Biomadison, Inc.

Timothy M Piazza

Ward C Tucker

Francis Mark Dunning

Compositions and methods for improved cell-based methods of characterizing botulinum neurotoxins are provided. Cells utilized in these methods include a reporting construct that is cleaved following uptake and processing of botulinum neurotoxin by the cell, resulting in proteolysis of the portion of the reporting construct that is released following cleavage. The released portion includes a fluorophore and amino acid substitutions or sequences that enhance the rate of proteolysis. A pair of reporting constructs can be utilized in which one member of the pair is modified to resist cleavage by the botulinum neurotoxin while co-localizing with the remaining member of the pair.

Reporting construct with synaptobrevin based moiety

Assay with synaptobrevin based moiety

Methods for increasing specific uptake of aneurotoxin are provided. Specific neurotoxin uptake by cells capable of being intoxicated byneurotoxin is enhanced by increasing temperature from about 37&#xb0; C. to up to about 41&#xb0; C., as indicated by a decrease in the ECfound for cells so treated. The effect requires the presence of both heavy and light chains of theneurotoxin, and is serotype selective.

Methods for improving uptake ofneurotoxin

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 ECof the cell-based assay relative to an analogous cell-based assay performed at physiological osmolarity and temperature.

Compositions and methods for improved cell-based botulinum neurotoxin assays

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 F&#xf6;rster 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.

Sensitized cultured cells for botulinum toxin characterization

Methods and compositions are provided where a transfected cell that produces a hybrid protein with a reporter-containing portion and atoxin cleavage site is contacted with atoxin 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 atoxin are also described.

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McFarland, WI, United States of America

Timothy M Piazza