DT neutralization assay
To test the minimal dose of active diphtheria toxin needed to
cause cell death in 16 h (1CTU100), cells were plated at 2 104
/well
in 96-well plates in presence of serially diluted DT. The CTU100 was
defined as 10 ng/ml in the tested experimental conditions. Post-2
mouse sera were two-fold serially diluted in cell medium, preincubated
with 4CTU100 of diphtheria toxin and then incubated
for 90 min at 37 C. Subsequently, 50ml of Vero cells at density of
2 105 cells/ml was incubated with 50ml of sera and toxin
mixtures for 16 h at 37 C. Endpoint titers were defined as the
reciprocal of the highest dilution able to inhibit cell death.