Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

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
View PDF23 Cites 10 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Fret protease assays for clostridial toxins
  • Fret protease assays for clostridial toxins
  • Fret protease assays for clostridial toxins

Examples

Experimental program
Comparison scheme
Effect test

example i

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...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

PropertyMeasurementUnit
absorbance spectrumaaaaaaaaaa
resonance energy transferaaaaaaaaaa
fluorescence intensityaaaaaaaaaa
Login to View More

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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
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
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com