Separation and purification of the hemagglutinins from Bordetella pertussis

Abstract

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.

Role of phosphatidylinositol 3-kinase in the binding ofBordetella pertussisto human monocytes

“…Wild-type FHA(RGD) and mutant FHA(RAD) proteins were isolated and purified from B. pertussis strains BP-TOX6 and BP1098-TOX6, respectively, using previously published techniques (Sato et al, 1983;Menozzi et al, 1991). These preparations were then concentrated with Centricon-3 concentrators (Amicon, Beverly, MA, USA).…”

Interaction of the Bordetella pertussis filamentous hemagglutinin with heparin

“…The hemagglutination activity of the B. pertussis culture supernatants and the heparin chromatography fractions were assayed in microtiter plates using a 3% fresh rabbit erythrocyte suspension in PBS as described elsewhere [19]. Standard assays contained 50 /~i of erythrocyte suspension, 50 ~1 of test sample, and 30/al of PBS containing various amounts of bovine intestinal mucosal heparin (Sigma, St. Louis, MO).…”

Genetic characterization of Bordetella pertussis filamentous haemagglutinin: a protein processed from an unusually large precursor

“…FHA pfOtem was prepared from B. /j(^rr(i,s\'75 W?8Slamer SchoiU) liquid (Ujlture si;perT*atant grown at 37 C with shaking, using pubiisfied techniques (Sato et al. 1983).…”