Purification of two high molecular weight toxins of Clostridium difficile which are antigenically related

Two Cl. difficile toxins were isolated from cultures of Cl. difficile strain VPI 10463. A purification procedure to prepare homogenous Cl. difficile toxins is given. This procedure allows purification of high molecular weight toxins A and B without using immunaffinity chromatography. The main step of the purification is the separation of a partially purified toxin preparation over a FPLC-Mono Q column by anion exchange chromatography. The experimental conditions for a rechromatography were determined to prepare the two major toxic activities as homogenous high molecular weight proteins. Our toxin A has a molecular weight (Mr) of ca. 300 kDa and an IP of 4.7. The Mr of our toxin B is ca. 250 kDa, the isoelectric focusing gives rise to two bands one at 4.7 and the other at 4.8. The two bands represent charge isomers as have been described for other bacterial toxins. Both toxins differ in cytotoxicity testing by a factor of 1000 but have the same activity when tested in vivo. Toxin specific monoclonal antibodies (mabs) were elicited by separate immunization of mice either with toxin A or toxin B, respectively. All of our mabs cross react with pure toxin A and toxin B when tested by ELISA or Western Blotting. Some mabs strongly cross react indicating that both toxins have major epitopes in common. A hypothesis for the structural and possible functional relatedness between the two toxins is discussed.