# US 10,768,178 B2 · Compositions and methods for improved cell-based botulinum neurotoxin assays

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- Number: US 10,768,178 B2
- Document ID: US10768178B2
- Status: Granted
- Kind: B2
- Application number: 16/246,201
- Filed: 2019-01-11
- Published: 2019-05-16
- Granted: 2020-09-08
- Earliest claimed: 2013-08-09
- Assignees: BioMadison, Inc.
- Applicants: Biomadison, Inc.
- Inventors: Ward C. Tucker; Timothy M. Piazza
- Web page: https://patentgrep.com/patent/US10768178B2

## CPC

- G01N 33/56911: {Bacteria}
- A47C 15/006: {Floating seats}
- C12Q 1/37: involving peptidase or proteinase
- C12Q 1/6897: involving reporter genes operably linked to promoters
- G01N 21/6408: {with measurement of decay time, time resolved fluorescence}
- G01N 21/6428: {Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" (in vivo A61B5/00; immunoassay G01N33/53)}
- G01N 2333/33: from Clostridium (G)
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- A47C: CHAIRS; SOFAS; BEDS
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES

## 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.

## Claims

### Claim 1

A method of increasing sensitivity of a cell based assay for Botulinum neurotoxin, comprising:
(i) providing, in a media having a sub-physiological osmolarity and a temperature of at least 39° C., a transfected cell comprising a reporting peptide cleavable by Botulinum neurotoxin;
(ii) contacting the cell with Botulinum neurotoxin; and
(iii) measuring a cleavage product derived from the reporting peptide,
thereby providing a synergistic decrease in EC50 for Botulinum neurotoxin relative to the cell-based assay performed at physiological temperature and physiological osmolarity.

### Claim 2 (depends on 1)

The method of claim 1, wherein the reporting peptide comprises:
a terminus comprising a reporter-containing portion, wherein the reporter-containing portion exhibits a signal; and,
a cleavage site that interacts with the Botulinum neurotoxin in a manner that produces a cleavage of the reporter-containing portion from a remainder of the reporting peptide, wherein measuring is performed by monitoring changes in the signal.

### Claim 3 (depends on 1)

The method of claim 1, wherein the media has an osmolarity of at least 250 mOsm.

### Claim 4 (depends on 1)

The method of claim 1, wherein the EC50 for Botulinum neurotoxin is decreased by at least a factor of three relative to the cell-based assay performed at physiological temperature and osmolarity.

### Claim 5 (depends on 1)

The method of claim 1, wherein the Botulinum neurotoxin is a recombinant toxin.

### Claim 6 (depends on 5)

The method of claim 5, wherein the recombinant toxin is directed to a non-neuronal cell.

### Claim 7 (depends on 1)

The method of claim 1, wherein the transfected cell is a non-neuronal cell.