title: Purification and immunological characterization of HPLC-purified pertussis toxin subunits.
abstract: Pertussis toxin (PT), an oligomeric exotoxin of Bordetella pertussis containing five dissimilar subunits, is considered to be an essential immunogen in acellular and component pertussis vaccines against whooping cough. A rapid single-step procedure for isolating PT subunits was developed using reverse-phase high-performance liquid chromatography. Recoveries of individual subunits were 75% (S1), 70% (S2), greater than 90% (S3), greater than 90% (S4), and 50% (S5), as judged by SDS-PAGE and amino acid analysis. Lyophilized subunits were solubilized in urea followed by step-wise dialysis to remove the urea. All subunits were inactive in histamine sensitization, lymphocytosis, and hemagglutination assays. However, purified S1 retained residual NAD-glycohydrolase and ADP-ribosyltransferase activity. A partially active holotoxin could be generated by mixing the five individual subunits. All subunits were immunogenic in rabbits and mice. Monospecific antisera raised in both animal species were able to neutralize the PT-mediated clustering of Chinese hamster ovary cells, but active immunization of mice with single subunits failed to protect them in the intracerebral challenge assay. These subunit preparations therefore retained neutralizing determinants, but did not contain protective epitopes.
authors.affiliation: Connaught Centre for Biotechnology Research, Willowdale, Ont., Canada.
authors.forename: P
authors.initials: P
authors.lastname: Chong
authors.forename: S
authors.initials: S
authors.lastname: Cockle
authors.forename: H
authors.initials: H
authors.lastname: Boux
authors.forename: M
authors.initials: M
authors.lastname: Klein
mesh_terms: D000818:Animals; D001886:Bordetella pertussis / Q000276:immunology* / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D003429:Cross Reactions; D006386:Hemagglutination Tests; D006632:Histamine / Q000494:pharmacology; D007114:Immunization; D015151:Immunoblotting; D008218:Lymphocytosis; D051379:Mice; D009244:NAD+ Nucleosidase / Q000378:metabolism; D037342:Pertussis Toxin; D011065:Poly(ADP-ribose) Polymerases / Q000276:immunology / Q000378:metabolism; D011817:Rabbits; D010566:Virulence Factors, Bordetella / Q000032:analysis / Q000276:immunology* / Q000302:isolation & purification / Q000378:metabolism; D014917:Whooping Cough / Q000517:prevention & control
chemical_list: D010566:Virulence Factors, Bordetella; D006632:Histamine; D011065:Poly(ADP-ribose) Polymerases; D037342:Pertussis Toxin; D009244:NAD+ Nucleosidase