Apr 30, 2026
Coxsackievirus A16 2A protease binding assay for compound screening using grating-coupled interferometry
- Eda Capkin1,2,3,
- Korvus Wang4,3,
- Michael Fairhead4,3,
- Milo Cooper1,2,3,
- Mathew Golding1,2,3,
- Tracy Keates1,2,3,
- Charlotte Chinn1,2,3,
- Peter Marples1,2,3,
- Ali Ebrahim1,2,3,
- Anu V. Chandran1,2,3,
- Cedric Vallee1,2,3,
- Eleanor Williams4,3,
- Lizbé Koekemoer4,3,
- Mary-Ann Xavier1,2,3,
- Warren Thompson1,2,3,
- Jasmin Aschenbrenner1,2,3,
- Frank von Delft4,1,2,5,3
- 1Diamond Light Source;
- 2Research Complex at Harwell;
- 3OpenBind Consortium;
- 4Centre of Medicines Discovery, University of Oxford;
- 5University of Johannesburg
- OpenBind Consortium
Protocol Citation: Eda Capkin, Korvus Wang, Michael Fairhead, Milo Cooper, Mathew Golding, Tracy Keates, Charlotte Chinn, Peter Marples, Ali Ebrahim, Anu V. Chandran, Cedric Vallee, Eleanor Williams, Lizbé Koekemoer, Mary-Ann Xavier, Warren Thompson, Jasmin Aschenbrenner, Frank von Delft 2026. Coxsackievirus A16 2A protease binding assay for compound screening using grating-coupled interferometry. protocols.io https://dx.doi.org/10.17504/protocols.io.kqdg3mn3ql25/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: March 31, 2026
Last Modified: April 30, 2026
Protocol Integer ID: 314192
Keywords: Grating coupled interferometry, Creoptix, Kinetics, binding assay, different protein target, active protein immobilization, protein, streptavidin chip surface, streptavidin chip, binding analysis, Enterovirus, Coxsackievirus, 2A Protease, Coxsackievirus A16, CVA16, Enterovirus A71 2A, Coxsackievirus A16 2A, EV-A71, compounds against coxsackievirus a16, surrogate for the related enterovirus a71, coxsackievirus a16, antiviral platform, binding assay, enabled antiviral platform, related enterovirus a71, cva16 2a protease, active protein immobilisation, different protein target, protein, binding analysis, assay for compound screening, target for openbind, protease, 2a protease
Funders Acknowledgements:
UK Department of Science, Innovation and Technology
Grant ID: G2-SCH-2025-06-16537
Abstract
This protocol aims to measure kinetic parameters (ka, kd, and KD) for compounds against coxsackievirus A16 (CVA16) 2A protease using the Creoptix Wave system. CVA16 2A protease is used as a surrogate for the related enterovirus A71 (EV-A71) 2A protease in the pan-enteroviral drug discovery efforts by our collaborator, the AI-Driven Structure-Enabled Antiviral Platform (ASAP) Discovery Center, and was chosen as the target for OpenBind's first data release package.
Grating-coupled interferometry (GCI) is a label-free technique to measure kinetics and affinity for different targets (proteins, small molecules, fragments, etc.) with enhanced sensitivity. Biotin-tagged CVA16 2A protease was captured on the streptavidin-coated chip surface via biotinylation. This immobilisation technique provides oriented and active protein immobilisation for binding assays and can be applied to different protein targets. Binding analysis was performed with the Repeated Analyte Pulses of Increasing Duration (RAPID) method, which involves the injection of samples at a single concentration with varied association times. Kinetic parameters were obtained from the Creoptix Wave software (v 4.5.18) and the in-house developed, open-source, Python-based tool 'SensoFit' (xchem/sensofit).
Protocol materials
Human Coxsackievirus A16 strain G10 2A protease 2A protease with non-cleavable C-terminal His AviTagaddgeneCatalog #228634
Protein expression and purification
2d
The coxsackievirus A16 2A protease used in this assay was purified and expressed using the plasmid and protocol below.
Protocol
CREATED BY
Michael Fairhead
Note
The construct nomenclature in Addgene has a different viral strain in the title. We are going to request Addgene to review it and change. After this, we are going to republish the protocol with the correct nomenclature.
Human Coxsackievirus A16 strain G10 2A protease 2A protease with non-cleavable C-terminal His AviTagaddgeneCatalog #228634
2d
Immobilisation
2h
The 2A protease was used at a concentration of 20 µg/µL in an immobilisation buffer that was selected based on high protein stability observed in nanoDSF assays. Immobilisation was run with an injection duration of 70 s and a capture threshold of 3000 pg/mm2. Flow cell temperature was maintained at 25 °C throughout the protocol.
EV 2A Immobilisation Buffer: 10 mM HEPES pH 7.0, 100 mM NaCl, 0.005% (v/v) Tween20, 0.5 mM TCEP.
Protocol
CREATED BY
Eda Capkin
2h
Binding assay
2h
Binding assays were performed with RAPID kinetics following the protocol below.
During the assay, the buffer composition was the same as the buffer during the Immobilisation stage, with the addition of 2% (v/v) DMSO. Samples were injected at varied concentrations depending on the response at the first trial, within the range of 0.5 micromolar (µM) to25 micromolar (µM) . The samples were injected for 25 s association and 90 s dissociation at 100 µL/min flow rate.
EV 2A Assay Buffer: 10 mM HEPES pH 7.0, 100 mM NaCl, 0.005% (v/v) Tween20, 0.5 mM TCEP, 2% (v/v) DMSO.
Protocol
CREATED BY
Eda Capkin
2h
Sample volumes were calculated as follows:
For 10 µM final concentration
(100,000 µM * V1) = (10 µM * 200 µL) = 20 nL transfer volume
For 25 µM final concentration
(100,000 µM * V1) = (25 µM * 200 µL) = 50 nL transfer volume
Sample volumes were transferred to 384-well plates.
The samples at 10 micromolar (µM) and 25 micromolar (µM) were directly diluted with 200 μL EV 2A Assay Buffer, while those at lower final volumes underwent an intermediate dilution step.
Data analysis
2h
Check the control and sample binding response on all flow channels (FC1, FC2, FC3, and FC4) and subtract the blank from the active flow channels (FC2-1, FC3-1,
2h
Protocol references
1. Kartal Ö, Andres F, Lai MP, Nehme R, Cottier K. waveRAPID—A Robust Assay for High-Throughput Kinetic Screens with the Creoptix WAVEsystem. SLAS DISCOVERY: Advancing the Science of Drug Discovery. 2021;26(8):995-1003. doi:10.1177/24725552211013827.
Acknowledgements
Diamond Light Source Ltd, Harwell Science and Innovation Campus, Didcot OX11 0QX, UK
Research Complex at Harwell, Harwell Science and Innovation Campus, Didcot OX11 0FA, UK
OpenBind received funding from the UK Department of Science, Innovation and Technology under grant number G2-SCH-2025-06-16537.