FURTHER PURIFICATION STUDIES ON THE PROTECTIVE ANTIGEN OFBACILLUS ANTHRACISPRODUCEDIN VITRO

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Abstract
A protective antigen of Bacillus anthracis was isolated in a high state of purity from culture filtrates after growth of a nonvirulent strain of the organism in a semisynthetic medium. The purified antigen has the physical and chemical properties of a protein, sediments essentially as a single component in the ultracentrifuge and migrates as a single protein component on paper electrophoresis. Given in 5 doses of 05 [mu]g, 2.5 [mu]g of purified antigen protected most rabbits and 0.4 ug also administered in 5 equal doses with 1% (v/v) of normal horse serum protected approximately 50% against a challenge of 250 average lethal doses of spores of B. anthracis. A solution (0.04 ml) containing antigen (0.5 [mu]/ml) gave a visible precipitation line in Ouchterlony plates with horse hyperimmune serum produced with spores of B. anthracis; more concentrated solutions gave a single precipitation line with 3 antisera produced by different methods. Another protein component with similar immunological activity has been isolated in smaller amounts; it could be a degradation product from the major component produced as a result of enzymic activity. The present study provides a valid basis for the Thorne and Belton method of titrating B. anthracis immunizing antigen.