Simplified purification method for Clostridium difficile toxin A.

Aim: To establish the purification method for Clostridium difficile (C. difficile) toxin A.

Methods: C. difficile VPI 10463 filtrate was cultured anaerobically by the dialysis bag methods. And then the toxin A was purified by precipitation with 500 g/L (NH4)2SO4 and acid precipitation at pH 5.5, followed by ion-exchange chromatography on DEAE-Toyopearl.

Results: Purified toxin A exhibited only one band on native polyacrylamide gel electrophoresis (native-PAGE) and Ouchterlony double immunodiffusion. The molecular weight of toxin A was estimated to be 550,000. The purified toxin A had a protein concentration of 0.881 mg/mL. The minimum lethal dose was 1X10(6) MLD/mL (i.p.mice). The cytotoxic titer was 10(7) CU/mg. The haemagglutinate activity was at a concentration of 1.72 microg/mL. The ratio of fluid volume (mL) accumulated to the length (cm) of the loop was 2.46.

Conclusion: The modified method for purification of toxin A of C. difficile was simple and convenient. It may be even more suitable for purification of toxin A on large scales.