Abstract
INTRODUCTION: Assessing the specificity of protein binders is an essential first step in protein biomarker assay development. Affimers are novel protein binders and can potentially replace antibodies in multiple protein capture-based assays. Affimers are selected for their high specificity against the target protein and have benefits over antibodies like batch-to-batch reproducibility and are stable across a wide range of chemical conditions. Here we mimicked a typical initial screening of affimers and commercially available monoclonal antibodies against two non-related proteins, IL-37b and proinsulin, to assess the potential of affimers as alternative to antibodies. METHODS: Binding specificity of anti-IL-37b and anti-proinsulin affimers and antibodies was investigated via magnetic bead-based capture of their recombinant protein targets in human plasma. Captured proteins were analyzed using SDS-PAGE, Coomassie blue staining, Western blotting and LC-MS/MS-based proteomics. RESULTS: All affimers and antibodies were able to bind their target protein in human plasma. Gel and LC-MS/MS analysis showed that both affimer and antibody-based captures resulted in co-purified background proteins. However, affimer-based captures showed the highest relative enrichment of IL-37b and proinsulin. CONCLUSIONS: For both proteins tested, affimers show higher specificity in purifying their target proteins from human plasma compared to monoclonal antibodies. These results indicate that affimers are promising antibody-replacement tools for protein biomarker assay development.
