Physico-chemical analysis of Bordetella pertussis antigens

Physico-chemical methods are being developed for use in the control and sta ndardization of acellular pertussis vaccines and their individual component s. We have compared native and detoxified preparations of the B, pertussis antigens, pertussis toxin (PT), filamentous haemagglutinin (FHA), and the 6 9-kDa outer membrane protein (P69) using circular dichroism (CD), fluoresce nce spectroscopy, SDS-PAGE and FPLC gel filtration chromatography. Upon ald ehyde detoxification, PT underwent a large change in its intrinsic fluoresc ence 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 o f higher M-r. FHA also changed conformation (5-nm red-shift in intrinsic fl uorescence) upon aldehyde detoxification, with a resultant increase in the M-r of its major constituent. The P69 protein appeared quite robust to form aldehyde 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-de sorbed DTaP preparation by these methods. When used in conjunction with imm unochemical and toxicological assays, these methods are informative and use ful in the characterization of candidate standards and should be valuable m ethods for ensuring the consistency of manufactured vaccines. (C) 1999 The International Association for Biologicals.