High-sensitivity Protein Detection Using Immuno-PCR Phage Construct
Litvinov, Julia 1974-
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Advances in the identification of novel biomarkers of cancer and infection are creating an increasing need for detection systems with superior sensitivity. Early detection and identification of infections speeds effective treatment, and cancers for which early diagnostic methods are available have distinctly higher 5-year survival rates than those for which early detection is lacking. Enzyme-linked immunosorbent assays (ELISAs) provide a high level of versatility, but often lack sensitivity for targets in the sub-picomolar concentration range. The current work describes the development of an ultrasensitive immuno-PCR protein detection assay. Bacteriophage biotin-coupled to off-the-shelf antibodies and quantified by real-time PCR are used as the reporter in the assay to detect the Vascular Endothelial Growth Factor (VEGF) in solution. VEGF promotes angiogenesis, is responsible for cell migration, inhibits cell death, and appears at elevated levels in cancer. The general experimental set-up can be modified based on top-down and bottom-up approaches. The top-down approach involves capturing VEGF on particles functionalized with anti-VEGF antibodies, recognition of VEGF by a monoclonal biotinylated antibody, attachment of NeutrAvidin, and the attachment of biotinylated phage onto the NeutrAvidin. The bottom-up approach involves binding a pre-made conjugate of biotinylated phage/NeutrAvidin/biotinylated antibody to VEGF captured on magnetic beads in a one-step reaction. The experiments are performed in phosphate buffer saline (PBS), 20% serum, and 50% bronchoalveolar lavage (BAL). VEGF can be reproducibly detected at concentrations down to 26 fM in PBS, 50% BAL, and 20% serum. In the second part of this work, VEGF is assayed on the sample-contact component of biosensors, amorphous pinhole-free aluminum oxide (alumina) coated surfaces, which represents an alternative way to detect phage reporters. This protective layer is usually used for insulation purposes in biosensors to protect them from corrosion in liquid, salty environments. Immobilization of biological agents on biosensors is a common practice and was achieved using TESBA–based silane chemistry coupled with protein A/G to properly orient the antibodies on the surface. VEGF is assayed at nano- and picomolar concentrations and the detection limit by ELISA is estimated to be 7.2 pM.