Army

Antibody assay for Bacillus Anthracis

Rapid and accurate identification of B. anthracis by simultaneously detecting the presence of cell wall antigen and capsule antigen in the same sample

Medical & Biotechnology

Scientists at the Army have recently developed a highly accurate method for identifying the presence of Bacillus anthracis in a sample by simultaneously detecting the presence of the capsule antigen and cell wall antigen in one sample. The patented technology is available via patent license agreement to companies that would make, use, or sell it commercially.

B. anthracis. Image CDC

Direct fluorescent antibody assay (DFA) is a procedure that uses antibodies tagged with fluorescent dyes to detect the presence of specific antigens on the surface of a cell or microorganism. Recognized antigens are visible when examined microscopically using a light source which causes the antibody-bound fluorophore to emit a specific wavelength.

A two-component DFA assay, using fluoroscein-labeled monoclonal antibodies specific to the Bacillus anthracis galactose-N-acetylglucosamine polysaccharide (Gal-NAG-PS) in the cell wall and poly-D-glutamic acid capsule (CAP) antigen is currently available for detection of Bacillus anthracis in a sample. Although other Bacillus species produce CAP capsular material and two B. cereus strains produce the Gal-NAG-Ps the combination of both traits is strongly indicative of B. anthracis.

Unfortunately, the current method requires that the test sample be incubated under two different growth conditions: one which greatly reduces capsule formation of B. anthracis (on sheep agar, at 35-37° C., under atmospheric CO2 conditions for 16-24 hours), and one allowing capsule formation (20% CO2 supplement is frequently used). Consequently, each antigen in the DFA assay is detected in a separate vial, each vial containing a different subculture of the sample. The necessity for different culture conditions of the sample and separate immunoassay vials for each antigen increases the time and cost required to complete the assay and allows for misinterpretation of results.

In response, Army researchers have developed a method for identifying the presence of Bacillus anthracis in a sample by simultaneously identifying the presence of capsule and cell wall antigens in one sample. The method encompasses the addition of detectably labeled antibody Fab fragment specific for a cell wall antigen in tandem with differentially detectably labeled anti-capsule antibody.

A major advantage of this tandem DFA protocol is that it encourages the immediate analysis of any incoming culture or blood sample, without further culturing. Much information can be gathered with a first look at the sample: if it contains B. anthracis, the cell wall will be detected and the sample will be pCHO positive. pCHO is B. anthracis plasmid encoding the synthesis of cell wall N-acetyl-D-glucosamine, Gal-NAG polysaccharide. If it contains capsule, the sample will be pXO2 positive, and the capsule laden cells will be detected. pXO2 is a B. anthracis plasmid encoding the synthesis of poly-D-glutamyl capsule. This is a quantum leap for early identification and verification of B. anthracis in a sample. Further confirmation follows about three hours after post-incubation under capsule producing conditions.

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