In vitro assay for quantifying clostridial neurotoxin activity
AssigneeMERZ PHARMA GmbH & CO. KGaA
InventorsThomas Wilk, Harold Victor Taylor, Karl-Heinz Eisele
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Abstract
Novel methods for determining the unknown biological activity of a clostridial neurotoxin in a sample with respect to the known biological activity of a clostridial neurotoxin in a reference sample, comprising the step of comparing the biological activity of a clostridial neurotoxin preparation with the biological activity of a standard preparation of a reference clostridial neurotoxin in certain in vitro systems.
Claims14
An in vitro method for determining the biological activity of a preparation of a clostridial neurotoxin, comprising the steps of:
- (a) contacting at least one test particle with a sample of the clostridial neurotoxin preparation;
- (b) measuring one or more parameters selected from time-dependent decrease of an amount of unchanged starting substrate within the test particle, time-dependent increase of an amount of one or more cleavage products resulting from proteolytic cleavage of a substrate within the test particle, time-dependent decrease of an amount of a compound released from the test particle;
- and
- (c) comparing the measurement of the parameter in step (b) of the sample of the clostridial neurotoxin preparation with the measurement of the parameter in step (b) of a reference sample, thereby determining the amount of the biological activity of the clostridial neurotoxin in the preparation;
- wherein the at least one test particle comprises at least a membrane enclosing a reaction volume, wherein the membrane carries one or more clostridial neurotoxin receptors which bind the clostridial neurotoxin and mediate internalization of the clostridial neurotoxin into the reaction volume, and wherein the reaction volume comprises a substrate for clostridial neurotoxin which is proteolytically cleaved by the clostridial neurotoxin in the reaction volume of the test particle.
A kit for determining the biological activity of a preparation of a clostridial neurotoxin comprising:
- (a) at least one test particle comprising at least a membrane enclosing a reaction volume, wherein the membrane carries one or more clostridial neurotoxin receptors which bind the clostridial neurotoxin and mediate internalization of the clostridial neurotoxin into the reaction volume, and wherein the reaction volume comprises a substrate for clostridial neurotoxin which is proteolytically cleaved by the clostridial neurotoxin in the reaction volume of the test particle;
- (b) one or more reagents for determining binding of the clostridial neurotoxin to the one or more receptors;
- and/or
- (c) one or more reagents for determining proteolytic cleavage of the substrate.
Dependencies are inferred from the claim text.