Unbinding forces of single pertussis toxin-antibody complexes measured by atomic force spectroscopy correlate with their dissociation rates determined by surface plasmon resonance.
An inactivated form of pertussis toxin (PTX) is the primary component of currently available acellular vaccines against Bordetella pertussis, the causative agent of whooping cough. The PTX analyzed here is purified at industrial scale and is subsequently inactivated using glutaraldehyde. The influence of this treatment on antibody recognition is of crucial importance and is analyzed in this study. Surface plasmon resonance (SPR) experiments using PTX and its inactivated form (toxoid) with 10 different monoclonal antibodies were conducted. PTX was found to recognize the antibodies with an average affinity of 1.34 ± 0.50 nM, and chemical inactivation caused only a modest decrease in affinity by a factor of approximately 4.5. However, glutaraldehyde treatment had contrary effects on the kinetic association constant k(a) and the dissociation constant k(d) . A significant reduction in k(a) was observed, whereas the dissociation of the toxoid from the bound antibody occurred slower than PTX. These data indicate that the chemical inactivation of PTX not only reduces the velocity of antibody recognition but also stabilizes the interaction with antibodies as shown by a reduction in k(d) . The same interactions were also studied by dynamic force spectroscopy (DFS). Data reveal a correlation between the k(d) values determined by SPR and the mean unbinding force as measured by DFS. The unbinding forces of one complex were determined as a function of the loading rate to directly estimate the k(d) value. Several interactions were impossible to be analyzed using SPR because of ultratight binding. Using DFS, the unbinding forces of these interactions were determined, which in turn could be used to estimate k(d) values. The use of DFS as a technique to study ultratight binding is discussed.
Sanofi Pasteur, Bâtiment X3, 1541 avenue Marcel Mérieux, 69280, Marcy l'Etoile, France.
Nadège
N
Moreno
Michel
M
Chevalier
Frédéric
F
Ronzon
Catherine
C
Manin
Monique
M
Dupuy
Tino
T
Krell
Jean-Paul
JP
Rieu
D000907:Antibodies, Bacterial / Q000737:chemistry* / Q000276:immunology; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000276:immunology; D000936:Antigen-Antibody Complex / Q000737:chemistry* / Q000276:immunology; D001886:Bordetella pertussis / Q000276:immunology*; D006801:Humans; D007700:Kinetics; D018625:Microscopy, Atomic Force / Q000379:methods; D037342:Pertussis Toxin / Q000737:chemistry* / Q000276:immunology; D010567:Pertussis Vaccine / Q000737:chemistry / Q000276:immunology; D011485:Protein Binding / Q000276:immunology; D013223:Statistics as Topic; D020349:Surface Plasmon Resonance / Q000379:methods; D022282:Vaccines, Acellular / Q000276:immunology; D014917:Whooping Cough / Q000276:immunology / Q000517:prevention & control
D000907:Antibodies, Bacterial; D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D010567:Pertussis Vaccine; D022282:Vaccines, Acellular; D037342:Pertussis Toxin