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Fret protease assays for clostridial toxins

a protease and clostridial technology, applied in the field of fluorescence resonance energy transfer and protease assays, can solve the problems that the botulinum serotype a/e substrate can also be susceptible to cleavage, and achieve the effect of increasing the fluorescence intensity of the donor and reducing the fluorescence intensity of the acceptor

Inactive Publication Date: 2008-02-14
ALLERGAN INC
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The clostridial toxin substrates provide a rapid, specific, and cost-effective method for determining toxin activity, eliminating the need for animal testing and offering precise quantification of toxin levels, enhancing food safety and pharmaceutical quality control.

Problems solved by technology

Such a botulinum serotype A / E substrate also can be susceptible to cleavage by both the BoNT / A and BoNT / E toxins.

Method used

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  • Fret protease assays for clostridial toxins
  • Fret protease assays for clostridial toxins
  • Fret protease assays for clostridial toxins

Examples

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Analysis of BoNT / A Activity Using Fluorescence Resonance Energy Transfer

[0189] This example describes the use of a FRET assay to analyze proteolytic activity of a botulinum toxin.

[0190] The FRET substrate X1-Asp-Ser-Asn-Lys-Thr-Arg-Ile-Asp-Glu-Ala-Asn-Gln-Arg-Ala-Thr-Lys-Met-Leu-Z2-NH2 (SEQ ID NO: 85) was synthesized by Alpha Diagnostics International (San Antonio, Tex.). This substrate contains a recognition sequence for BoNT / A flanked by a fluorescein-modified lysine residue (“X1”) and a tetramethylrhodamine-modified lysine residue (“Z2”) followed by a carboxy-terminal amide. Following proteolysis by botulinum toxin serotype A, the cleavage products X1-Asp-Ser-Asn-Lys-Thr-Arg-Ile-Asp-Glu-Ala-Asn-Gln (SEQ ID NO: 86) and Arg-Ala-Thr-Lys-Met-Leu-Z2-NH2 (SEQ ID NO: 87) are produced.

[0191] Additional FRET substrates also are synthesized: X1-Asp-Ser-Asn-Lys-Thr-Arg-Ile-Asp-Glu-Ala-Asn-Gln-Arg-Ala-Thr-Lys-Met-Leu-Gly-Ser-Gly-Z2-NH2 (SEQ ID NO: 88); X1-Ala-Asp-Ser-Asn-Lys-Thr-Arg-Ile-A...

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Abstract

The present invention provides clostridial toxin substrates useful in assaying for the protease activity of any clostridial toxin, including botulinum toxins of all serotypes as well as tetanus toxins. A clostridial toxin substrate of the invention contains a donor fluorophore; an acceptor having an absorbance spectrum overlapping the emission spectrum of the donor fluorophore; and a clostridial toxin recognition sequence that includes a cleavage site, where the cleavage site intervenes between the donor fluorophore and the acceptor and where, under the appropriate conditions, resonance energy transfer is exhibited between the donor fluorophore and the acceptor.

Description

[0001] This application is a divisional and claims priority pursuant to 35 U.S.C. § 120 to U.S. patent application Ser. No. 09 / 942,098, filed Aug. 28, 2001, 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, for example, assays for protease activity of clostridial toxins such botulinum toxins and tetanus toxins, and more specifically, to intramolecularly quenched substrates and methods for assaying for clostridial toxin protease 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 micrograms. Thus,...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): G01N33/569C07K14/33C12Q1/04G01N21/78C07K7/00C07K7/06C07K7/08C07K14/00C07K14/435C07K14/47C12Q1/37
CPCC07K14/001C07K14/435G01N2333/33G01N33/56911C12Q1/37
Inventor STEWARD, LANCE E.FERNANDEZ-SALAS, ESTERAOKI, KEI ROGER
Owner ALLERGAN INC
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