ELISA estimation of the binding of epidermolytic toxin to neonatal mouse skin

A sandwich ELISA, with antisera from rat and rabbit, was used to determine epidermolytic toxin (ET) to a limit of about 0.01 ng at 0.1 ng/ml. The binding of ET to the epidermis of skin discs was measured in vitro. The ability of the assay to discriminate between the two forms of the toxin was used to demonstrate that there was a saturable component of toxin binding to the epidermis. The rate of uptake, the effect of the inhibitor EGTA and comparative experiments with the inactive nitrated toxin confirmed that the observed binding is associated with toxigenesis. From measurements at toxin concentrations from 0.25 microgram/ml to 100 micrograms/ml, it was calculated that the saturable binding component has a Kd of about 2 micrograms/ml (approximately 60 nM) and a capacity of 0.5 ng per skin disc (1 ng per cm2 of epidermis).