Isolation ofEscherichia coli heat-labile enterotoxin by affinity chromatography: Characterization of subunits

This paper describes the isolation ofEscherichia coli heat-labile enterotoxin (LT) by affinity chromatography on an anti-cholera toxin immunoglobulin-Sepharose column, and the subunit composition of crude and affinity-isolated LT. LT and its subunits were assayed with ganglioside (G_M1)-ELISA, immunodiffusion, skin toxicity, and broken cell adenylate cyclase activation methods. The results show that the immunoaffinity method, applied to LT of different strains and batches, yielded about 100-fold purification with approximately 50% recovery of LT antigen. LT was shown to contain a G_M1-ganglioside binding subunit as well as another subunit which does not bind to G_M1 but activates adenylate cyclase. Immunodiffusion tests showed that the two LT subunits were immunologically related to but not identical with, respectively, the B and A subunits of cholera toxin. The LT “A” and “B” subunits were present in similar proportions in the affinity-isolated and crude LT preparations, but in the purified fraction they had only partially reassociated into holotoxin.