Means and methods for the determination of the biological activity of neurotoxin polypeptides in cells

Abstract

The invention pertains to a method for directly determining the biological activity of a Neurotoxin polypeptide in cells, comprising the steps of: a) incubating cells susceptible to Neurotoxin intoxication with a Neurotoxin polypeptide for a time and under conditions which allow for the Neurotoxin polypeptide to exert its biological activity; b) fixing the cells and, optionally, permeabilizing the cells with a detergent; c) contacting the cells with at least a first capture antibody specifically binding to the non-cleaved and Neurotoxin-cleaved substrate and with at least a second capture antibody specifically binding to the cleavage site of the Neurotoxin-cleaved substrate, under conditions which allow for binding of the capture antibodies to the substrates; d) contacting the cells with at least a first detection antibody specifically binding to the first capture antibody, under conditions which allow for binding of the first detection antibody to the first capture antibody, thus forming first detection complexes, and at least a second detection antibody specifically binding to the second capture antibody, under conditions which allow for binding of the second detection antibody to the second capture antibody, thus forming second detection complexes; e) determining the amount of the first and second detection complexes of step d), and f) calculating the amount of substrate cleaved by the Neurotoxin polypeptide in the cells by means of the second detection complexes, thereby determining the biological activity of the Neurotoxin polypeptide in the cells. The invention further provides for a kit for carrying out the method of the invention.