Nanopore Biosensor for Kinetics of Reparative Antibodies on Plasma Membranes

描述（由申请人提供）：对膜结合抗原进行体外无标记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（如BIAcoreTM）实现。对于跨膜蛋白如G蛋白偶联受体（GPCR），该问题是复杂的，因为与固体基质直接接触的蛋白质，特别是与BIAcore TM中使用的金基质直接接触的蛋白质，通常失去其功能或变性。本文提出的纳米孔感测架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并形成微小的孔以限制所支撑的脂质膜，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。该提案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IgM促进脱髓鞘病变的髓鞘再生并保护轴突。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，单克隆抗体不结合到一个单一的膜分子，但信号复合物内的脂质微域（脂筏）的细胞。如果该复合物被破坏，则mAb结合被消除。IgM仅在与完整的质膜结合时保持其细胞特异性。任何固定方法（甲醇、甲醛、冷冻）都会破坏复合膜抗原。当候选抗原以分离的形式存在时，IgM非特异性地结合全部或不结合。因此，重要的是将细胞膜抗原维持在其天然状态以保持适当的mAb结合动力学。需要一种新的抗原筛选技术来研究这些困难但关键的质膜脂质和碳水化合物分子。不幸的是，没有无标记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-目前用于测量结合动力学的金标准-与固定在金膜基底上的纯化分子（主要是蛋白质）一起工作。然而，该仪器不适合于定量mAb和细胞表面抗原在其天然膜插入状态下的相互作用。在这里，我们建议使用一种新的仪器，纳米LAMP（激光照射金属孔）阵列，以量化的单克隆抗体的结合动力学的抗原锚定在细胞膜在一个高的空间分辨率。我们已经验证了这个平台与无膜系统和人工膜结合动力学测量。本文提出的工作将通过在金属纳米孔上重建少突胶质细胞和神经元细胞膜来进一步优化该平台，以测量和量化它们与人类治疗性IgMs的结合亲和力，从而识别候选抗原。一旦开发出来，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发挥重要作用。作为未来的方向，我们还提出了重建悬挂在独立金属纳米孔基底上的独立脂质膜的可能性，将跨膜蛋白如GPCR并入，并证明了用人工膜系统进行动力学传感的可行性，该人工膜系统可以整合跨膜蛋白与两侧的缓冲溶液接触。 公共卫生相关性：该提议旨在确定纳米多孔金膜检测平台是否可以以保留与完整细胞相似的正确质膜功能的方式呈递细胞抗原。标准结合测定表面等离子体共振（SPR）检测器如BIAcore仅使用单个纯化分子就能很好地工作，但不能模拟膜内多个分子的呈现。我们建议通过测量人IgM与从髓鞘、少突胶质细胞和神经元分离的膜的相互作用来验证纳米孔SPR平台。这些修复性IgM明显结合神经系统的细胞，但试图确定其膜抗原使用常规的结合技术一直没有定论。如果成功，基于纳米孔的结合检测器将适用于基础生物学和药物发现的各种配体/抗原结合研究。

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. PUBLIC HEALTH RELEVANCE: 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.