US 12,422,428 B2
Compositions and methods for cell-based assays
Dhammika Atapattu, Madison, WI (US); and Ward C. Tucker, Monona, WI (US)
Assigned to BIOMADISON, INC., Del Mar, CA (US)
Filed by BIOMADISON, INC., Del Mar, CA (US)
Filed on Apr. 13, 2022, as Appl. No. 17/719,742.
Application 17/719,742 is a continuation in part of application No. 15/817,065, filed on Nov. 17, 2017, granted, now 11,325,954.
Application 15/817,065 is a continuation in part of application No. 15/018,621, filed on Feb. 8, 2016, granted, now 10,100,094, issued on Oct. 16, 2018.
Application 15/018,621 is a continuation of application No. 13/485,537, filed on May 31, 2012, granted, now 9,274,121, issued on Mar. 1, 2016.
Claims priority of provisional application 61/492,237, filed on Jun. 1, 2011.
Prior Publication US 2022/0260555 A1, Aug. 18, 2022
This patent is subject to a terminal disclaimer.
Int. Cl. G01N 33/542 (2006.01); C07K 14/00 (2006.01); C12N 15/85 (2006.01); G01N 33/58 (2006.01)
CPC G01N 33/542 (2013.01) [C07K 14/00 (2013.01); C12N 15/85 (2013.01); G01N 33/582 (2013.01); G01N 2333/33 (2013.01)] 7 Claims
 
1. A method of identifying a reporting construct suitable for use in a cell-based assay for characterization of a botulinum neurotoxin, comprising:
transforming a cell susceptible to intoxication with the botulinum neurotoxin with an expression vector encoding a hybrid protein comprising (a) a membrane binding domain, (b) a reporter-containing domain comprising a degradable reporter that is degraded in the cell's cytosol within a time course of the cell-based assay, (c) a linking region comprising a soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor (SNARE) or fragment thereof comprising a cleavage site that interacts with the botulinum toxin in a manner that cleaves the reporter-containing domain from remainder of the hybrid protein and is interposed between the membrane binding domain and the reporter-containing domain, and (d) a control domain comprising a control reporter domain coupled to the membrane binding domain;
characterizing targeting of a plasma membrane of the cell by the reporting construct by obtaining an image of the cell by fluorescence microscopy;
characterizing background fluorescence intensity and fluorescence intensity of the cell;
contacting a plurality of the cells with the botulinum neurotoxin at a plurality of concentrations, wherein each of the plurality of concentrations us from 10 pM to 30 nM;
after contacting the plurality of the cells with the botulinum neurotoxin, obtaining a first fluorescence measurement at a first wavelength corresponding to an emission wavelength of the degradable reporter and a second fluorescence measurement at a second wavelength corresponding to an emission wavelength of the control reporter; and
plotting an emission ratio comprising the first fluorescence measurement divided by the second fluorescence measurement against concentration of the botulinum neurotoxin,
wherein plasma membrane targeting comprises uniform fluorescence over the plasma membrane and wherein fluorescence intensity of the cell is at least twice background intensity.