CA2451909A1 - High throughput assays for the proteolytic activities of clostridial neurotoxins - Google Patents

High throughput assays for the proteolytic activities of clostridial neurotoxins Download PDF

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Publication number
CA2451909A1
CA2451909A1 CA002451909A CA2451909A CA2451909A1 CA 2451909 A1 CA2451909 A1 CA 2451909A1 CA 002451909 A CA002451909 A CA 002451909A CA 2451909 A CA2451909 A CA 2451909A CA 2451909 A1 CA2451909 A1 CA 2451909A1
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Prior art keywords
neurotoxin
substrate
seq
peptide
serotype
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Granted
Application number
CA002451909A
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French (fr)
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CA2451909C (en
Inventor
James J. Schmidt
Robert G. Stafford
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
US Army Medical Research Institute of Infectious Diseases
Original Assignee
James J. Schmidt
Robert G. Stafford
U.S. Army Medical Research Institute Of Infectious Diseases
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Publication of CA2451909A1 publication Critical patent/CA2451909A1/en
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Anticipated expiration legal-status Critical
Expired - Fee Related legal-status Critical Current

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Classifications

    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/33Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Clostridium (G)
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/37Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/536Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
    • G01N33/542Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with steric inhibition or signal modification, e.g. fluorescent quenching
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911Bacteria
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/33Assays involving biological materials from specific organisms or of a specific nature from bacteria from Clostridium (G)
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10STECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00Chemistry: molecular biology and microbiology
    • Y10S435/8215Microorganisms
    • Y10S435/822Microorganisms using bacteria or actinomycetales
    • Y10S435/842Clostridium

Abstract

In this application is described substrates for high-throughput assays of clostridial neurotoxin proteolytic activities. Two types of substrates are described for use in assays for the proteolytic activities of clostridial neurotoxins: (1) modified peptides or proteins that can serve as FRET substrates and (2) modified peptides or proteins that can serve as immobiliz ed substrates. In both types a fluorescent molecules is present in the substrat e, eliminating the requirement for the addition of a fluorigenic reagent. The assays described can be readily adapted for use in automated or robotic systems.

Claims (54)

1. A clostridial neurotoxin substrate comprising any peptide or protein that can serve as a substrate for the proteolytic activity of any clostridial neurotoxin, said protein or peptide having been modified to contain a signal moiety on one side of the cleavage site, and a moiety on the other side of the cleavage site that quenches the magnitude of that signal such that when the substrate is cleaved, an increase in signal is produced.
2. The substrate according to claim 1 wherein said clostridial neurotoxin is botulinum neurotoxin serotype A.
3. The substrate according to claim 2 wherein said substrate is a peptide identified in SEQ ID NO:1 or SEQ ID NO:2.
4. The substrate according to claim 1 wherein said clostridial neurotoxin is botulinum neurotoxin serotype B or tetanus toxin.
5. The substrate of claim 4 wherein said substrate is identified in SEQ ID NO:3 and SEQ ID
NO:4.
6. The substrate according to claim 1 wherein said clostridial neurotoxin is botulinum neurotoxin serotype D or botulinum neurotoxin serotype F.
7. The substrate of to claim 6 wherein said substrate is chosen from the group consisting of a peptide identified in SEQ ID NO:5, SEA ID NO:6, and SEQ ID NO:7.
8. A method for detecting the presence of clostridial neurotoxin proteolytic acitivity in a sample said method comprising mixing the sample with a peptide substrate according to claim 1, and detecting an increase in signal produced from proteolytic cleavage of said substrate.
9. A method for measuring concentration of neurotoxin in a sample, comprising mixing the sample with a peptide substrate according to claim 1, measuring an increase in signal with time produced from proteolytic cleavage of said substrate and, determining the concentration of said neurotoxin by correlation to a standard.
10. The method according to claim 9 wherein said neurotoxin is botulinum neurotoxin serotype A.
11. The method according to claim 10 wherein said peptide substrate is a peptide identified in SEQ ID
NO:1 or SEQ ID NO:2.
12. The method according to claim 9 wherein said neurotoxin is botulinum neurotoxin serotype B or tetanus toxin.
13. The method according to claim 12 wherein said peptide substrate is a peptide identified in SEQ ID
NO:3 or SEQ ID NO:4.
14. The method according to claim 9 wherein said neurotoxin is botulinum neurotoxin serotype D or F.
15. The method according to claim 14 wherein said peptide substrate is chosen from the group consisting of a peptide identified in SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7.
16. A kit for determining the concentration of a clostridial neurotoxin in a sample, the kit containing in close confinement, (i) one or more peptide substrates according to claim 1 cleavable by said clostridial neurotoxin;
(ii) said clostridial neurotoxin standard.
17. The kit according to claim 16 wherein said clostridial neurotoxin is botulinum neurotoxin serotype A and the peptide substrate is one or both of the peptides identified in SEQ ID NO:1 and SEQ ID
NO:2.
18. The kit according to claim 16 wherein said clostridial neurotoxin is botulinum neurotoxin serotype B or tetanus toxin and the peptide substrate is one or both of the peptides identified in SEQ ID
NO:3 and SEQ ID NO:4.
19. The kit according to claim 16 wherein said clostridial neurotoxin is botulinum neurotoxin serotype D or F and the peptide substrate is one or more of the peptides identified in SEQ ID NO:5, SEQ ID
NO:6, and SEQ ID NO:7.
20. A botulinum neurotoxin substrate comprising any peptide or protein that can serve as a substrate for the proteolytic activity of any clostridial neurotoxin, said protein or peptide having been modified so that it can be attached on one side of the proteolytic cleavage site to a solid material.
21. The substrate according to claim 20 wherein said clostridial neurotoxin is botulinum neurotoxin serotype A.
22. The substrate according to claim 21 wherein said substrate is a peptide identified in SEQ ID NO:8 or SEQ ID NO:11.
23. The substrate according to claim 20 wherein said clostridial neurotoxin is botulinum neurotoxin serotype B or tetanus toxin.
24. The substrate of claim 23 wherein said substrate is identified in SEQ ID NO:9.
25. The substrate according to claim 20 wherein said clostridial neurotoxin is botulinum neurotoxin serotype D or serotype F.
26. The substrate of claim 25 wherein said substrate is a peptide identified in SEQ ID NO:10.
27. The substrate of claim 20 wherein said botulinum neurotoxin is botulinum neurotoxin serotype E.
28. The substrate of claim 27 wherein said substrate is a peptide identified in SEQ ID NO:11 and 12.
29. A method for detecting the presence of clostridial neurotoxin proteolytic acitivity in a sample said method comprising mixing the sample with a peptide substrate according to claim 20, and detecting an increase in signal produced from proteolytic cleavage of said substrate.
30. A method for measuring concentration of neurotoxin in a sample, comprising mixing the sample with a peptide substrate according to claim 20, measuring an increase in signal with time produced from proteolytic cleavage of said substrate and, determining the concentration of said neurotoxin by correlation to a standard.
31. The method according to claim 30 wherein said neurotoxin is botulinum neurotoxin serotype A.
32. The method according to claim 31 wherein said peptide substrate is a peptide identified in SEQ ID
NO:8 or SEQ ID NO:11.
33. The method according to claim 30 wherein said neurotoxin is botulinum neurotoxin serotype B or tetanus toxin.
34. The method according to claim 33 wherein said peptide substrate is a peptide identified in SEQ ID
NO:9.
35. The method according to claim 30 wherein said neurotoxin is botulinum neurotoxin serotype D or F.
36. The method according to claim 35 wherein said peptide substrate is a peptide identified in SEQ ID
NO:10.
37. The method according to claim 30 wherein said neurotoxin is botulinum neurotoxin serotype E.
38. The method according to claim 37 wherein said peptide substrate is a peptide identified in SEQ ID
NO:11 or SEQ ID NO:12.
39. A kit for determining the concentration of a clostridial neurotoxin in a sample, the kit containing in close confinement, (i) one or more peptide substrates according to claim 20 cleavable by said clostridial neurotoxin;
(ii) said clostridial neurotoxin standard.
40. The kit according to claim 39 wherein said clostridial neurotoxin is botulinum neurotoxin serotype A and the peptide substrate is one or both of the peptides identified in SEQ ID NO:8 and SEQ ID
NO:11.
41. The kit according to claim 39 wherein said clostridial neurotoxin is botulinum neurotoxin serotype B or tetanus toxin and the peptide substrate is a peptide identified in SEQ ID NO:9.
42. The kit according to claim 39 wherein said clostridial neurotoxin is botulinum neurotoxin serotype D or F and the peptide substrate is a peptide identified in SEQ ID NO:10.
43. The kit according to claim 39 wherein said clostridial neurotoxin is botulinum neurotoxin serotype E and the peptide substrate is one or more of the peptides identified in SEQ ID NO:11 or SEQ ID
NO:12.
44. A method for identifying inhibitors or enhancers of proteolytic activity of a clostridial neurotoxin comprising:
preincubating a neurotoxin with a test compound to make a neurotoxin-compound solution, exposing said solution to a substrate of said neurotoxin according to claim 20, measuring signal resulting from the proteolysis of said substrate by said neurotoxin, and comparing said signal with controls, wherein an increase in signal indicates a compound which enhances neurotoxin activity and a decrease in signal indicates a compound with inhibits said neurotoxin.
45. The method according to claim 44 wherein said neurotoxin is botulinum neurotoxin serotype A and the substrate is a peptide identified in SEQ ID NO:8 or SEQ ID NO:11.
46. The method according to claim 44 wherein said neurotoxin is botulinum neurotoxin serotype B or tetanus toxin and the substrate is a peptide identified in SEQ ID NO:9.
47. The method according to claim 44 wherein said neurotoxin is botulinum neurotoxin serotype D or F and the substrate is a peptide identified in SEQ ID NO:10.
48. The method according to claim 44 wherein said neurotoxin is botulinum neurotoxin serotype E and the substrate is one or more of the peptides identified in SEQ ID NO:11 or SEQ ID NO:12.
49. A method for identifying a serotype of a clostridial neurotoxin in a sample suspected of containing a neurotoxin, the method comprising incubating the sample with antibodies against each clostridial neurotoxin such that a neurotoxin is bound to its serotype-specific antibody, removing unbound components, adding activation solution such that clostridial protease is activated, adding solutions containing clostridial neurotoxin peptide substrates according to claim 1 to said activated protease, detecting signal generated from proteolysis of said substrate by said protease, wherein a signal above control indicates presence of a neurotoxin, and determining the serotype of the clostridial neurotoxin by noting the specificity of the antibody.
50. The method of claim 49 wherein the antibodies are bound to a solid material.
51. The method of claim 50 wherein the solid material is a multiwell plate.
52. The method of claim 51 wherein each well contains an antibody specific for a different neurotoxin serotype.
53. The method of claim 49 wherein each peptide substrate is labeled with a different signal.
54. A kit for identifying a serotype of a clostridial neurotoxin in a sample suspected of containing a neurotoxin, comprising serotype-specific antibodies clostridial neurotoxin standards, and peptide substrates according to claim 1.
CA2451909A 2000-09-25 2001-09-25 High throughput assays for the proteolytic activities of clostridial neurotoxins Expired - Fee Related CA2451909C (en)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US23505000P 2000-09-25 2000-09-25
US60/235,050 2000-09-25
PCT/US2001/030188 WO2002025284A2 (en) 2000-09-25 2001-09-25 High-throughput assays for the proteolytic activities of clostridial neurotoxins

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CA2451909A1 true CA2451909A1 (en) 2002-03-28
CA2451909C CA2451909C (en) 2010-12-21

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US (3) US6762280B2 (en)
EP (1) EP1419390B1 (en)
AT (1) ATE378596T1 (en)
AU (1) AU2001294771A1 (en)
CA (1) CA2451909C (en)
DE (1) DE60131468D1 (en)
WO (1) WO2002025284A2 (en)

Families Citing this family (36)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7563874B2 (en) * 1998-08-31 2009-07-21 The Regents Of The University Of California Therapeutic monoclonal antibodies that neutralize botulinum neurotoxins
US20040219619A1 (en) * 2000-07-21 2004-11-04 Ester Fernandez-Salas Methods of identifying compounds that alter toxin persistence and/or protease activity
ATE378596T1 (en) * 2000-09-25 2007-11-15 U S Medical Res Inst Of Infect HIGH-THROUGHPUT ASSAY FOR PROTEOLYTIC ACTIVITIES OF CLOSTRIDIUM NEUROTOXINS
US8148141B2 (en) * 2001-05-07 2012-04-03 Hipep Laboratories Peptide-immobilized substrate and method for measuring target protein
US7208285B2 (en) 2001-08-28 2007-04-24 Allergan, Inc. Fret protease assays for botulinum serotype A/E toxins
US7332567B2 (en) * 2001-08-28 2008-02-19 Allergan, Inc. Fret protease assays for clostridial toxins
US8022172B2 (en) 2001-08-28 2011-09-20 Allergan, Inc. Luminescence resonance energy transfer (LRET) assays for clostridial toxin activity
US7374896B2 (en) 2001-08-28 2008-05-20 Allergan, Inc. GFP-SNAP25 fluorescence release assay for botulinum neurotoxin protease activity
US7183066B2 (en) 2002-09-27 2007-02-27 Allergan, Inc. Cell-based fluorescence resonance energy transfer (FRET) assays for clostridial toxins
WO2004031355A2 (en) * 2002-10-01 2004-04-15 University Of Maryland Methods for identifying inhibitors of botulinum neurotoxins
WO2004052934A1 (en) * 2002-12-12 2004-06-24 National Institute Of Advanced Industrial Science And Technology Monitor protein for measuring processing of protein
WO2004102215A1 (en) * 2003-05-07 2004-11-25 The United States Of America, As Represented By The Secretary Of The Navy Naval Research Laboratory Assay for testing neurotoxin vaccine efficacy
US20050079566A1 (en) * 2003-09-10 2005-04-14 Caterer Nigel Robert Analytical methods for determination of proteolytic cleavage at specific sites
US7611856B2 (en) * 2003-11-05 2009-11-03 Los Alamos National Security, Llc Mass spectrometry-based methods for detection and differentiation of botulinum neurotoxins
JP5178009B2 (en) 2003-12-19 2013-04-10 ウィスコンシン アルムニ リサーチ ファンデイション Methods and complexes for botulinum neurotoxin detection
US7399607B2 (en) * 2004-09-22 2008-07-15 Allergan, Inc. Fluorescence polarization assays for determining clostridial toxin activity
JP2008528060A (en) * 2005-01-27 2008-07-31 ザ リージェンツ オブ ザ ユニバーシティ オブ カリフォルニア Therapeutic monoclonal antibodies that neutralize botulinum neurotoxins
CA2605160A1 (en) 2005-04-05 2006-10-05 Allergan, Inc. Clostridial toxin activity assays
DK1869459T3 (en) * 2005-04-05 2010-09-27 Allergan Inc Lipophilic color-based assays for clostridial toxin activity
CA2610103A1 (en) 2005-09-19 2007-03-19 Allergan, Inc. Clostridial toxin activatable clostridial toxins
DE102006019447A1 (en) * 2006-04-24 2007-10-25 Dressler, Dirk, Dr. Medicament, useful to treat diseases e.g. headache, hyperhidrosis and muscular pain, comprises botulinum toxin and at least a marker, preferably a saline-solution
AU2007256364A1 (en) * 2006-06-09 2007-12-13 Bio Pur Ag A method for the detection of enzymatic reactions
US8598321B2 (en) 2007-03-22 2013-12-03 The Regents Of The University Of California Therapeutic monoclonal antibodies that neutralize botulinum neurotoxins
US8753831B2 (en) 2007-06-05 2014-06-17 City Of Hope Methods for detection of botulinum neurotoxin
US8067192B2 (en) * 2007-06-05 2011-11-29 City Of Hope Methods for detection of botulinum neurotoxin
US20110098309A1 (en) * 2007-07-12 2011-04-28 Acumen Pharmaceuticals, Inc. Methods of inhibiting the formation of amyloid-beta diffusable ligands using acylhydrazide compounds
US8962677B2 (en) * 2007-07-12 2015-02-24 Acumen Pharmaceuticals, Inc. Methods of restoring cognitive ability using non-peptidic compounds
US9006283B2 (en) 2007-07-12 2015-04-14 Acumen Pharmaceuticals, Inc. Methods of modifying amyloid β oligomers using non-peptidic compounds
US9217024B2 (en) 2007-12-18 2015-12-22 Acumen Pharmaceuticals, Inc. ADDL receptor polypeptides, polynucleotides and host cells for recombinant production
WO2010014854A2 (en) 2008-07-31 2010-02-04 The Regents Of The University Of California Antibodies that neutralize botulinum neurotoxins
US20120202754A1 (en) * 2009-10-18 2012-08-09 Schmidt James J Enhanced substrates for the protease activity of serotype a botulinum neurotoxin
US9243057B2 (en) 2010-08-31 2016-01-26 The Regents Of The University Of California Antibodies for botulinum neurotoxins
RU2549463C1 (en) * 2013-12-03 2015-04-27 Федеральное бюджетное учреждение науки Государственный научный центр прикладной микробиологии и биотехнологии (ФБУН ГНЦ ПМБ) Method of determining botulinum neurotoxin of type a based immunodetection, combined with polymerase chain reaction
WO2019212946A1 (en) 2018-04-30 2019-11-07 Ribon Therapeutics Inc. Screening methods for parp modulators
IL272002A (en) * 2020-01-13 2021-07-29 The Israel Institute Of Biological Res Iibr Methods for identifying anti clostridial neurotoxin compounds
CN114539362B (en) * 2022-04-25 2022-07-26 中国疾病预防控制中心传染病预防控制所 Botulinum toxin specific substrate peptide, detection kit and detection method

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB9411138D0 (en) * 1994-06-03 1994-07-27 Microbiological Res Authority Toxin assay
US5605809A (en) * 1994-10-28 1997-02-25 Oncoimmunin, Inc. Compositions for the detection of proteases in biological samples and methods of use thereof
US5965699A (en) * 1996-11-06 1999-10-12 The United States Of America As Represented By The Secretary Of The Army Assay for the proteolytic activity of serotype a from clostridium botulinum
FR2809733B1 (en) 2000-06-02 2004-04-30 Inst Nat Sante Rech Med PEPTIDE SUBSTRATE RECOGNIZED BY BONT / B TYPE BOTULINUM TOXIN AND ITS USE FOR DETERMINING AND / OR DETECTING SAID TOXIN OR CORRESPONDING INHIBITORS
ATE378596T1 (en) * 2000-09-25 2007-11-15 U S Medical Res Inst Of Infect HIGH-THROUGHPUT ASSAY FOR PROTEOLYTIC ACTIVITIES OF CLOSTRIDIUM NEUROTOXINS

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EP1419390A2 (en) 2004-05-19
US20050287622A1 (en) 2005-12-29
CA2451909C (en) 2010-12-21
US7157553B2 (en) 2007-01-02
US20030077685A1 (en) 2003-04-24
US20040146963A1 (en) 2004-07-29
WO2002025284A3 (en) 2003-09-18
EP1419390B1 (en) 2007-11-14
US7034107B2 (en) 2006-04-25
WO2002025284A2 (en) 2002-03-28
DE60131468D1 (en) 2007-12-27
US6762280B2 (en) 2004-07-13
AU2001294771A1 (en) 2002-04-02
ATE378596T1 (en) 2007-11-15

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