Immunoelectrophoretic analysis, toxicity, and kinetics of in vitro production of the protective antigen and lethal factor components of Bacillus anthracis toxin

Abstract

The kinetics of Bacillus anthracis toxin production in culture and its lethal activity in rats, mice, and guinea pigs were investigated. Lethal toxin activity was produced in vitro throughout exponential growth at essentially identical rates in both encapsulated virulent and nonencapsulated avirulent strains. The two toxin proteins which produce lethality when in combination, lethal factor (LF) and protective antigen (PA), could be quantitated directly from culture fluids by rocket immunoelectrophoresis. Using purified preparations of these proteins, we determined that a combination of 8 micrograms of LF and 40 micrograms of PA was required for a maximal rate of killing (39 to 40 min) in Fischer 344 rats (250 to 300 g). Conversely, a minimum of 0.6 microgram of LF and 3 micrograms of PA was required for lethality. The 50% lethal dose for Hartley guinea pigs was 50 micrograms of LF and 250 micrograms of PA, and for Swiss mice it was 2.5 micrograms of LF and 12.5 micrograms of PA. Analyses classically reserved for enzyme kinetic studies were used to study the kinetics of lethal activity in the rat model after intravenous injection of LF-PA mixtures. The amounts of LF and PA which were required to give half the rate of killing (i.e., double the minimum time to death) were 1.2 and 5.8 micrograms, respectively. A theoretical minimum time to death was determined to be 38 min. A third anthrax toxin component, edema factor, was shown to inhibit lethal toxin activity. Edema factor could not be quantitated by rocket immunoelectrophoresis because the protein did not form distinct precipitin bands with available antisera.