Technical Abstract: Shiga toxin (Stx) 2 variants, Stx2a, Stx2c, Stx2d and Stx2g were purified to homogeneity from bacterial culture supernatants by a one-step monoclonal anti-Stx affinity chromatography method. The method was based on the binding affinity of these Stxs for a monoclonal antibody against the Stx2 A-subunit. The final yields of Stx2a, 2c, 2d, and 2g after this affinity purification ranged from 220 to 470 ng/mL of the culture supernatant. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of proteins from different Stx preparations revealed two protein bands, each with molecular weights of 32 and 7 kDa, corresponding to the values for the Stx2 A- and B-subunit, respectively. Electrophoresis of these proteins under native conditions, however, resulted in different protein banding patterns. The single major protein band from the Stx2a and Stx2g preparations had a MW of 72 kDa, corresponding to the predicted size of a Stx2 holotoxin. In contrast, the protein bands from the Stx2c and Stx2d preparations had a MW at least 8-fold larger; protein mobility was unchanged by the addition of a reducing agent to the samples. The change in Stx2c and Stx2d protein mobility coincided with decreases of their cytotoxicities for Hela cells. These experimental conditions corresponded to 50% cytotoxic activities of Stx2a, Stx2c, Stx2d and Stx2g for Hela cells of 100, 1000, 1000, and 10 pg, respectively. Most Stx activity was lost after heating at 80°C for one hour, but not at 60°C. Stx2g was less heat stable compared to the other Stx2 types tested. An anti-Stx2 B-subunit mouse monoclonal antibody neutralized the cytotoxicities of all Stxs for Hela cells.