Purification of Bacillus anthracis lethal factor by immunosorbent chromatography.
Lethal factor from B. anthracis (Vollum 1B strain) has been purified 1130-fold by immunosorbent chromatography using a mouse anti-lethal factor monoclonal antibody Sepharose-4B column. The antibody was covalently attached to CNBr activated Sepharose-4B. Lethal factor bound at pH 7 (0.05 M sodium phosphate buffer) and was eluted with buffer containing 4 M NaSCN with 77% recovery of the immunological activity. Pre-elution with 4 M NaCl was effective in eluting non-biospecifically bound proteins. Migration of lethal factor on SDS-polyacrylamide gel electrophoresis indicated a single component (greater than 99% pure) with a molecular weight of 82,000. The effect of a number of dissociation buffers on the antigen-antibody complex has been investigated. Sodium thiocyanate (4 M) in 0.05 M sodium phosphate buffer, pH 7, was the most effective eluting solution, causing complete dissociation of the antigen-antibody complex, while 4M NaI and 4 M NaCl caused 93% and 15% dissociation, respectively. The antigen-antibody complex was found to undergo a reversible dissociation at moderately high pH values. The ionizable group(s) responsible for this dissociation exhibited a pKa value of 9.90. Purified lethal factor exhibited a significant decrease in immunological activity upon freeze-thawing, with 52% loss in potency observed after 3 freeze-thaw cycles.
D000818:Animals; D000942:Antigens, Bacterial; D001408:Bacillus anthracis / Q000032:analysis*; D001427:Bacterial Toxins / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D007163:Immunosorbent Techniques; D051379:Mice; D008807:Mice, Inbred BALB C
D000942:Antigens, Bacterial; D001427:Bacterial Toxins; C030325:anthrax toxin