Applications of project in healthcare diagnostics and therapy
The general category we envision our project impacting is that of Point of Care Serology-based assays for diagnostics in healthcare. This was developed through feedback from the community (see Human Practices). The prominent real-world example of such an assay that has been in use is Lateral Flow Tests/Rapid Antigen Tests during the COVID-19 pandemic. This is where we have mainly focussed our attention.
Our real world therapeutic implementation of our project would be used to anticipate novel variants by creating a catalogue of shuffled nanobodies. By fusing the shuffled nanobodies to CBD binding domains, the hope is that we can more quickly create new rapid antigen testing for new variants.
The general design of the biosensor involves a cellulose test strip with mRFP as a visual indicator. It will include mRFP-nb(fuGFP) added to the start of the test strip or in sample buffer, a test line of immobilised CBD-nb(fuGFP), and a control line of immobilised CBD-nbRFP (Figure 1). When fuGFP is present in the sample, it will bind to the primary antibody, move along the cellulose and result in red fluorescence on the test and control line.
By creating a collection of CBD-bound fluoroproteins, we hope that multiplex rapid antigen testing may be a future implementation of our project. By developing multiple nanobodies for different variants of a disease, or various infectious agents, we could create a rapid antigen test capable of differentiating between different variants or pathogens with shared symptoms.
One of the major disadvantages for at-home rapid antigen testing is a lack of epidemiological data on specific variants and strains within the population so creating a single test that could differentiate between variants or strains with similar presentations would be a significant improvement to the utility of rapid antigen testing.
While the recent SARS-CoV-2 pandemic highlighted the demand and utility for rapid antigen testing, our framework was designed to have broad utility that may be used for novel disease outbreaks.
Our methodology for using CBDs and recombinant nanobodies hoped to decrease the manufacturing costs for producing rapid antigen tests to increase the availability and access of these to the general population.
Applications of our project in research
We propose that cellulose binding domains could have utility as a cost effective protein purification method, using our favourite, CBDcipA as a fusion tag which may be implemented in research by biochemistry and protein research labs who operate on lower budgets or are looking to decrease expenditure.
Nanobodies are regularly used in structural biology to characterise protein structure and selectively block protein-protein interaction sites for use in understanding biochemical systems (Rivera-Calzada et al. 2013; Westfield et al. 2011). While selecting for specific conformational epitopes for these implementations would likely be inefficient with our current screening procedure, adapting screening pressures to best suit the specific antigen and epitope binding site for the desired nanobody would be a natural part of this or any similar implementation.
References
Hadfield, J., Megill, C., Bell, S.M., Huddleston, J., Potter, B., Callender, C., Sagulenko, P., Bedford, T. and Neher, R.A., 2018. Nextstrain: real-time tracking of pathogen evolution. Bioinformatics, 34(23), pp.4121-4123.
Pardon, E., Laeremans, T., Triest, S., Rasmussen, S.G.F., Wohlkönig, A., Ruf, A., Muyldermans, S., Hol, W.G.J., Kobilka, B.K., and Steyaert, J., 2014. A general protocol for the generation of Nanobodies for structural biology. Nature Protocols, 9(3), pp.74-693.
Rivera‐Calzada, A., Fronzes, R., Savva, C.G., Chandran, V., Lian, P.W., Laeremans, T., Pardon, E., Steyaert, J., Remaut, H., Waksman, G. and Orlova, E.V., 2013. Structure of a bacterial type IV secretion core complex at subnanometre resolution. The EMBO journal, 32(8), pp.1195-1204.
Westfield, G.H., Rasmussen, S.G., Su, M., Dutta, S., DeVree, B.T., Chung, K.Y., Calinski, D., Velez-Ruiz, G., Oleskie, A.N., Pardon, E. and Chae, P.S., 2011. Structural flexibility of the Gαs α-helical domain in the β2-adrenoceptor Gs complex. Proceedings of the National Academy of Sciences, 108(38), pp.16086-16091.