The capability to perform in vitro, label-free dynamic assays for membrane-bound antigens is a highly desired task, but is rarely achieved using standard commercialized technology such as BIAcoreTM. The problem is compounded for transmembrane proteins such as G protein coupled receptors (GPCR) because proteins in direct contact with a solid substrate, in particular with the gold substrate used in BIAcoreTM, often lose their functionality or denature. The nanopore- sensing architecture proposed here has the unique potential to overcome these challenges, since each nanopore sits on a glass substrate and forms a tiny well to confine the supported lipid membranes, while the surrounding gold film provides surface plasmon resonance effects to dynamically monitor binding of molecules onto the membrane. This proposal will validate these membrane biosensing concepts by characterizing the binding of therapeutic human monoclonal antibodies to candidate antigens. These human IgMs promote remyelination of demyelinated lesions and preserve axons. These are ideal molecules in which to test this system because the IgM antigen binding appears to require an intact membrane environment. A major challenge in moving these reparative IgMs to clinical trial is to understand the kinetics of binding to the cell-surface antigens. Our hypothesis and preliminary data suggests that the mAbs do not bind to a single membrane molecule, but to a signaling complex within lipid micro-domains (lipid rafts) of cells. If this complex is disrupted, mAb binding is eliminated. The IgMs maintain their cell specificity only when bound to intact plasma membranes. Fixation of any kind (methanol, formaldehyde, freezing) destroys the complex membrane antigen. When candidate antigens are presented in isolated form, the IgMs bind non-specifically to all or to none. Therefore, it is important to maintain the cell membrane antigens in their native state to preserve appropriate mAb binding kinetics. A new antigen screening technology is required to study these difficult but critical lipid and carbohydrate molecules of the plasma membrane. Unfortunately, there are no label-free kinetic screening and quantification methods to measure the binding affinity between cell plasma membranes and mAbs. The commercial BIAcore"""""""" instrument - currently the gold standard for measuring binding kinetics - works with purified molecules, primarily proteins, immobilized on a gold film substrate. However, this instrument is not suitable for quantification of interactions between mAbs and cell-surface antigens in their native membrane inserted state. We propose here to use a novel instrument, a nano-LAMP (LAser-illuminated Metallic Pore) array, to quantify the binding kinetics of mAbs to antigens anchored within a cell membrane at a high spatial resolution. We have validated this platform with membrane-free systems and with artificial membranes for binding kinetics measurements. The work proposed here will further optimize the platform by reconstituting oligodendrocytes and neuronal cell membranes on metallic nanopores to measure and quantify their binding affinity with human therapeutic IgMs, to identify candidate antigens. Once developed, this technology will likely prove important in the study of complex molecular interactions and signals transduced by cell receptors. As a future direction, we also propose the possibility of reconstituting free-standing lipid membranes hanging over a free-standing metallic nanopore substrate, incorporating transmembrane proteins such as GPCRs, and demonstrating the feasibility of kinetic sensing with an artificial membrane system that can integrate transmembrane proteins in contact with a buffer solution on both sides.
This proposal is designed to determine whether a nanoporous gold film detection platform can present cell antigens in manner that preserves the correct plasma membrane functionality similar to that of an intact cell. Standard binding assay Surface Plasmon Resonance (SPR) detectors such as BIAcore work well using only a single purified molecule, but cannot model the presentation of multiple molecules within a membrane. We propose to validate the nanopore SPR platform by measuring the interaction of human IgMs with membranes isolated from myelin, oligodendrocytes and neurons. These reparative IgMs clearly bind to cells of the nervous system, but attempts to identify their membrane antigens using conventional binding technology have been inconclusive. If successful, a nanopore based binding detector will be applicable to a wide variety of ligand/antigen binding studies for basic biology and drug discovery.
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