Cell-based fluorescence resonance energy transfer (FRET) assays for clostridial toxins

Abstract

The present invention provides a method of determining clostridial toxin activity by (a) contacting with a sample a cell containing a clostridial toxin substrate that includes a donor fluorophore; an acceptor having an absorbance spectrum overlapping the emission spectrum of the donor fluorophore; and a clostridial toxin recognition sequence containing a cleavage site that intervenes between the donor fluorophore and the acceptor, where resonance energy transfer is exhibited between the donor fluorophore and the acceptor under the appropriate conditions; (b) exciting the donor fluorophore; and (c) determining resonance energy transfer of the contacted cell relative to a control cell, where a difference in resonance energy transfer of the contacted cell as compared to the control cell is indicative of clostridial toxin activity.

Description

[0001] This application is a divisional and claims priority pursuant to 35 U.S.C. §120 to U.S. patent application Ser. No. 10 / 261,161, filed Sep. 27, 2002, which is hereby incorporated by reference in its entirety.BACKGROUND OF THE INVENTION [0002] 1. Field of the Invention [0003] The present invention relates generally to fluorescence resonance energy transfer and protease assays and, more specifically, to cell-based methods for assaying for clostridial toxin activity. [0004] 2. Background Information [0005] The neuroparalytic syndrome of tetanus and the rare but potentially fatal disease, botulism, are caused by neurotoxins produced by bacteria of the genus Clostridium. These clostridial neurotoxins are highly potent and specific poisons of neural cells, with the human lethal dose of the botulinum toxins on the order of nanograms. Thus, the presence of even minute levels of botulinum toxins in foodstuffs represents a public health hazard that must be avoided through rigorous testi...

AI Technical Summary

Benefits of technology

The present invention provides a substrate composition that includes a delivery agent and a clostridial toxin substrate containing a donor fluorophore and an acceptor. The delivery agent can be covalently linked to the substrate or non-covalently associated with it. The substrate can be a variety of clostridial toxin substrates containing a recognition sequence for the toxin. The invention also provides a method for delivering a substrate composition to a target cell using a delivery agent. The technical effect of the invention is to provide a substrate composition that can be used to detect and measure the activity of clostridial toxins in a variety of biological samples.

Problems solved by technology

However, in spite of their potentially deleterious effects, low controlled doses of botulinum neurotoxins have been successfully used as therapeutics and for some cosmetic applications.

Unfortunately, the mouse lethality assay suffers from several drawbacks: cost due to the large numbers of laboratory animals required; lack of specificity; the potential for inaccuracy unless large animal groups are used; and the necessary sacrifice of animal life.

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