Invited Speaker

Sensitive and High Throughput Multiplexed Immunoassays for Biomarker Discovery in Biobanked Samples using Proximity Ligation Assays and qPCR
Simon Fredriksson
Sweden

Methods consuming small sample amounts and allow for sensitive screening of large numbers of putativebiomarkers indicative of disease are required to advance biomarker discovery and validation. We present multiplex Proximity Ligation Assays (PLA) were protein analytes are converted to unique DNA amplicons and subsequently detected by high throughput quantitative PCR. These multiplexed profiling panels solve many analytical problems found in biomarker verification, such as sensitivity (low femto Molar), multiplexing, throughput, and sample consumption (one micro Litre of plasma). As the assay can use either matched monoclonal antibody pairs or even single batches of polyclonal antibodies both with high detection specificity, a wider range of potential biomarkers can be targeted compared to conventional immunoassay methods. In another incarnation of PLA performed in situ, the assay can precisely quantify proteins and protein interactions in FFPE tissue providing localized data through subjective single molecule counting and fluorescence microscopy. The ability to quantify protein modifications and protein-protein interactions in situ using pairs of primary target specific antibodies opens a new realm of biomarker opportunities particularly with in drug response markers.

Olink Bioscience in Uppsala is coordinating a EU-funded effort to expand our capabilities to profile hundreds of putative plasma biomarkers in minute amounts of biobanked samples in a project called PROACTIVE. A high throughput and multiplexed platform for biomarker research will be built based on the proximity ligation assay (PLA). Proof of principle data has recently been published for multiplexed PLA as a profiling tool for low abundance cancer biomarkers. The enabling power of this technology will make this project comprise the largest immunoassay based effort to find novel biomarkers for colorectal cancer ever performed in a single sample collection. Once in place, the technology platform will become a valuable resource for the diagnostics and pharmaceutical communities in need of capable tools to find novel biomarkers and biomarker panels.