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Article ; Online: Isolation and quantification of botulinum neurotoxin from complex matrices using the BoTest matrix assays.

Dunning, F Mark / Piazza, Timothy M / Zeytin, Füsûn N / Tucker, Ward C

Journal of visualized experiments : JoVE

2014 , Issue 85

Abstract: Accurate detection and quantification of botulinum neurotoxin (BoNT) in complex matrices is required for pharmaceutical, environmental, and food sample testing. Rapid BoNT testing of foodstuffs is needed during outbreak forensics, patient diagnosis, and ... ...

Abstract	Accurate detection and quantification of botulinum neurotoxin (BoNT) in complex matrices is required for pharmaceutical, environmental, and food sample testing. Rapid BoNT testing of foodstuffs is needed during outbreak forensics, patient diagnosis, and food safety testing while accurate potency testing is required for BoNT-based drug product manufacturing and patient safety. The widely used mouse bioassay for BoNT testing is highly sensitive but lacks the precision and throughput needed for rapid and routine BoNT testing. Furthermore, the bioassay's use of animals has resulted in calls by drug product regulatory authorities and animal-rights proponents in the US and abroad to replace the mouse bioassay for BoNT testing. Several in vitro replacement assays have been developed that work well with purified BoNT in simple buffers, but most have not been shown to be applicable to testing in highly complex matrices. Here, a protocol for the detection of BoNT in complex matrices using the BoTest Matrix assays is presented. The assay consists of three parts: The first part involves preparation of the samples for testing, the second part is an immunoprecipitation step using anti-BoNT antibody-coated paramagnetic beads to purify BoNT from the matrix, and the third part quantifies the isolated BoNT's proteolytic activity using a fluorogenic reporter. The protocol is written for high throughput testing in 96-well plates using both liquid and solid matrices and requires about 2 hr of manual preparation with total assay times of 4-26 hr depending on the sample type, toxin load, and desired sensitivity. Data are presented for BoNT/A testing with phosphate-buffered saline, a drug product, culture supernatant, 2% milk, and fresh tomatoes and includes discussion of critical parameters for assay success.
MeSH term(s)	Animals ; Botulinum Toxins/isolation & purification ; Complex Mixtures/analysis ; High-Throughput Screening Assays/methods ; Mice
Chemical Substances	Complex Mixtures ; Botulinum Toxins (EC 3.4.24.69)
Language	English
Publishing date	2014-03-03
Publishing country	United States
Document type	Journal Article ; Research Support, U.S. Gov't, Non-P.H.S. ; Video-Audio Media
ZDB-ID	2259946-0
ISSN	1940-087X ; 1940-087X
ISSN (online)	1940-087X
ISSN	1940-087X
DOI	10.3791/51170
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article: Isolation and quantification of botulinum neurotoxin from complex matrices using the botest matrix assays

Dunning, F. Mark / Piazza, Timothy M / Zeytin, Füsûn N / Tucker, Ward C

Journal of visualized experiments. 2014 Mar. 03, , no. 85

2014

Abstract	Accurate detection and quantification of botulinum neurotoxin (BoNT) in complex matrices is required for pharmaceutical, environmental, and food sample testing. Rapid BoNT testing of foodstuffs is needed during outbreak forensics, patient diagnosis, and food safety testing while accurate potency testing is required for BoNT-based drug product manufacturing and patient safety. The widely used mouse bioassay for BoNT testing is highly sensitive but lacks the precision and throughput needed for rapid and routine BoNT testing. Furthermore, the bioassay's use of animals has resulted in calls by drug product regulatory authorities and animal-rights proponents in the US and abroad to replace the mouse bioassay for BoNT testing. Several in vitro replacement assays have been developed that work well with purified BoNT in simple buffers, but most have not been shown to be applicable to testing in highly complex matrices. Here, a protocol for the detection of BoNT in complex matrices using the BoTest Matrix assays is presented. The assay consists of three parts: The first part involves preparation of the samples for testing, the second part is an immunoprecipitation step using anti-BoNT antibody-coated paramagnetic beads to purify BoNT from the matrix, and the third part quantifies the isolated BoNT's proteolytic activity using a fluorogenic reporter. The protocol is written for high throughput testing in 96-well plates using both liquid and solid matrices and requires about 2 hr of manual preparation with total assay times of 4-26 hr depending on the sample type, toxin load, and desired sensitivity. Data are presented for BoNT/A testing with phosphate-buffered saline, a drug product, culture supernatant, 2% milk, and fresh tomatoes and includes discussion of critical parameters for assay success.
Keywords	animal use replacement ; bioassays ; botulinum toxin ; buffers ; drugs ; food safety ; foods ; forensic sciences ; liquids ; manufacturing ; mice ; milk ; patients ; precipitin tests ; proteolysis ; safety testing ; tomatoes ; United States
Language	English
Dates of publication	2014-0303
Size	p. e51170.
Publishing place	Journal of Visualized Experiments
Document type	Article
ZDB-ID	2259946-0
ISSN	1940-087X
ISSN	1940-087X
DOI	10.3791/51170
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Article: Role of synaptotagmin in Ca2+-triggered exocytosis.

Tucker, Ward C / Chapman, Edwin R

The Biochemical journal

2002 Volume 366, Issue Pt 1, Page(s) 1–13

Abstract: The Ca(2+)-binding synaptic-vesicle protein synaptotagmin I has attracted considerable interest as a potential Ca(2+) sensor that regulates exocytosis from neurons and neuroendocrine cells. Recent studies have shed new light on the structure, biochemical/ ...

Abstract	The Ca(2+)-binding synaptic-vesicle protein synaptotagmin I has attracted considerable interest as a potential Ca(2+) sensor that regulates exocytosis from neurons and neuroendocrine cells. Recent studies have shed new light on the structure, biochemical/biophysical properties and function of synaptotagmin, and the emerging view is that it plays an important role in both exocytosis and endocytosis. At least a dozen additional isoforms exist, some of which are expressed outside of the nervous system, suggesting that synaptotagmins might regulate membrane traffic in a variety of cell types. Here we provide an overview of the members of this gene family, with particular emphasis on the question of whether and how synaptotagmin I functions during the final stages of membrane fusion: does it regulate the Ca(2+)-triggered opening and dilation of fusion pores?
MeSH term(s)	Animals ; Calcium/metabolism ; Calcium-Binding Proteins ; Cell Membrane/metabolism ; Endocytosis ; Exocytosis ; Humans ; Kinetics ; Membrane Fusion ; Membrane Glycoproteins/chemistry ; Membrane Glycoproteins/physiology ; Membrane Proteins/metabolism ; Models, Biological ; Nerve Tissue Proteins/chemistry ; Nerve Tissue Proteins/physiology ; Nervous System/metabolism ; Protein Isoforms ; Protein Structure, Tertiary ; SNARE Proteins ; Synaptotagmin I ; Synaptotagmins ; Vesicular Transport Proteins
Chemical Substances	Calcium-Binding Proteins ; Membrane Glycoproteins ; Membrane Proteins ; Nerve Tissue Proteins ; Protein Isoforms ; SNARE Proteins ; SYT1 protein, human ; Synaptotagmin I ; Vesicular Transport Proteins ; Synaptotagmins (134193-27-4) ; Calcium (SY7Q814VUP)
Language	English
Publishing date	2002-08-15
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't ; Research Support, U.S. Gov't, P.H.S. ; Review
ZDB-ID	2969-5
ISSN	1470-8728 ; 0264-6021 ; 0006-2936 ; 0306-3275
ISSN (online)	1470-8728
ISSN	0264-6021 ; 0006-2936 ; 0306-3275
DOI	10.1042/BJ20020776
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Article: Synaptotagmin isoforms couple distinct ranges of Ca2+, Ba2+, and Sr2+ concentration to SNARE-mediated membrane fusion.

Bhalla, Akhil / Tucker, Ward C / Chapman, Edwin R

Molecular biology of the cell

2005 Volume 16, Issue 10, Page(s) 4755–4764

Abstract: Ca2+-triggered exocytosis of synaptic vesicles is controlled by the Ca2+-binding protein synaptotagmin (syt) I. Fifteen additional isoforms of syt have been identified. Here, we compared the abilities of three syt isoforms (I, VII, and IX) to regulate ... ...

Abstract	Ca2+-triggered exocytosis of synaptic vesicles is controlled by the Ca2+-binding protein synaptotagmin (syt) I. Fifteen additional isoforms of syt have been identified. Here, we compared the abilities of three syt isoforms (I, VII, and IX) to regulate soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-mediated membrane fusion in vitro in response to divalent cations. We found that different isoforms of syt couple distinct ranges of Ca2+, Ba2+, and Sr2+ to membrane fusion; syt VII was approximately 400-fold more sensitive to Ca2+ than was syt I. Omission of phosphatidylserine (PS) from both populations of liposomes completely abrogated the ability of all three isoforms of syt to stimulate fusion. Mutations that selectively inhibit syt.target-SNARE (t-SNARE) interactions reduced syt stimulation of fusion. Using Sr2+ and Ba2+, we found that binding of syt to PS and t-SNAREs can be dissociated from activation of fusion, uncovering posteffector-binding functions for syt. Our data demonstrate that different syt isoforms are specialized to sense different ranges of divalent cations and that PS is an essential effector of Ca2+.syt action.
MeSH term(s)	Animals ; Barium/pharmacology ; Calcium/metabolism ; Calcium/pharmacology ; Cations, Divalent ; Ethylmaleimide/chemistry ; Humans ; In Vitro Techniques ; Liposomes/metabolism ; Membrane Fusion/drug effects ; Membrane Fusion/physiology ; Phosphatidylserines/metabolism ; Protein Binding ; Protein Isoforms/physiology ; Rats ; SNARE Proteins/metabolism ; Strontium/pharmacology ; Synaptotagmin I/physiology
Chemical Substances	Cations, Divalent ; Liposomes ; Phosphatidylserines ; Protein Isoforms ; SNARE Proteins ; Synaptotagmin I ; Syt1 protein, rat ; Barium (24GP945V5T) ; Ethylmaleimide (O3C74ACM9V) ; Calcium (SY7Q814VUP) ; Strontium (YZS2RPE8LE)
Language	English
Publishing date	2005-10
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't ; Research Support, U.S. Gov't, P.H.S.
ZDB-ID	1098979-1
ISSN	1939-4586 ; 1059-1524
ISSN (online)	1939-4586
ISSN	1059-1524
DOI	10.1091/mbc.E05-04-0277
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Article ; Online: Reconstitution of Ca2+-regulated membrane fusion by synaptotagmin and SNAREs.

Tucker, Ward C / Weber, Thomas / Chapman, Edwin R

Science (New York, N.Y.)

2004 Volume 304, Issue 5669, Page(s) 435–438

Abstract: We investigated the effect of synaptotagmin I on membrane fusion mediated by neuronal SNARE proteins, SNAP-25, syntaxin, and synaptobrevin, which were reconstituted into vesicles. In the presence of Ca2+, the cytoplasmic domain of synaptotagmin I (syt) ... ...

Abstract	We investigated the effect of synaptotagmin I on membrane fusion mediated by neuronal SNARE proteins, SNAP-25, syntaxin, and synaptobrevin, which were reconstituted into vesicles. In the presence of Ca2+, the cytoplasmic domain of synaptotagmin I (syt) strongly stimulated membrane fusion when synaptobrevin densities were similar to those found in native synaptic vesicles. The Ca2+ dependence of syt-stimulated fusion was modulated by changes in lipid composition of the vesicles and by a truncation that mimics cleavage of SNAP-25 by botulinum neurotoxin A. Stimulation of fusion was abolished by disrupting the Ca2+-binding activity, or by severing the tandem C2 domains, of syt. Thus, syt and SNAREs are likely to represent the minimal protein complement for Ca2+-triggered exocytosis.
MeSH term(s)	Animals ; Binding Sites ; Calcium/metabolism ; Calcium-Binding Proteins ; Exocytosis ; Fluorescence Resonance Energy Transfer ; Lipid Bilayers ; Lipids/analysis ; Liposomes/chemistry ; Liposomes/metabolism ; Membrane Fusion ; Membrane Glycoproteins/chemistry ; Membrane Glycoproteins/metabolism ; Membrane Proteins/chemistry ; Membrane Proteins/metabolism ; Mice ; Mutation ; Nerve Tissue Proteins/chemistry ; Nerve Tissue Proteins/metabolism ; Protein Structure, Tertiary ; Qa-SNARE Proteins ; R-SNARE Proteins ; Rats ; Synaptic Vesicles/chemistry ; Synaptic Vesicles/metabolism ; Synaptosomal-Associated Protein 25 ; Synaptotagmin I ; Synaptotagmins
Chemical Substances	Calcium-Binding Proteins ; Lipid Bilayers ; Lipids ; Liposomes ; Membrane Glycoproteins ; Membrane Proteins ; Nerve Tissue Proteins ; Qa-SNARE Proteins ; R-SNARE Proteins ; Snap25 protein, mouse ; Snap25 protein, rat ; Synaptosomal-Associated Protein 25 ; Synaptotagmin I ; Syt1 protein, mouse ; Syt1 protein, rat ; Synaptotagmins (134193-27-4) ; Calcium (SY7Q814VUP)
Language	English
Publishing date	2004-04-16
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't ; Research Support, U.S. Gov't, P.H.S.
ZDB-ID	128410-1
ISSN	1095-9203 ; 0036-8075
ISSN (online)	1095-9203
ISSN	0036-8075
DOI	10.1126/science.1097196
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Article: PIP2 increases the speed of response of synaptotagmin and steers its membrane-penetration activity toward the plasma membrane.

Bai, Jihong / Tucker, Ward C / Chapman, Edwin R

Nature structural & molecular biology

2004 Volume 11, Issue 1, Page(s) 36–44

Abstract: Synaptotagmin-1 (syt), the putative Ca2+ sensor for exocytosis, is anchored to the membrane of secretory organelles. Its cytoplasmic domain is composed of two Ca2+-sensing modules, C2A and C2B. Syt binds phosphatidylinositol 4,5-bisphosphate (PIP2), a ... ...

Abstract	Synaptotagmin-1 (syt), the putative Ca2+ sensor for exocytosis, is anchored to the membrane of secretory organelles. Its cytoplasmic domain is composed of two Ca2+-sensing modules, C2A and C2B. Syt binds phosphatidylinositol 4,5-bisphosphate (PIP2), a plasma membrane lipid with an essential role in exocytosis and endocytosis. We resolved two modes of PIP2 binding that are mediated by distinct surfaces on the C2B domain of syt. A novel Ca2+-independent mode of binding predisposes syt to penetrate PIP2-harboring target membranes in response to Ca2+ with submillisecond kinetics. Thus, PIP2 increases the speed of response of syt and steers its membrane-penetration activity toward the plasma membrane. We propose that syt-PIP2 interactions are involved in exocytosis by facilitating the close apposition of the vesicle and target membrane on rapid time scales in response to Ca2+.
MeSH term(s)	Animals ; Calcium Signaling ; Calcium-Binding Proteins ; Exocytosis ; In Vitro Techniques ; Kinetics ; Membrane Glycoproteins/chemistry ; Membrane Glycoproteins/metabolism ; Membrane Lipids/metabolism ; Membrane Proteins/metabolism ; Nerve Tissue Proteins/chemistry ; Nerve Tissue Proteins/metabolism ; Phosphatidylinositol 4,5-Diphosphate/metabolism ; Protein Binding ; Protein Structure, Tertiary ; Proteolipids/metabolism ; R-SNARE Proteins ; Rats ; Recombinant Proteins/chemistry ; Recombinant Proteins/metabolism ; Synaptotagmin I ; Synaptotagmins ; Thermodynamics
Chemical Substances	Calcium-Binding Proteins ; Membrane Glycoproteins ; Membrane Lipids ; Membrane Proteins ; Nerve Tissue Proteins ; Phosphatidylinositol 4,5-Diphosphate ; Proteolipids ; R-SNARE Proteins ; Recombinant Proteins ; Synaptotagmin I ; Syt1 protein, rat ; proteoliposomes ; Synaptotagmins (134193-27-4)
Language	English
Publishing date	2004-01
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't ; Research Support, U.S. Gov't, P.H.S.
ZDB-ID	2126708-X
ISSN	1545-9985 ; 1545-9993
ISSN (online)	1545-9985
ISSN	1545-9993
DOI	10.1038/nsmb709
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Article: Ca(2+)-synaptotagmin directly regulates t-SNARE function during reconstituted membrane fusion.

Bhalla, Akhil / Chicka, Michael C / Tucker, Ward C / Chapman, Edwin R

Nature structural & molecular biology

2006 Volume 13, Issue 4, Page(s) 323–330

Abstract: In nerve terminals, exocytosis is mediated by SNARE proteins and regulated by Ca(2+) and synaptotagmin-1 (syt). Ca(2+) promotes the interaction of syt with anionic phospholipids and the target membrane SNAREs (t-SNAREs) SNAP-25 and syntaxin. Here, we ... ...

Abstract	In nerve terminals, exocytosis is mediated by SNARE proteins and regulated by Ca(2+) and synaptotagmin-1 (syt). Ca(2+) promotes the interaction of syt with anionic phospholipids and the target membrane SNAREs (t-SNAREs) SNAP-25 and syntaxin. Here, we have used a defined reconstituted fusion assay to determine directly whether syt-t-SNARE interactions couple Ca(2+) to membrane fusion by comparing the effects of Ca(2+)-syt on neuronal (SNAP-25, syntaxin and synaptobrevin) and yeast (Sso1p, Sec9c and Snc2p) SNAREs. Ca(2+)-syt aggregated neuronal and yeast SNARE liposomes to similar extents via interactions with anionic phospholipids. However, Ca(2+)-syt was able to bind and stimulate fusion mediated by only neuronal SNAREs and had no effect on yeast SNAREs. Thus, Ca(2+)-syt regulates fusion through direct interactions with t-SNAREs and not solely through aggregation of vesicles. Ca(2+)-syt drove assembly of SNAP-25 onto membrane-embedded syntaxin, providing direct evidence that Ca(2+)-syt alters t-SNARE structure.
MeSH term(s)	Animals ; Calcium/metabolism ; Exocytosis ; Fungal Proteins/genetics ; Fungal Proteins/metabolism ; In Vitro Techniques ; Liposomes ; Membrane Fusion/physiology ; Models, Biological ; Mutagenesis, Site-Directed ; Nerve Endings/metabolism ; Rats ; Recombinant Proteins/genetics ; Recombinant Proteins/metabolism ; SNARE Proteins/genetics ; SNARE Proteins/metabolism ; Synaptosomal-Associated Protein 25/genetics ; Synaptosomal-Associated Protein 25/metabolism ; Synaptotagmin I/genetics ; Synaptotagmin I/metabolism
Chemical Substances	Fungal Proteins ; Liposomes ; Recombinant Proteins ; SNARE Proteins ; Synaptosomal-Associated Protein 25 ; Synaptotagmin I ; Syt1 protein, rat ; Calcium (SY7Q814VUP)
Language	English
Publishing date	2006-04
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ZDB-ID	2126708-X
ISSN	1545-9985 ; 1545-9993
ISSN (online)	1545-9985
ISSN	1545-9993
DOI	10.1038/nsmb1076
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Article ; Online: Detection of botulinum neurotoxin serotype A, B, and F proteolytic activity in complex matrices with picomolar to femtomolar sensitivity.

Dunning, F Mark / Ruge, Daniel R / Piazza, Timothy M / Stanker, Larry H / Zeytin, Füsûn N / Tucker, Ward C

Applied and environmental microbiology

2012 Volume 78, Issue 21, Page(s) 7687–7697

Abstract: Rapid, high-throughput assays that detect and quantify botulinum neurotoxin (BoNT) activity in diverse matrices are required for environmental, clinical, pharmaceutical, and food testing. The current standard, the mouse bioassay, is sensitive but is low ... ...

Abstract	Rapid, high-throughput assays that detect and quantify botulinum neurotoxin (BoNT) activity in diverse matrices are required for environmental, clinical, pharmaceutical, and food testing. The current standard, the mouse bioassay, is sensitive but is low in throughput and precision. In this study, we present three biochemical assays for the detection and quantification of BoNT serotype A, B, and F proteolytic activities in complex matrices that offer picomolar to femtomolar sensitivity with small assay volumes and total assay times of less than 24 h. These assays consist of magnetic beads conjugated with BoNT serotype-specific antibodies that are used to purify BoNT from complex matrices before the quantification of bound BoNT proteolytic activity using the previously described BoTest reporter substrates. The matrices tested include human serum, whole milk, carrot juice, and baby food, as well as buffers containing common pharmaceutical excipients. The limits of detection were below 1 pM for BoNT/A and BoNT/F and below 10 pM for BoNT/B in most tested matrices using 200-μl samples and as low as 10 fM for BoNT/A with an increased sample volume. Together, these data describe rapid, robust, and high-throughput assays for BoNT detection that are compatible with a wide range of matrices.
MeSH term(s)	Animals ; Antibodies/immunology ; Biological Assay ; Botulinum Toxins/analysis ; Botulinum Toxins/immunology ; Botulinum Toxins/metabolism ; Botulinum Toxins, Type A/analysis ; Botulinum Toxins, Type A/immunology ; Botulinum Toxins, Type A/metabolism ; Clostridium botulinum ; Daucus carota ; High-Throughput Screening Assays ; Humans ; Immunomagnetic Separation ; Infant ; Infant Formula ; Limit of Detection ; Magnetite Nanoparticles ; Milk ; Sensitivity and Specificity ; Serum
Chemical Substances	Antibodies ; Magnetite Nanoparticles ; rimabotulinumtoxinB (0Y70779M1F) ; Botulinum Toxins (EC 3.4.24.69) ; Botulinum Toxins, Type A (EC 3.4.24.69) ; botulinum toxin type F (U1R2P71O7G)
Language	English
Publishing date	2012-08-24
Publishing country	United States
Document type	Journal Article ; Research Support, U.S. Gov't, Non-P.H.S.
ZDB-ID	223011-2
ISSN	1098-5336 ; 0099-2240
ISSN (online)	1098-5336
ISSN	0099-2240
DOI	10.1128/AEM.01664-12
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Article: Molecular regulation of membrane resealing in 3T3 fibroblasts.

Shen, Sheldon S / Tucker, Ward C / Chapman, Edwin R / Steinhardt, Richard A

The Journal of biological chemistry

2004 Volume 280, Issue 2, Page(s) 1652–1660

Abstract: Membrane resealing in mammalian cells after injury depends on Ca(2+)-dependent fusion of intracellular vesicles with the plasma membrane. When cells are wounded twice, the subsequent resealing is generally faster. Physiological and biochemical studies ... ...

Abstract	Membrane resealing in mammalian cells after injury depends on Ca(2+)-dependent fusion of intracellular vesicles with the plasma membrane. When cells are wounded twice, the subsequent resealing is generally faster. Physiological and biochemical studies have shown the initiation of two different repair signaling pathways, which are termed facilitated and potentiated responses. The facilitated response is dependent on the generation and recruitment of new vesicles, whereas the potentiated response is not. Here, we report that the two responses can be differentially defined molecularly. Using recombinant fragments of synaptobrevin-2 and synaptotagmin C2 domains we were able to dissociate the molecular requirements of vesicle exocytosis for initial membrane resealing and the facilitated and potentiated responses. The initial resealing response was blocked by fragments of synaptobrevin-2 and the C2B domain of synaptotagmin VII. Both the facilitated and potentiated responses were also blocked by the C2B domain of synaptotagmin VII. Although the initial resealing response was not blocked by the C2AB domain of synaptotagmin I or the C2A domain of synaptotagmin VII, recruitment of new vesicles for the facilitated response was inhibited. We also used Ca2+ binding mutant studies to show that the effects of synaptotagmins on membrane resealing are Ca(2+)-dependent. The pattern of inhibition by synaptotagmin C2 fragments that we observed cannot be used to specify a vesicle compartment, such as lysosomes, in membrane repair.
MeSH term(s)	Animals ; Calcium/metabolism ; Calcium/pharmacology ; Calcium-Binding Proteins/antagonists & inhibitors ; Calcium-Binding Proteins/chemistry ; Calcium-Binding Proteins/pharmacology ; Cell Membrane/drug effects ; Cell Membrane/metabolism ; Cell Membrane/pathology ; Exocytosis/drug effects ; Fibroblasts/cytology ; Kinetics ; Membrane Fusion/drug effects ; Membrane Glycoproteins/antagonists & inhibitors ; Membrane Glycoproteins/chemistry ; Membrane Glycoproteins/pharmacology ; Membrane Proteins/chemistry ; Membrane Proteins/pharmacology ; Mice ; NIH 3T3 Cells ; Nerve Tissue Proteins/antagonists & inhibitors ; Nerve Tissue Proteins/chemistry ; Nerve Tissue Proteins/pharmacology ; Peptide Fragments/chemistry ; Peptide Fragments/pharmacology ; R-SNARE Proteins ; Swiss 3T3 Cells ; Synaptotagmin I ; Synaptotagmins
Chemical Substances	Calcium-Binding Proteins ; Membrane Glycoproteins ; Membrane Proteins ; Nerve Tissue Proteins ; Peptide Fragments ; R-SNARE Proteins ; Synaptotagmin I ; Syt1 protein, mouse ; Syt7 protein, mouse ; Synaptotagmins (134193-27-4) ; Calcium (SY7Q814VUP)
Language	English
Publishing date	2004-11-09
Publishing country	United States
Document type	Journal Article ; Research Support, U.S. Gov't, P.H.S.
ZDB-ID	2997-x
ISSN	1083-351X ; 0021-9258
ISSN (online)	1083-351X
ISSN	0021-9258
DOI	10.1074/jbc.M410136200
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Article ; Online: In vitro detection and quantification of botulinum neurotoxin type e activity in avian blood.

Piazza, Timothy M / Blehert, David S / Dunning, F Mark / Berlowski-Zier, Brenda M / Zeytin, Füsûn N / Samuel, Michael D / Tucker, Ward C

Applied and environmental microbiology

2011 Volume 77, Issue 21, Page(s) 7815–7822

Abstract: Botulinum neurotoxin serotype E (BoNT/E) outbreaks in the Great Lakes region cause large annual avian mortality events, with an estimated 17,000 bird deaths reported in 2007 alone. During an outbreak investigation, blood collected from bird carcasses is ... ...

Abstract	Botulinum neurotoxin serotype E (BoNT/E) outbreaks in the Great Lakes region cause large annual avian mortality events, with an estimated 17,000 bird deaths reported in 2007 alone. During an outbreak investigation, blood collected from bird carcasses is tested for the presence of BoNT/E using the mouse lethality assay. While sensitive, this method is labor-intensive and low throughput and can take up to 7 days to complete. We developed a rapid and sensitive in vitro assay, the BoTest Matrix E assay, that combines immunoprecipitation with high-affinity endopeptidase activity detection by Förster resonance energy transfer (FRET) to rapidly quantify BoNT/E activity in avian blood with detection limits comparable to those of the mouse lethality assay. On the basis of the analysis of archived blood samples (n = 87) collected from bird carcasses during avian mortality investigations, BoTest Matrix E detected picomolar quantities of BoNT/E following a 2-h incubation and femtomolar quantities of BoNT/E following extended incubation (24 h) with 100% diagnostic specificity and 91% diagnostic sensitivity.
MeSH term(s)	Animals ; Bird Diseases/diagnosis ; Birds ; Blood Chemical Analysis/methods ; Botulinum Toxins/blood ; Botulism/diagnosis ; Botulism/veterinary ; Chemistry Techniques, Analytical/methods ; Fluorescence Resonance Energy Transfer/methods ; Great Lakes Region ; Immunoprecipitation/methods ; Sensitivity and Specificity ; Time Factors
Chemical Substances	Botulinum Toxins (EC 3.4.24.69) ; botulinum toxin type E (T579M564JY)
Language	English
Publishing date	2011-09-09
Publishing country	United States
Document type	Evaluation Study ; Journal Article ; Research Support, Non-U.S. Gov't ; Research Support, U.S. Gov't, Non-P.H.S.
ZDB-ID	223011-2
ISSN	1098-5336 ; 0099-2240
ISSN (online)	1098-5336
ISSN	0099-2240
DOI	10.1128/AEM.06165-11
Database	MEDical Literature Analysis and Retrieval System OnLINE

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