NC3Rs funded PhD: Affimer – The Next Generation Diagnostic

Animal products are important reagents used in many research settings but little research is being performed to help reduce the number of these reagents in general laboratories. Antibodies are prime examples of such reagents. Polyclonal anti-immunoglobulin G (anti-IgG) secondary antibodies are essential tools in boosting the sensitivity of primary antibodies in a wide range of applications in research and diagnostics. They are established reagents in any molecular biology laboratory around the world. Despite some progress in developing alternative formats of antibodies (recombinant antibodies) their generation relies mainly on animal immunization, bleeding and sacrifice. The ultimate aim of this exciting PhD proposal is the replacement of animal-produced secondary antibodies by novel artificial binding proteins, termed Affimers. Affimers out perform antibodies with a) short development times (10 days), b) robustness including high stability, c) no batch-to-batch variations and d) high expression levels in bacterial expression system. Our group has generated large Affimer phage libraries and screened more than 600 target proteins, peptides and small compounds successfully.
Affimers are now established as versatile and renewable reagents and have been used in numerous high-impact publications such as eLife, PNAS, Molecular Cell, Nature Structural & Molecular Biology, Science Signalling, EMBO and Blood.


Objectives:

(1) Select new highly-specific and cross-reactive Affimer proteins against the constant regions of mouse and rabbit immunoglobulin subclasses IgG1, IgG2 and IgG3 by phage display.
(2) Identify Affimer proteins binding to different epitopes on the constant regions of mouse and rabbit immunoglobulin subclasses
(3) Engineer and produce selected Affimer proteins from Objective 2 as Affimer-Fc fusion proteins using human Fc IgG1 as a scaffold and label them with horseradish peroxidase (HRP) or biotin.
(4) Perform direct side-by-side comparisons of Affimer reagents from Objective 3 with commercial secondary antibodies to determine efficacy in different applications such as ELISA, Immunohistochemistry, cell imaging and flow cytometry.
(5) Generate a single “polyclonal” secondary Affimer reagent able to detect mouse and rabbit IgG1, IgG2 and IgG3 subclasses. Direct side-by-side comparisons with commercial secondary antibodies will be undertaken to determine efficacy.

The PhD student will be trained in diverse techniques such as phage display, gene cloning, recombinant protein production using mammalian and bacterial expression systems, protein purification including affinity purification and Äkta gelfiltration, protein labelling, ELISA, cell imaging, flow cytometry and IHC.