(T1030-01-07) Evaluation of Formation of SpyTag Form of Botulinum Toxin A (BoNT/A) by Electrochemiluminescence Sandwich Immunoassay

Purpose: BiTox is a novel genetically engineered form of Botulinum Toxin A, which is being developed as a non-opiate alternative therapy for neuropathic pain. In the BiTox system, BoNT/A heavy chain and light chain can be expressed recombinantly and purified as separate non-toxic proteins. The formation of a SpyTag covalent bond which results in pharmacologically active BiTox molecule, which is capable of both translocating into neurons and cleaving SNAP-25, a component of the SNARE protein complex and an endogenous target of BoNT/A. Evaluation of the kinetics of the SpyTag+SpyCatcher reaction has been monitored via SDS-PAGE and SEC chromatography. An immunoassay which can quantitate the formed product, but not the components, offers greater sensitivity and the ability to measure the formation of product in small volumes required for working with a select toxin.

Methods: An ECL assay was developed with commercially available anti-BoNT/A antibodies

Results: Here we report a ruthenium based electrochemiluminescence sandwich assay which has minimal cross reactivity with the individual components (2.79% cross reaction with SpyCatcher Lc, < 1% cross reactivity with SpyTag Hc), and is capable of quantitating BiTox formation at a lower limit of quantitation (LLOQ) of 5 ng/ml (3µl sample) with analytical recovery within ±20% error and an upper limit of quantitation (ULOQ) of 600 ng/ml.

Conclusion: This ECL immunoassay is suitable for quantitating BiTox from 5-600 ng/ml in the presence of excess Spytag or Spycatcher reactant.

References: Antonina S Mangione 1, Ilona Obara, Maria Maiarú, Sandrine M Geranton, Cristina Tassorelli, Enrico Ferrari, Charlotte Leese, Bazbek Davletov, Stephen P Hunt, Nonparalytic botulinum molecules for the control of pain, Pain, 2016 May; 157(5);1045-1055, doi: 10.1097/j.pain.0000000000000478
Leese C, Bresnahan R, Doran C, Simsek D, Fellows AD, Restani L, Caleo M, Schiavo G, Mavlyutov T, Henke T, Binz T, Davletov B., Duplication of clostridial binding domains for enhanced macromolecular delivery into neurons, Toxicon X,. 2020 Mar;5:100019. doi: 10.1016/j.toxcx.2019.100019


Calibrator Curve of Purified BiTox


Comparison of Formed Product Lots and Reactants

Acknowledgements: B. Davletov and A. Maximov are Principals at Neuresta, INC.