Separation and purification of the hemagglutinins from Bordetella pertussis

The role of the filamentous hemagglutinin (FHA) and the lymphocytosis-promoting factor hemagglutinin (LPF) in pertussis pathogenesis and immunity is the subject of active investigation. To be certain of their role as protective antigens, the hemagglutinins must be pure and free of each other. This report describes procedures to separate and purify FHA and LPF from the culture supernatant of stationary cultures of Bordetella pertussis Tohama, using hydroxylapatite, haptoglobin-Sepharose, and Sepharose CL-6B filtration chromatography. Purified FHA contained less than 0.002% active LPF assayed by histamine-sensitizing activity, and both hemagglutinins contained less than 0.01% of each other based on antigenic activity measured by an enzyme-linked immunosorbent assay, using affinity chromatography-purified antibody to each hemagglutinin. LPF and FHA were also shown to be antigenically distinct by immunodiffusion and were judged to be highly purified proteins by polyacrylamide gel electrophoresis. In addition, the purification procedures yielded milligram amounts of each hemagglutinin with very good recovery of starting activities.