US 2014/0235490 A1Published application

Methods for detection of botulinum neurotoxin

AssigneeCity of Hope

InventorsMarkus KALKUM, Karine BAGRAMYAN

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Abstract

Provided herein is a large immuno-sorbent surface area assay (ALISSA) for the rapid and sensitive detection of botulinum neurotoxins (BoNTs) and anthrax toxin. This assay is designed to capture a low number of toxin molecules and to measure their intrinsic protease activity via conversion of a fluorogenic or luminescent substrate. Also provided herein are novel peptides that can be specifically cleaved by BoNT and novel peptides that are resistant to cleavage by BoNT. The combination of these cleavable and control peptides can be used for implementation of an exemplary ALISSA used to specifically detect BoNT enzymatic activity. Furthermore, the ALISSA as described herein may also be used in a column based format for use in a high-throughput system for testing large quantities of samples.

Claims20

Dependent claims

A method for detecting the presence of a BoNT toxin in a sample comprising:

a) exposing the sample putatively containing a BoNT toxin to an enrichment matrix comprising a substrate fusion protein capable of eliciting a detectable luminogenic signal following modification by the BoNT toxin, the exposure occurring under conditions permitting the modification of the substrate fusion protein by the BoNT toxin,
the substrate fusion protein comprising a BoNT cleavage site wherein:
(i) if the BoNT toxin is BoNT/A, the substrate fusion protein comprises a fragment of the synaptosome-associated protein of 25 kDa (SNAP25) containing the BoNT/A cleavage site;
(ii) if the BoNT toxin is BoNT/B, the substrate fusion protein comprises a fragment of vesicle-associated membrane protein (VAMP) containing the BoNT/B cleavage site;
(iii) if the BoNT toxin is BoNT/C, the substrate fusion protein comprises a fragment of VAMP containing the BoNT/C cleavage site or a fragment of SNAP25 containing the BoNT/C cleavage site;
(iv) if the BoNT toxin is BoNT/D, the substrate fusion protein comprises a fragment of VAMP containing the BoNT/D cleavage site;
(v) if the BoNT toxin is BoNT/E, the substrate fusion protein comprises a fragment of SNAP25 containing the BoNT/E cleavage site;
(vi) if the BoNT toxin is BoNT/F, the substrate fusion protein comprises a fragment of VAMP containing the BoNT/F cleavage site;
and
(vii) if the BoNT toxin is BoNT/G, the substrate fusion protein comprises a fragment of VAMP containing the BoNT/G cleavage site;
and
b) detecting the presence of the BoNT toxin by measuring a change in light emission in the sample.

9 more elements

A method for detecting the presence of a BoNT toxin in a sample comprising:

a) exposing the sample putatively containing a BoNT toxin to an enrichment matrix comprising a substrate fusion protein comprising one or more firefly luciferase proteins or a fragment thereof, the substrate fusion protein capable of being modified by the BoNT toxin, the exposure occurring under conditions permitting the modification of the substrate fusion protein by the BoNT toxin,
the substrate fusion protein comprising a BoNT cleavage site wherein:
(i) if the BoNT toxin is BoNT/A, the substrate fusion protein comprises a fragment of the synaptosome-associated protein of 25 kDa (SNAP25) containing the BoNT/A cleavage site;
(ii) if the BoNT toxin is BoNT/B, the substrate fusion protein comprises a fragment of vesicle-associated membrane protein (VAMP) containing the BoNT/B cleavage site;
(iii) if the BoNT toxin is BoNT/C, the substrate fusion protein comprises a fragment of VAMP containing the BoNT/C cleavage site or a fragment of SNAP25 containing the BoNT/C cleavage site;
(iv) if the BoNT toxin is BoNT/D, the substrate fusion protein comprises a fragment of VAMP containing the BoNT/D cleavage site;
(v) if the BoNT toxin is BoNT/E, the substrate fusion protein comprises a fragment of SNAP25 containing the BoNT/E cleavage site;
(vi) if the BoNT toxin is BoNT/F, the substrate fusion protein comprises a fragment of VAMP containing the BoNT/F cleavage site;
and
(vii) if the BoNT toxin is BoNT/G, the substrate fusion protein comprises a fragment of VAMP containing the BoNT/G cleavage site;
and
b) exposing the modified substrate fusion protein to a detection fusion protein, the exposure occurring under conditions permitting the binding of the substrate fusion protein to the detection fusion protein;
and
c) detecting the presence of the BoNT toxin by measuring a change in light emission.

11 more elements

A method for detecting the presence of a BoNT toxin in a sample comprising:

a) exposing the sample putatively containing a BoNT toxin to an enrichment matrix provided in a column comprising a substrate fusion protein comprising one or more firefly luciferase proteins or a fragment thereof, the substrate fusion protein capable of eliciting a detectable luminogenic signal following modification by the BoNT toxin, the exposure occurring under conditions permitting the modification of the substrate fusion protein by the BoNT toxin,
the substrate fusion protein further comprising a BoNT cleavage site wherein:
(i) if the BoNT toxin is BoNT/A, the substrate fusion protein comprises a fragment of the synaptosome-associated protein of 25 kDa (SNAP25) containing the BoNT/A cleavage site;
(ii) if the BoNT toxin is BoNT/B, the substrate fusion protein comprises a fragment of vesicle-associated membrane protein (VAMP) containing the BoNT/B cleavage site;
(iii) if the BoNT toxin is BoNT/C, the substrate fusion protein comprises a fragment of VAMP containing the BoNT/C cleavage site or a fragment of SNAP25 containing the BoNT/C cleavage site;
(iv) if the BoNT toxin is BoNT/D, the substrate fusion protein comprises a fragment of VAMP containing the BoNT/D cleavage site;
(v) if the BoNT toxin is BoNT/E, the substrate fusion protein comprises a fragment of SNAP25 containing the BoNT/E cleavage site;
(vi) if the BoNT toxin is BoNT/F, the substrate fusion protein comprises a fragment of VAMP containing the BoNT/F cleavage site;
and
(vii) if the BoNT toxin is BoNT/G, the substrate fusion protein comprises a fragment of VAMP containing the BoNT/G cleavage site;
and
b) detecting the presence of the BoNT toxin by measuring a change in light emission of the sample.

9 more elements

Dependencies are inferred from the claim text.

Facts

Application: 14/272,005Filed 7 May 2014
Priority: 5 Jun 2007
Published: 21 Aug 2014
Term ends: Not estimatedThe source records don’t support an estimate.

Classification15 codes

G01N 33/5308
Immunoassay; Biospecific binding assay; Materials therefor: for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
C07K 7/08
Linear peptides containing only normal peptide links: having 12 to 20 amino acids
C07K 14/705
From animals; from humans: receptors; Cell surface antigens; Cell surface determinants
C07K 16/1282
Gram-positive bacteria: clostridium

Family

Application60/942,199Us provisional application · filed 5 Jun 2007
Application12/134,092Division · filed 5 Jun 2008
Application13/306,769Continuation in part · filed 29 Nov 2011
Application13/600,186Continuation · filed 30 Aug 2012
Application14/272,005Continuation
Application13/600,186Continuation in part
Application13/306,769Division
US 2009/0176259 A1Published application
US 2012/0322081 A1Published application
US 2013/0065259 A1Published application
US 8,067,192 B2Related patent
US 8,753,831 B2Related patent
US 9,562,903 B2Related patent
US 2014/0235490 A1This patent

From the US filing history. Foreign family members aren’t listed.