Identification of Bacillus anthracis by using monoclonal antibody to cell wall galactose-N-acetylglucosamine...

Guanidine extracts of crude Bacillus anthracis cell wall were used to vaccinate BALB/c mice and to develop monoclonal antibody (MAb) to vegetative cell surface antigens. Two hybridomas selected during this study produced immunoglobulin M immunoglobulins, which appear to be directed to an epitope associated with the galactose-N-acetyl-D-glucosamine polysaccharide. Both demonstrated specificity in their binding to purified B. anthracis cell wall, o-stearoyl-polysaccharide conjugates, and intact, nonencapsulated vegetative cells. The interaction of the MAbs with purified polysaccharide was inhibited by 0.5 M galactose and lactose but not by N-acetylglucosamine, glutamate, glycine, or glycerol. Inhibition by glucose or sucrose was approximately 75% of that seen with galactose. Electron microscopy showed that both MAbs interacted with the cell wall of vegetative cells as well as with the cortex of spores. Neither MAb reacted with encapsulated vegetative cells, such as those from infected guinea pigs, nor did they react with intact spores. After conjugation to fluorescein isothiocyanate, the MAbs stained intensely all B. anthracis strains tested, whereas with two exceptions, none of the strains of 20 other Bacillus spp. was stained. The exceptions, strains of Bacillus cereus, could be differentiated from B. anthracis by being beta-hemolytic on blood agar.