Physico-chemical analysis of Bordetella pertussis antigens

Physico-chemical methods are being developed for use in the control and standardization of acellular pertussis vaccines and their individual components. We have compared native and detoxified preparations of the B. pertussis antigens, pertussis toxin (PT), filamentous haemagglutinin (FHA), and the 69-kDa outer membrane protein (P69) using circular dichroism (CD), fluorescence spectroscopy, SDS-PAGE and FPLC gel filtration chromatography. Upon aldehyde detoxification, PT underwent a large change in its intrinsic fluorescence maximum (8-10 nm red-shift) and a large increase in its apparent size, detected by chromatography. Polyacrylamide gels showed individual subunits of the same apparent molecular weight (M(r)) as well as some polypeptides of higher M(r). FHA also changed conformation (5-nm red-shift in intrinsic fluorescence) upon aldehyde detoxification, with a resultant increase in the M(r)of its major constituent. The P69 protein appeared quite robust to formaldehyde treatment as measured by the same methods. Its near-UV CD spectrum contains a prominent tryptophan band; so this method may be more suitable for observing differences in conformation. We also examined an aluminium-desorbed DTaP preparation by these methods. When used in conjunction with immunochemical and toxicological assays, these methods are informative and useful in the characterization of candidate standards and should be valuable methods for ensuring the consistency of manufactured vaccines.