Recognition of pertussis toxin by antibodies to synthetic peptides.
Eight synthetic peptides, selected from the amino acid sequence of pertussis toxin (PT) subunits S1, S2, S3 and S4, were assessed for their ability to induce protein-recognizing and neutralizing antibodies. Seven of these peptides, prepared as conjugates of either keyhole limpet haemocyanin or tetanus toxoid, induced significant levels of antibody, all of which reacted with SDS-denatured PT on Western blots. Six of the antibodies bound to PT-coated ELISA plates; this binding was inhibited by homologous peptide antigen. However, none of the antibodies, including those directed against the N-terminus of subunit S1, were able to attenuate in vivo or in vitro toxin-dependent activity. Further investigation revealed that only one antibody, specific for the C-terminus of S1 (peptide Slc, 237-255), could recognize the conformation of native PT in solution. The other five antipeptide antibodies which reacted with PT-coated ELISA plates did not recognize PT when captured onto ELISA plates via either a monoclonal antibody or fetuin, unless the conformation of the toxin had been relaxed by reduction with dithiothreitol. Conversely, the native PT-recognizing response of peptide Slc did not bind the conformationally relaxed PT molecule. From this study, it appears likely that a peptide capable of inducing PT-neutralizing antibody must closely resemble the conformation of the cognate sequence in the native protein.
Division of Biotechnology, PHLS Center for Applied Microbiology and Research, Porton Down, Salisbury, U.K.
R N
RN
Seabrook
A
A
Robinson
R P
RP
Sharma
L I
LI
Irons
L A
LA
Ashworth
C P
CP
Price
T
T
Atkinson
D000595:Amino Acid Sequence; D000818:Animals; D000918:Antibody Specificity; D001428:Bacterial Vaccines / Q000276:immunology; D003432:Cross-Linking Reagents; D006433:Hemocyanins; D007106:Immune Sera; D051379:Mice; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis / Q000276:immunology; D037342:Pertussis Toxin; D011817:Rabbits; D010566:Virulence Factors, Bordetella / Q000276:immunology*
D001428:Bacterial Vaccines; D003432:Cross-Linking Reagents; D007106:Immune Sera; D010455:Peptides; D010566:Virulence Factors, Bordetella; D006433:Hemocyanins; D037342:Pertussis Toxin; C032808:keyhole-limpet hemocyanin