Abstract 170: Development of a Novel Protein Nanobody Detection System for Serum Soluble Lectin-like Oxidised Low Density Lipoprotein Receptor-1

Abstract

Introduction: The Lectin-like Oxidised Low Density Lipoprotein (OxLDL) Receptor 1 (LOX-1) is a scavenger receptor found on vascular endothelial cells. LOX-1 is proteolysed at the cell surface and its soluble fragments (sLOX-1) are shed into the extracellular space. Clinical studies have demonstrated a link between serum sLOX-1 concentration and cardiovascular disease. Current technologies for the detection of sLOX-1 rely on costly monoclonal antibodies. Adhirons are antibody mimetics which can be directed against a wide range of molecules. We aimed to investigate whether Adhirons isolated against human sLOX-1 could be used to measure its concentration in solution. Methods: BL21 Star DE3 Escheriae Coli underwent transformation with plasmids for 5 different cysteine-tagged Adhirons. Protein expression was induced and, following bacterial lysis, these were purified using nickel-agarose columns. The binding of Adhirons to the extracellular domain of immobilised, recombinant human LOX-1 was tested by modified direct enzyme linked immunosorbance assay (ELISA). Adhiron-based, sandwich ELISA and Chemiluminescence Enzyme Immunoassay (CLEIA) were developed to detect sLOX-1 in solution. The application of these techniques in the detection of LOX-1 in biological buffers was tested. Results: Direct ELISA demonstrated a limit of detection (LOD) of 5 nanograms per millilitre. A complimentary binding pair of Adhirons (H 1 capture, A 1 detection and vice versa ) was identified. Colorimetry-based ELISA using these Adhirons detected sLOX-1 in phosphate buffered saline at an LOD of 150 nanograms per millilitre. CLEIA enabled a detection limit of 50 nanograms per millilitre, these results were reproduced in the presence of protein blockers (Bovine Serum Albumin, Casein). Conclusion: These experiments demonstrate proof of concept for the use of Adhirons as a viable platform for the detection of sLOX-1 in solution. Further refinement and optimisation is needed for the detection sLOX-1 levels in human blood samples (500 - 3000 picogram per millilitre concentration) in order to relate these to human disease.