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

• 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
• 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

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）：对膜结合抗原进行体外无标记动态分析的能力是一项非常需要的任务，但很少使用标准的商业化技术（如BIAcoreTM）来实现。对于G蛋白偶联受体（GPCR）等跨膜蛋白来说，这个问题更加复杂，因为与固体底物直接接触的蛋白质，特别是与BIAcoreTM中使用的金底物直接接触的蛋白质，通常会失去其功能或变性。这里提出的纳米孔传感结构具有克服这些挑战的独特潜力，因为每个纳米孔位于玻璃基板上，形成一个微小的孔来限制支持的脂质膜，而周围的金膜提供表面等离子体共振效应，以动态监测分子与膜的结合。该建议将通过表征治疗性人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IgMs促进脱髓鞘病变的再脱髓鞘和保存轴突。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igm转移到临床试验的一个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，单克隆抗体不与单个膜分子结合，而是与细胞脂质微域（脂筏）内的信号复合物结合。如果这个复合体被破坏，单抗结合就被消除。igm只有在与完整的质膜结合时才能维持其细胞特异性。任何固定方法（甲醇、甲醛、冷冻）都会破坏复合膜抗原。当候选抗原以分离形式呈现时，igm非特异性地与所有抗原结合或不与抗原结合。因此，保持细胞膜抗原的天然状态以保持适当的单抗结合动力学是很重要的。需要一种新的抗原筛选技术来研究这些质膜上困难但关键的脂质和碳水化合物分子。遗憾的是，目前还没有无标记的动力学筛选和定量方法来测量细胞膜与单克隆抗体之间的结合亲和力。商用BIAcore“仪器——目前测量结合动力学的金标准——与固定在金膜底物上的纯化分子（主要是蛋白质）一起工作。然而，该仪器不适合定量单克隆抗体与细胞表面抗原在天然插膜状态下的相互作用。我们建议使用一种新型仪器，纳米lamp（激光照射金属孔）阵列，以高空间分辨率量化单克隆抗体与固定在细胞膜内的抗原的结合动力学。我们已经用无膜系统和人工膜验证了这个平台的结合动力学测量。本文提出的工作将进一步优化平台，通过在金属纳米孔上重建少突胶质细胞和神经元细胞膜，测量和量化它们与人类治疗性IgMs的结合亲和力，以鉴定候选抗原。一旦开发出来，这项技术可能会在复杂分子相互作用和细胞受体转导信号的研究中被证明是重要的。作为未来的发展方向，我们还提出了重建悬挂在独立金属纳米孔底物上的独立脂质膜的可能性，并结合跨膜蛋白（如gpcr），并展示了用人工膜系统进行动态传感的可行性，该系统可以将跨膜蛋白与两侧的缓冲溶液接触。

项目成果

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.

毫米大小的悬浮等离子体纳米荷尔阵列，用于表面张力驱动的流动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

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

Effect of Nanohole Spacing on the Self-Imaging Phenomenon Created by the Three-Dimensional Propagation of Light through Periodic Nanohole Arrays.

• DOI: 10.1021/jp306179d
• 发表时间: 2012-09-20
• 期刊: JOURNAL OF PHYSICAL CHEMISTRY C
• 影响因子: 3.7
• 作者: Chowdhury, Mustafa H.;Lindquist, Nathan C.;Lesuffleur, Antoine;Oh, Sang-Hyun;Lakowicz, Joseph R.;Ray, Krishanu
• 通讯作者: Ray, Krishanu