Quantitative study of the binding and hemolytic efficiency of Escherichia coli hemolysin

Mono- and polyclonal antibodies were used to construct a sandwich enzyme-linked immunosorbent assay that permitted quantitation of Escherichia coli hemolysin in soluble and membrane-bound forms. Toxin concentrations of 4 to 14 micrograms/ml were measured in culture supernatants of E. coli LE 2001 at times of peak hemolytic activity. Quantitative studies on the binding of E. coli hemolysin to rabbit erythrocytes were conducted at 0 and 37 degrees C. At 37 degrees C, 85 to 95% of bindable toxin was cell bound after 60 min, and no saturability of binding was observed in the studied range of concentrations, which resulted in deposition of approximately 100 to 50,000 toxin molecules per cell. Binding was slower and less effective at 0 degrees C; however, hemolysis did occur at low temperature. The number of cell-bound toxin molecules required to generate a hemolytic lesion within 60 min was estimated to be approximately 100 molecules per cell at 37 degrees C and 800 to 1,000 molecules per cell at 0 degrees C. Upon prolonged incubation (5 to 20 h, 37 degrees C), the number of molecules evoking a functional lesion decreased to approximately 5 to 20 per cell. These results are compatible with the concept that E. coli hemolysin first adsorbs to the cell surface, with membrane insertion and pore formation following in a second step that may be temporally dissociated from that of binding. The data support the pore concept of toxin action by showing that attachment of a low and finite number of toxin molecules to an erythrocyte will ultimately generate a cytolytic lesion.