Evaluate P-EIM technology for membrane protein binding affinities measurement

Project: Research project

Project Details

Description

Evaluate P-EIM technology for membrane protein binding affinities measurement Evaluate P-EIM technology for membrane protein binding affinities measurement Research Plan 1. Project 1: Interaction of anti-EGFR antibody with EGFR on live cell surface 1. Background and rationale: The binding kinetics of an antibody to its cell surface target is often an important parameter underlying in vivo efficacy of an antibody therapeutic. Epidermal growth factor receptor (EGFR) is a membrane protein significantly overexpressed in colorectal cancer cells. Although surface plasmon resonance (SPR) can be employed to determine binding kinetics between EGFR and its antibodies, there are two limitations of the SPR measurements that have been generated to date using protein chips. First, the extracellular domain of EGFR, rather than intact EGFR receptor, was used due to the challenge of isolating and purifying intact membrane-bound receptor. Secondly, the truncated protein is immobilized onto a sensor chip, such that the orientation and configuration are not guaranteed, perhaps even unlikely, to replicate that of EGFR as embedded in a live cell membrane. The current project is intended to avoid these limitations and provide valuable kinetic data on the antibody-membrane receptor interaction in a live cell environment. The Center for Bioelectronics and Biosensors at Arizona State University has established plasmonicbased electrochemical impedance microscopy (P-EIM).1-8 This new analytical chemistry method can perform simultaneous plasmonic, impedance and fluorescence imaging, thus making it possible to combine the strengths of both label-based and label-free techniques in one system. P-EIM capable of acquiring images of a single live cell.5 Binding events taking place on the target cell that increase the local mass density can be quantitatively detected by P-EIM. As a proof of concept, this system has been used to measure the binding kinetics of lectin to glycoproteins in the intact cell surface.8 In addition to providing kinetic data on EGFR-antibody binding, the proposed research project will further efforts to establish livecell P-EIM as a standard platform for determining biologically-relevant measurement of antibody affinity to membrane receptors, a powerful alternative to the present protein chip-based SPR measurements. The Kinetic Exclusion Assay (KinExA) platform from Sapidyne Instruments will be used as an alternative method for measuring equilibrium affinity of the EGFR-antibody binding to validate the SPR data
StatusFinished
Effective start/end date11/13/125/12/15

Funding

  • INDUSTRY: Domestic Company: $50,000.00

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