title: Development of a Highly Sensitive Cell-Based Assay for Detecting Botulinum Neurotoxin Type A through Neural Culture Media Optimization.
abstract: Botulinum neurotoxin (BoNT) is the most lethal naturally produced neurotoxin. Due to the extreme toxicity, BoNTs are implicated in bioterrorism, while the specific mechanism of action and long-lasting effect was found to be medically applicable in treating various neurological disorders. Therefore, for both public and patient safety, a highly sensitive, physiologic, and specific assay is needed. In this paper, we show a method for achieving a highly sensitive cell-based assay for BoNT/A detection using the motor neuron-like continuous cell line NG108-15. To achieve high sensitivity, we performed a media optimization study evaluating three commercially available neural supplements in combination with retinoic acid, purmorphamine, transforming growth factor β1 (TGFβ1), and ganglioside GT1b. We found nonlinear combinatorial effects on BoNT/A detection sensitivity, achieving an EC50 of 7.4 U ± 1.5 SD (or ~7.9 pM). The achieved detection sensitivity is comparable to that of assays that used primary and stem cell-derived neurons as well as the mouse lethality assay.
authors.affiliation: Department of Biomedical Engineering, University of Wisconsin, Madison, WI, USA Wisconsin Institute for Medical Research and University of Wisconsin Carbone Cancer Center, Madison, WI, USA.
authors.forename: Won S
authors.initials: WS
authors.lastname: Hong
authors.affiliation: Department of Biomedical Engineering, University of Wisconsin, Madison, WI, USA Wisconsin Institute for Medical Research and University of Wisconsin Carbone Cancer Center, Madison, WI, USA.
authors.forename: Hannah M
authors.initials: HM
authors.lastname: Pezzi
authors.affiliation: Department of Biomedical Engineering, University of Wisconsin, Madison, WI, USA.
authors.forename: Andrea R
authors.initials: AR
authors.lastname: Schuster
authors.affiliation: Department of Biomedical Engineering, University of Wisconsin, Madison, WI, USA Wisconsin Institute for Medical Research and University of Wisconsin Carbone Cancer Center, Madison, WI, USA.
authors.forename: Scott M
authors.initials: SM
authors.lastname: Berry
authors.affiliation: Department of Biomedical Engineering, University of Wisconsin, Madison, WI, USA Wisconsin Institute for Medical Research and University of Wisconsin Carbone Cancer Center, Madison, WI, USA.
authors.forename: Kyung E
authors.initials: KE
authors.lastname: Sung
authors.affiliation: Department of Biomedical Engineering, University of Wisconsin, Madison, WI, USA Wisconsin Institute for Medical Research and University of Wisconsin Carbone Cancer Center, Madison, WI, USA djbeebe@wisc.edu.
authors.forename: David J
authors.initials: DJ
authors.lastname: Beebe
mesh_terms: D000818:Animals; D001681:Biological Assay / Q000379:methods*; D019274:Botulinum Toxins, Type A / Q000633:toxicity*; D045744:Cell Line, Tumor; D003470:Culture Media / Q000378:metabolism*; D053595:Embryonic Stem Cells / Q000187:drug effects; D005732:Gangliosides / Q000378:metabolism; D051379:Mice; D009025:Morpholines / Q000378:metabolism; D009046:Motor Neurons / Q000187:drug effects*; D009498:Neurotoxins / Q000633:toxicity*; D011687:Purines / Q000378:metabolism; D051381:Rats; D012680:Sensitivity and Specificity; D053773:Transforming Growth Factor beta1 / Q000378:metabolism; D014212:Tretinoin / Q000378:metabolism
chemical_list: D003470:Culture Media; D005732:Gangliosides; D009025:Morpholines; D009498:Neurotoxins; D011687:Purines; D053773:Transforming Growth Factor beta1; D014212:Tretinoin; C016481:trisialoganglioside GT1; D019274:Botulinum Toxins, Type A; C470893:purmorphamine
keywords: botulinum neurotoxin; cell-based assays; media optimization; toxicology