Simultaneous determination of Bordetella pertussis toxin and filamentous haemagglutinin concentrations by hydroxyapatite high-performance liquid chromatography.
A simple and rapid method for the simultaneous determination of Bordetella pertussis toxin (PT) and filamentous haemagglutinin (FHA) concentrations in fermentation broths has been developed. The rapid single-step analysis performed by hydroxyapatite high-performance liquid chromatography using a salt gradient with UV detection allows both the separation of PT from FHA and the measurement of their respective concentrations. The assay is highly reproducible. Over 35 lots of acellular B. pertussis vaccine production lots were examined and PT concentrations measured by high-performance liquid chromatography were found to be in good agreement with the values obtained by sodium dodecyl sulphate-polyacrylamide gel electrophoresis densitometry. The chromatographic conditions have been optimized to separate the intact holotoxin from its B-oligomer subunits.
Chong
P
P
Connaught Centre for Biotechnology Research, Willowdale, Ontario, Canada.
Jackson
G
G
Cwyk
W
W
Klein
M
M
D000596:Amino Acids / Q000032:analysis; D000942:Antigens, Bacterial / Q000032:analysis; D001426:Bacterial Proteins / Q000032:analysis; D002851:Chromatography, High Pressure Liquid; D017886:Durapatite; D004591:Electrophoresis, Polyacrylamide Gel; D005285:Fermentation; D006388:Hemagglutinins / Q000032:analysis*; D006882:Hydroxyapatites; D008970:Molecular Weight; D037342:Pertussis Toxin; D012015:Reference Standards; D013056:Spectrophotometry, Ultraviolet; D010566:Virulence Factors, Bordetella / Q000032:analysis*
D000596:Amino Acids; D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D006388:Hemagglutinins; D006882:Hydroxyapatites; D010566:Virulence Factors, Bordetella; D017886:Durapatite; D037342:Pertussis Toxin