin. In view of the importance of the protein as a surface antigen, the present study was designed to purify and analyze P2 with particular emphasis on detection of antigenic determinants expressed on the bacterial surface and identification of bactericidal targets on P2. The P2 protein was purified by using detergent solubility, anion-exchange chromatography, and gel-filtration chromatography sequentially. Two monoclonal antibodies to P2 were developed. One antibody (2E6) recognized a determinant expressed on the bacterial surface, whereas the other antibody (3F3) recognized an internal epitope. The surface-exposed 2E6 determinant was present on 12% of strains from a nationwide collection. P2 is a bactericidal target for antibody 2E6. Cyanogen bromide cleavage of P2 resulted in two fragments, as in type b strains. Both monoclonal antibodies recognized epitopes on the larger fragment. These observations have potentially important implications regarding the development of vaccines to prevent H. influenzae infections and the development of a serotyping system for epidemiologic studies.
D000907:Antibodies, Bacterial / Q000276:immunology; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity; D001425:Bacterial Outer Membrane Proteins / Q000302:isolation & purification*; D002846:Chromatography, Affinity; D003488:Cyanogen Bromide; D003602:Cytotoxicity, Immunologic; D000939:Epitopes; D006193:Haemophilus influenzae / Q000145:classification / Q000276:immunology*; D008854:Microscopy, Electron; D010446:Peptide Fragments / Q000276:immunology; D013045:Species Specificity
D000907:Antibodies, Bacterial; D000911:Antibodies, Monoclonal; D001425:Bacterial Outer Membrane Proteins; D000939:Epitopes; D010446:Peptide Fragments; D003488:Cyanogen Bromide