Simple, efficient purification of filamentous hemagglutinin and pertussis toxin from Bordetella pertussis by hydrophobic and affinity interaction.
Two major antigens of Bordetella pertussis, filamentous hemagglutinin (FHA) and pertussis toxin (PT), were efficiently purified from culture filtrate by exploiting their relative hydrophobicities and differences in affinity to sialic acid-containing protein. High yields of FHA (40 to 80 mg/liter) and PT (8 to 16 mg/liter) were first produced by growing the bacteria in the modified CL medium. The FHA and PT in the culture filtrate were adsorbed onto butyl-Sepharose by hydrophobic interaction at appropriately high ionic strength. Elution of the antigens was effected by decreasing their hydrophobicities with a buffer of low ionic strength. FHA was then separated from PT with an affinity column of fetuin-Sepharose. The fraction passing through the column contained purified FHA, and the fetuin-bound PT was eluted with buffered MgCl2. The FHA and PT purified by these steps were electrophoretically and serologically identical to the reference purified FHA and PT preparations. Approximately 16 to 32 mg of purified FHA and 4 to 8 mg of purified PT were obtained from 1 liter of culture filtrate. The described procedure for making FHA and PT antigens from B. pertussis for serologic and immunologic use is very simple, efficient, and reproducible.
Skelton
S K
SK
Center for Infectious Diseases, Centers for Disease Control, Atlanta, Georgia 30333.
Wong
K H
KH
D000942:Antigens, Bacterial / Q000302:isolation & purification; D001886:Bordetella pertussis / Q000032:analysis* / Q000276:immunology; D002846:Chromatography, Affinity; D006388:Hemagglutinins / Q000302:isolation & purification*; D009994:Osmolar Concentration; D037342:Pertussis Toxin; D010566:Virulence Factors, Bordetella / Q000302:isolation & purification*
D000942:Antigens, Bacterial; D006388:Hemagglutinins; D010566:Virulence Factors, Bordetella; D037342:Pertussis Toxin