Nanopore Biosensor for Kinetics of Reparative Antibodies on Plasma Membranes

用于质膜上修复抗体动力学的纳米孔生物传感器

基本信息

批准号：
8475616
负责人：
Sang-Hyun Oh
金额：
$ 39.13万
依托单位：
UNIVERSITY OF MINNESOTA
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-06-01 至 2015-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8475616
关键词：
Affinity Antibodies Antigen-Presenting Cells Antigens Architecture Artificial Membranes Autoimmune Process Axon Binding Biological Biological Assay Biology Biosensing Techniques Biosensor Buffers Carbohydrates Cell membrane Cells Clinical Trials Complex Data Detection Environment Film Formaldehyde Freezing Future G-Protein-Coupled Receptors Glass Glycolipids Gold Human Immunoglobulin M In Vitro Integral Membrane Protein Kinetics Label Lasers Lesion Life Ligands Lipid Bilayers Lipids Measurement Measures Mechanics Membrane Membrane Lipids Membrane Microdomains Membrane Proteins Methanol Methods Modeling Molecular Conformation Monitor Monoclonal Antibodies Myelin Nerve Tissue Nervous system structure Neurons Oligodendroglia Proteins Recombinant Immune Globulin Research Resolution Side Signal Transduction Solid Solutions Specificity Structure Supporting Cell Surface Antigens Surface Plasmon Resonance System Technology Testing Therapeutic Time Vesicle Virus Work antigen binding base cellular transduction design detector drug discovery high throughput screening human monoclonal antibodies instrument miniaturize nano nanopore novel public health relevance receptor reconstitution remyelination sample fixation screening standard measure

项目摘要

DESCRIPTION (provided by applicant): 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.

描述（由申请人提供）：对膜结合的抗原进行体外，无标签的动态测定的能力是一项高度期望的任务，但很少使用标准商业化技术（例如Biacoretm）实现。该问题对于跨膜蛋白（例如G蛋白偶联受体（GPCR））而更复杂，因为与固体底物直接接触的蛋白，尤其是与biacoretm中使用的金底物，通常会失去其功能或剥夺。此处提出的纳米孔感应架构具有克服这些挑战的独特潜力，因为每个纳米孔都位于玻璃基板上，并形成了一个微小的良好，以限制受支持的脂质膜，而周围的金膜则提供表面等离子体的谐振效果，以使分子在膜上的结合到膜上。该建议将通过表征治疗性人类单克隆抗体与候选抗原的结合来验证这些膜生物传感概念。这些人类IgM促进了脱髓鞘病变并保存轴突的再髓。这些是测试该系统的理想分子，因为IgM抗原结合似乎需要完整的膜环境。将这些修复性IgM转移到临床试验的主要挑战是了解与细胞表面抗原结合的动力学。我们的假设和初步数据表明，mAb不与单个膜分子结合，而是与细胞脂质微层（脂质筏）内的信号传导复合物结合。如果该复合物被破坏，则消除了mAb结合。仅当与完整的质膜结合时，IgM才能保持其细胞特异性。任何种类的固定（甲醇，甲醛，冷冻）会破坏复杂的膜抗原。当候选抗原以孤立形式呈现时，IgMS与所有或无特异性结合。因此，将细胞膜抗原保持在其天然状态很重要，以保留适当的mAb结合动力学。需要一种新的抗原筛选技术来研究质膜的这些困难但关键的脂质和碳水化合物分子。不幸的是，没有无标签的动力学筛选和定量方法来测量细胞质膜和mAb之间的结合亲和力。商业biacore“仪器 - 目前是测量结合动力学的金标准 - 与纯化的分子（蛋白质是蛋白质）一起起作用，固定在黄金膜底物上。但是，该仪器主要不适合量化mabs和细胞表面抗原之间的相互作用。为了量化在高空间分辨率的细胞膜内的mAb的结合动力学，我们已经用无膜系统验证了这个平台，并使用人工膜进行了人工膜，以实现在此处提出的工作，以进一步调整量子的范围。与人类治疗的IgM相结合，一旦开发了候选抗原，这项技术可能会在研究复杂的分子相互作用和细胞受体转导的信号中很重要。通过人造膜系统展示动力学传感的可行性，该系统可以整合跨膜蛋白与两侧的缓冲溶液接触。

项目成果

期刊论文数量（14）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Ultrasmooth metallic films with buried nanostructures for backside reflection-mode plasmonic biosensing.

DOI：
10.1002/andp.201200144
发表时间：
2012-11
期刊：
ANNALEN DER PHYSIK
影响因子：
2.4
作者：
Lindquist, Nathan C.;Johnson, Timothy W.;Jose, Jincy;Otto, Lauren M.;Oh, Sang-Hyun
通讯作者：
Oh, Sang-Hyun

Millimeter-Sized Suspended Plasmonic Nanohole Arrays for Surface-Tension-Driven Flow-Through SERS.

DOI：
10.1021/cm5031848
发表时间：
2014-11-25
期刊：
CHEMISTRY OF MATERIALS
影响因子：
8.6
作者：
Kumar, Shailabh;Cherukulappurath, Sudhir;Johnson, Timothy W.;Oh, Sang-Hyun
通讯作者：
Oh, Sang-Hyun

Lipid Membrane Deformation Accompanied by Disk-to-Ring Shape Transition of Cholesterol-Rich Domains.

DOI：
10.1021/jacs.5b04559
发表时间：
2015-07
期刊：
Journal of the American Chemical Society
影响因子：
15
作者：
Yong-Sang Ryu;Daehan Yoo;Nathan J. Wittenberg;Luke R. Jordan;S. Lee;A. Parikh;Sang-Hyun Oh
通讯作者：
Yong-Sang Ryu;Daehan Yoo;Nathan J. Wittenberg;Luke R. Jordan;S. Lee;A. Parikh;Sang-Hyun Oh

Reconstituting ring-rafts in bud-mimicking topography of model membranes.

DOI：
10.1038/ncomms5507
发表时间：
2014-07-24
期刊：
NATURE COMMUNICATIONS
影响因子：
16.6
作者：
Ryu, Yong-Sang;Lee, In-Ho;Suh, Jeng-Hun;Park, Seung Chul;Oh, Soojung;Jordan, Luke R.;Wittenberg, Nathan J.;Oh, Sang-Hyun;Jeon, Noo Li;Lee, Byoungho;Parikh, Atul N.;Lee, Sin-Doo
通讯作者：
Lee, Sin-Doo

Atomic layer deposition (ALD): A versatile technique for plasmonics and nanobiotechnology.