High-quality and high-performance antibodies for multiplex imaging research and diagnosis.

Modern biomedical and clinical research relies on specific detection reagents. These reagents are used to provide information on a particularcomponent present in a biological sample. For historical reasons, antibodies are by far the most widely used class of specific detection reagents.However, differently to other reagents used in research or clinics (e.g.: genomic sequences), most antibodies are undefined at the molecular level.In the absence of alternatives, researchers have reconciled themselves to the inadequate definition and characterization of these traditionalreagents, even in an era when working with oligonucleotides, genes or vectors of unknown sequence is inconceivable. Progress in research relieson reproducibility, the ability to repeat experiments with reagents identical to those used in publications is an essential part of that system; a goalthat is impossible with today’s commercial antibodies. When researchers use antibodies, they make assumptions about antibody recognitionproperties, even though recent publications indicate that many commercial antibodies do not function as advertised. Under the most optimisticscenarios, no more than 50% of antibodies actually recognize their targets specifically. We propose here to create a tiered high throughput recombinant renewable antibody selection pipeline that will harness the advantages of in vitroantibody selection to select high quality validated antibodies against proteins of interest rapidly and cheaply. We pioneered the use of high throughput phage display selection that now we can combine with yeast display to select different antibodies againsttargets of interest. This approach combines the advantages of selecting from enormous phage antibody libraries (>10exp10 clones) in volumescompatible with microtiter plates, with the subsequent ability to precisely define individual selections using fluorescence activated cell sorting(FACS) of derivative yeast libraries. Furthermore, it gives the possibility to modulate selection conditions in order to drive the selection towardsantibodies working in specific assay conditions. Our idea is to use the selected antibodies to build a “diagnostic panel” for Hyperion Imaging System. This is a system designed to enable highlymultiplexed immunohistochemistry (IHC) to provide a comprehensive view of the tissue microenvironment, making it possible to perform in-depthcharacterization of protein biomarkers, cellular composition and signaling. Our focus will specifically address some unmeet diagnostic needs in two well-defined model diseases, HCC and glioblastoma, for which somespecific detection reagents are yet missing to perform multiplexing analyzes. The combination of high-quality reagents and the use of mass cytometry is expected to advance the state of the art in high-multiplex imaging forprecision medicine and diagnostics.