Direct replacement of secondary antibodies by affimer proteins in lateral flow devices

Christian will collaborate with an industry partner to validate the replacement of secondary antibodies in diagnostic lateral flow tests with synthetic proteins, called affimers, developed as part of an NC3Rs-funded studentship. Lateral flow tests use secondary antibodies that are typically produced in a variety of species including goats, sheep and donkeys. This proof-of-concept study alone could lead to the replacement of hundreds of animals if affimer proteins could be applied to lateral flow tests.

This award was made as part of the 2024 non-animal derived product validation grants supported with funding from the Department for Science, Innovation and Technology (DSIT).

Antibodies are the gold standard for affinity reagents in therapy, research and diagnostics. While monoclonal antibodies can be produced by hybridoma cell lines or recombinantly, polyclonal antibodies are still produced almost exclusively in animals, relying on animal immunisation, bleeding and killing of the animals. Therefore, they represent a major challenge for animal welfare. In addition, polyclonal antibodies are subject to batch variations and require costly batch validation each time. Hence, as part of an NC3Rs studentship, we developed animal-free affinity reagents named Affimers as direct replacement of polyclonal secondary antibodies. Both anti-mouse and anti-rabbit Affimer proteins were generated and showed comparable results to secondary antibodies in flow cytometry, ELISA, cell imaging and western blot. We propose to take pre-characterised Affimer proteins to the next level and validate them in diagnostic settings. We will produce anti-mouse and anti-rabbit Affimer proteins, print them onto the nitrocellulose membranes and test them in side-by-side comparison with secondary antibodies in lateral flow devices (LFDs). At the same time, we will also test the reverse approach with immobilised Affimer proteins on gold particles to detect the printed primary antibody. We have selected SureScreen Diagnostics, a leading developer of LFDs in the UK, as our partner. We believe SureScreens' expertise and know-how will accelerate the implementation and dissemination of Affimer proteins in commercial products. Our goal is to provide animal-free, reproducible, easy-to-manufacture and cost saving reagents that have at least equivalent operational sensitivity to existing antibody systems. We aim to enable diagnostic developers to replace secondary antibodies with Affimer reagents without having to change their standard operational protocols (SOPs). For example, SureScreen Diagnostics currently use secondary antibodies in the control lines for many of their LFDs. This alone requires hundreds of mg of secondary antibody per year, which could otherwise be replaced with Affimer proteins. If successful, this project has the potential to save immediately three goats and could also be the starting point for saving of hundreds of animal lives every year in United Kingdom alone.