Technological Advancements in Affinity Proteomics : From planar antibody microarrays towards a solution-based platform

Abstract: Abstract: Proteomics has the potential to deliver disease-associated biomarkers that could provide diagnostic, prognostic and predictive information to enable precision medicine. Affinity proteomics, most commonly based on antibodies and their ability to specifically capture target proteins, has emerged has a valuable tool in biomarker discovery. Our group has developed a recombinant antibody microarray platform that can be used for protein expression profiling of serum samples to define multiplex biomarker signatures. This thesis is focused on antibody engineering and assay development to further improve the current microarray platform and also present proof-of-concept for a novel solution-based platform.In Paper I we evaluated the novel detection reagent PID in search for increased signal-to-noise ratio and improved sensitivity of the microarray assay. PID is a fluorophore-packed nanoparticle and was here used to replace the currently employed single fluorophore molecule. The result showed that it was possible to use PID as a detection reagent in our assay and even higher signals were achieved, although accompanied by a heterogeneous background that will require further optimization.In Paper II and III we explored the Dock’n’Flash method for site-specific conjugation to enable oriented immobilization or functionalization of scFvs. Immobilizing the scFvs in an oriented configuration on the slide could lead to increased sensitivity and performance of the microarray assay. Functionalization could enable novel scFv applications. The scFvs were equipped with the unnatural amino acid pBpa and photocrosslinked to beta-cyclodextrin on a coated slide or in solution. Proof-of-concept was demonstrated for one scFv in Paper II and the study was expanded and the pBpa position optimized in Paper III. In Paper IV we sought to overcome some inherent limitations associated with planar microarrays for global serum profiling by developing the solution-based MIAS platform. In MIAS, proteins were displayed on beads and quantified via DNA-barcoded scFvs using next generation sequencing (NGS). Sortase A was used to site-specifically conjugate the oligonucleotide barcode to scFvs. Proof-of-concept for the assay steps was demonstrated using barcoded scFvs targeting three different serum proteins.In summary, the work presented in this thesis can be used to improve the performance of our current antibody microarray platform and also provides the first steps towards a novel solution-based platform. This could in turn enable improved development of disease-associated protein biomarkers.