Ganglioside GM 1 -binding activities of cholera toxin variants. Relative binding activity of each holotoxin preparation with from zero to five binding sites (and with zero, one, or two tagged B subunits) to ganglioside GM 1 receptor was measured by ELISA with a fixed amount of toxin (5 ng, 60 fmol) added to individual wells coated previously with serial dilutions of GM 1 , and bound toxin was detected with polyclonal rabbit anti-CTB and horserad- ish peroxidase (HRP)-conjugated secondary antibody. Figure 3 shows that at high GM 1 density, toxins with more than one native GM 1 BS bound almost as well as native cholera toxin. The single-BS holotoxin showed a lower plateau signal at high GM 1 density than holotoxins with more than one BS, which we interpret to be due to a less favorable equilibrium between binding and release for the holotoxin with one BS for GM 1 . With more than one BS, a toxin molecule is expected to exhibit faster initial binding to immobilized GM 1 , multivalent binding as the density of immobilized GM 1 increases, and slower dissociation from GM 1 , resulting in increased avidity of binding. In wells coated with 75 nM GM 1 (50 l, 3.75 pmol), toxins with 2 or more native GM 1 BS bound at more than 90% of the wt level, toxin with a single BS bound significantly less at 60% of the wt level, and toxin with no BS gave a minimal signal (4% or less of the wt level). At lower GM 1 densities, there were significant differences between all variants, and in wells coated with 1.2 nM GM 1 (50 l, 60 fmol), toxins with four or three BS bound at 54 and 49% of wt levels, respectively, and toxins with 2, 1, or no BS bound at 25, 12, and 1% of wt levels, respectively. The amount of GM 1 required to coat each well and give 50% of the maximal signal was calculated to be 65, 100, 150, 240, and 850 fmol per well for wt holotoxin and for holotoxin variants with 4, 3, 2, or 1 wt BS, respectively.