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In a previous investigation (Strange and Belton, 1954), a protein fraction which was active in immunizing rabbits against artificially induced anthrax infection was isolated from culture filtrates of Bacillus anthracis. For the production of protective antigen, the synthetic medium of Wright et al. (1954) or a modified medium containing casein hydrolyzate (Belton and Strange, 1954) was used. No acceptable criteria of purity were given for the preparations and the presence of other nonspecific bacterial proteins could not be excluded. Antigenic material can be tested for immunological homogeneity by the agar diffusion technique of Ouchterlony (1953). Recently a method for titrating a B. anthracis immunizing antigen, based on this technique, was reported (Thorne and Belton, 1957). A culture filtrate containing a protective antigen produced a characteristic precipitation line with antiserum and the titer was defined as the highest dilution of sample which would give a visible reaction. Excellent correlation was found between the titers of culture filtrates and their immunizing activities in rabbits. It seemed probable that the line observed in agar diffusion plates was a precipitate of protective antigen and its specific antibody but this could be proved only by isolating the antigen component in a pure state and demonstrating its protective activity. Previously isolated material from culture filtrates (Strange and Belton, 1954) was immunologically heterogeneous since preparations produced up to four precipitation lines with antiserum. The predominant line was equivalent to the characteristic line produced by culture filtrates. This report presents a new fractionation procedure for the isolation of two components from culture filtrates of B. anthracis, each of which is a potent protective antigen and gives only one

FURTHER PURIFICATION STUDIES ON THE PROTECTIVE ANTIGEN OFBACILLUS ANTHRACISPRODUCEDIN VITRO