Abstract: Compositions and methods are disclosed that provide a rapid, sensitive, and accurate cell-based assay for enzyme activity, particularly for enzyme activities associated with botulinum toxins. A cell is provided that expresses a construct that includes an anchor region, a cleavage site, and a reporting region having two or more identical reporter peptides. Enzymatic activity at the cleavage site releases the reporter region into the cytosol, where multiple degradation events occur. The observed change in the signal is proportional to the enzymatic activity.

Abstract: Vesicles that incorporate reporting constructs for characterizing Botulinum neurotoxin protease activity and suitable for use in an assay are provided. The reporting constructs are a pair of recombinant hybrid proteins that act in concert. The reporting constructs are a pair of recombinant hybrid proteins that act in concert, and that include a Botulinum neurotoxin protease recognition and cleavage sequence positioned to release a fluorophore upon cleavage.

Abstract: Compositions and methods are described in which a primary detergent or surfactant in an aqueous solution is removed by the addition of a secondary detergent or surfactant in concentrations that exceed the critical micellar concentration (CMC) of the secondary detergent or surfactant. These compositions and methods are particularly applicable to protein-containing solutions. Typical primary detergents/surfactants include polysorbate 20, polysorbate 80, and Triton X-100. Suitable secondary detergents or surfactants can be ionic, nonionic, or zwitterionic. Typical secondary detergents/surfactants include, but are not limited to, galactoside detergents (e.g. octyl-?-galactoside), glucamide detergents (e.g. MEGA 8, MEGA 9, MEGA 10), cholamide detergents (e.g. CHAPS, CHAPSO, BIGCHAPS), and sulfobetaine detergents (such as sulfobetaine 3-10).

Abstract: Compositions and methods for determining the efficacy and/or potency of a vaccine preparation are described herein. Splenocytes from immunized animals are isolated and frozen. Upon thawing aliquots these cells are activated by exposure to a series of dilutions of q vaccine preparation being tested and a series of dilutions of a reference vaccine with known characteristics. Cells secreting immunogen-specific antibody and cells secreting nonspecific antibody are enumerated, as is the amount of immunogen-specific and nonspecific antibody produced. Comparison between the results from the vaccine preparations provides a measure of relative vaccine efficacy and/or potency.

Abstract: A protease directed to a non-neuronal SNARE protein is described. The protease is produced by selective mutation of a botulinum neurotoxin light chain, and is characterized utilizing a reporting construct that includes all or part of the non-neuronal SNARE protein. Such a protease has utility in the treatment of diseases associated with hypersecretion, where the hypersecretion is mediated by a non-neuronal SNARE protein.

Abstract: Methods for increasing specific uptake of a Botulinum neurotoxin are provided. Specific neurotoxin uptake by cells capable of being intoxicated by Botulinum neurotoxin is enhanced by increasing temperature from about 37° C. to up to about 41° C., as indicated by a decrease in the EC50 found for cells so treated. The effect requires the presence of both heavy and light chains of the Botulinum neurotoxin, and is serotype selective.

Abstract: Compositions for characterization of botulinum toxin (BoNT) are described that include a genetically modified cell that is transfected with an artificial construct comprising a nucleic acid sequence that encodes for a hybrid protein having (a) a reporter-containing portion chemically coupled to (b) a cleavage site and (c) a control fluorophore. The cleavage site interacts with a BoNT in a manner that cleaves the reporter-containing portion from remainder of the construct. The cleaved portion is destroyed or otherwise degraded by the local environment, and presence of BoNT is evidenced by reduction in signal from the reporter. The cleavage sequence is all or part of a SNARE protein, the cleavable reporter-containing portion is preferably Yellow Fluorescent Protein (YFP), Citrine, Venus, or a YPet protein and the control fluorophore is preferably CFP, mStrawberry, or a mCherry protein.

Abstract: Compositions and methods are provided that reduce the time required for detection of botulinum neurotoxins in cell-based assays. In one aspect an isoquinolynyl compound can be used for this purpose. In such cell-based assays the cell can include an enzyme that facilitates degradation of the reporter significantly faster after the cleavage than before the cleavage, and presence of the Botulinum toxin correlates with reduction of the signal from a baseline signal. The cell can advantageously express both the construct that includes the reporter, and an enzyme that facilitates the degradation.

Abstract: Reporting constructs for characterizing Botulinum neurotoxin protease activity and suitable for use in a cell-based assay are provided. The reporting construct can be a single recombinant hybrid protein or a pair of recombinant hybrid proteins that act in concert. The recombinant hybrid protein(s) include a fluorophore and an N-terminal non-peptide membrane anchoring portion. The recombinant hybrid protein or at least one of a pair of recombinant hybrid proteins that act in concert include a Botulinum neurotoxin protease recognition and cleavage sequence positioned to release a fluorophore upon cleavage.

Abstract: Compositions and methods are provided that improve detection of botulinum neurotoxins in cell-based assays. In one aspect an isoquinolynyl compound can be used to enhance the sensitivity of both Förster resonance energy transfer (FRET) and non-FRET cell-based assays. Osmolarity of the cell culture media can be adjusted to optimize the effect of the compound. In that subject matter an environment cell can include an enzyme that facilitates degradation of the reporter significantly faster after the cleavage than before the cleavage, and presence of the Botulinum toxin correlates with reduction of the signal from a baseline signal. Where the environment is a cell, the cell can advantageously express both the construct that includes the reporter, and an enzyme that facilitates the degradation.

Abstract: Methods for a cell-based assay for Botulinum neurotoxin are provided in which a transfected cell that produces a reporting peptide is contacted with a Botulinum neurotoxin in media having a sub-physiological osmolarity and a temperature that is above physiological temperature. This combination provides an unexpected synergistic effect in reducing the EC50 of the cell-based assay relative to an analogous cell-based assay performed at physiological osmolarity and temperature.

Abstract: Methods for improving the specificity of assays for botulinum neurotoxins are described. Reporting constructs are provided that include cleavable linker sequence with genetically modified recognition and/or cleavage sites. These linker sequences act as a substrate for a botulinum neurotoxin, with cleavage of the reporting construct providing a detectable signal. When first and second neurotoxins have activity with the same substrate protein, mutation and/or deletion of a recognition and/or cleavage site associated with the second neurotoxin improves the specificity of the detectable signal for the first neurotoxin.

Abstract: Compositions and methods for analyzing protease activity, and especially BoNT/B, BoNT/G, BoNT/D, and/or BoNT/F protease activity, using a cell based assay are provided. Cells express at least two recombinant hybrid proteins each of which includes a fluorophore and a membrane anchoring peptide, and at least one of which includes a BoNT protease recognition and cleavage sequence positioned to release a fluorophore upon cleavage. Analysis is performed by monitoring fluorescence following exposure to a BoNT. The fluorophores are positioned so that no useful FRET occurs between them, permitting fluorescence produced by the non-released fluorophore to be used in data normalization.

Abstract: A protease directed to a non-neuronal SNARE protein is described. The protease is produced by selective mutation of a botulinum neurotoxin light chain, and is characterized utilizing a reporting construct that includes all or part of the non-neuronal SNARE protein. Such a protease has utility in the treatment of diseases associated with hypersecretion, where the hypersecretion is mediated by a non-neuronal SNARE protein.

Abstract: Apparatus, systems and methods can provide improved detection of botulinum neurotoxins. In one aspect an isoquinolynyl compound can be used to enhance the sensitivity of both Förster resonance energy transfer (FRET) and non-FRET cell-based assays. In another aspect, non-FRET assays and constructs utilize a reporter that is not coupled with the second fluorophore in a manner that produces significant FRET. In that subject matter an environment cell can include an enzyme that facilitates degradation of the reporter significantly faster after the cleavage than before the cleavage, and presence of the Botulinum toxin correlates with reduction of the signal from a baseline signal. Where the environment is a cell, the cell can advantageously express both the construct that includes the reporter, and an enzyme that facilitates the degradation.

Abstract: Compositions and methods for analyzing intracellular BoNT protease activity, and especially BoNT/B, BoNT/G, BoNT/D, and/or BoNT/F protease activity are provided. Cells express at least two recombinant hybrid proteins each of which includes a fluorophore and a membrane anchoring peptide, and at least one of which includes a BoNT protease recognition and cleavage sequence positioned to release a fluorophore upon cleavage. Analysis is performed by monitoring fluorescence following exposure to a BoNT.

Abstract: Methods and compositions are provided where a transfected cell that produces a hybrid protein with a reporter-containing portion and a botulinum toxin cleavage site is contacted with a botulinum toxin in media having a reduced sodium concentration, thereby increasing the sensitivity. Such media can also be used in combination with elevated cell culture temperatures. Kits that include such media and a botulinum toxin are also described.

Abstract: Compositions for characterization of Botulinum toxin (BoNT) are described that include a genetically modified cell that is transfected with an artificial construct comprising a nucleic acid sequence that encodes for a hybrid protein having (a) a reporter-containing portion chemically coupled to (b) a cleavage site and (c) a control fluorophore. The cleavage site interacts with a BoNT in a manner that cleaves the reporter-containing portion from remainder of the construct. The cleaved portion is destroyed or otherwise degraded by the local environment, and presence of BoNT is evidenced by reduction in signal from the reporter. The cleavage sequence is all or part of a SNARE protein, the cleavable reporter-containing portion is preferably Yellow Fluorescent Protein (YFP), Citrine, Venus, or a YPet protein and the control fluorophore is preferably CFP, mStrawberry, or a mCherry protein.

Abstract: Methods for the identification of inhibitors of botulinum neurotoxins are described. Cells are provided that are genetically engineered to express peptides that act as a substrate for a botulinum neurotoxin and the provide reporting groups. Spacer sequences between the reporting groups serve to optimize energy transfer between the reporting groups. Characterization of the energy transfer-dependent signal prior to and following exposure to a botulinum neurotoxin in the presence of a candidate inhibitor provides a measure of the effectiveness of the candidate inhibitor.

Abstract: A molecular construct comprises a donor label, an acceptor label, a linker peptide disposed between the donor and the acceptor, the linker having a cleavage site sequence, and a spacer between at least one of (a) the donor and the cleavage site sequence and (b) the acceptor and the cleavage site sequence. Preferably, the construct is selected from the group consisting of CFP-(SGLRSRA)(SEQ ID NO. 9)-SNAP-25-(SNS)-YFP, and CFP-(SGLRSRA)(SEQ ID NO. 9)-synaptobrevin-(SNS)-YFP. In preferred embodiments, the linker peptide is a substrate of a botulinum neurotoxin selected from the group consisting of synaptobrevin (VAMP), syntaxin and SNAP-25, or a fragment thereof that can be recognized and cleaved by the botulinum neurotoxin. Advantageously, the spacer increases the electronic coupling between the donor label and the acceptor label relative to a corresponding construct without the spacer.