An Agar-Diffusion Method for Titrating Bacillus anthracis Immunizing Antigen and its Application to a Study of Antigen Production

This study describes an agar-diffusion method based on Ouchterlony's technique for titrating Bacillus anthracis protective antigen and antibodies. The method is simpler and more sensitive than previously available rabbit skin tests. Researchers developed and optimized this assay using the avirulent Weybridge strain, primarily grown in defined medium and Casamino acids medium. Results were reproducible and correlated closely with toxin neutralization tests and rabbit immunization studies. The authors used this assay to systematically improve antigen yields by optimizing culture conditions. Key modifications included reducing ferrous sulfate concentration from 0.0005 M to 0.000025 M, increasing sodium bicarbonate to 0.6%, adding charcoal, and enriching the medium with amino acids. Antigen yields increased at least eightfold in the defined medium and several-fold in Casamino acids medium. In shaken flask cultures, antigen production peaked rapidly (16-18 hours) then declined, with degradation products appearing as additional precipitation lines. The agar-diffusion method proved valuable for detecting antigen concentration changes and could titrate antisera from multiple species, including 250 human sera, with results agreeing closely with skin tests. This straightforward assay enabled fundamental studies on antigen biosynthesis and degradation.

Key findings

• An agar-diffusion method based on Ouchterlony's technique was developed for simple, sensitive titration of B. anthracis protective antigen and antibodies, with reproducible results correlating with animal protection tests.
• Antigen yields increased at least 8-fold in defined medium by reducing FeSO₄ concentration to 0.000025 M, increasing NaHCO₃ to 0.6%, adding charcoal, and enriching with amino acids.
• The assay successfully titrated antisera from multiple animal species and 250 human sera, with results agreeing closely with toxin neutralization tests.
• In shaken flask cultures, peak antigen production occurred at 16-18 hours followed by rapid degradation, accompanied by appearance of multiple precipitation lines suggesting antigen fragmentation.
• Normal horse serum or gelatin additives approximately doubled the assay sensitivity, though the effect was non-specific.