Nanopore Biosensor for Kinetics of Reparative Antibodies on Plasma Membranes

描述(由申请人提供)：能够对膜结合抗原进行体外、无标记的动态分析是一项非常理想的任务，但很少使用标准的商业化技术，如BIAcoreTM。对于G蛋白偶联受体(GPCR)这样的跨膜蛋白来说，这个问题更加复杂，因为与固体底物，特别是BIAcoreTM中使用的黄金底物直接接触的蛋白质通常会失去功能或变性。这里提出的纳米孔传感体系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衬底上并形成一个微小的井来限制支撑的类脂膜，而周围的金膜提供表面等离子体共振效应来动态监测分子与膜的结合。这项建议将通过表征治疗性人类单抗与候选抗原的结合来验证这些膜生物传感概念。这些人类免疫球蛋白促进脱髓鞘病变的重新髓鞘形成，并保护轴突。这些分子是测试这一系统的理想分子，因为IgM抗原的结合似乎需要完整的膜环境。将这些修复性免疫球蛋白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，单抗不与单个膜分子结合，而是与细胞内脂质微域(脂筏)内的信号复合体结合。如果这个复合体被破坏，mAb结合就会被消除。只有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)都会破坏复合膜抗原。当候选抗原以分离形式呈现时，IgM与所有或不结合非特异性结合。因此，重要的是保持细胞膜抗原的天然状态，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类和碳水化合物分子。遗憾的是，目前还没有一种无标记的动力学筛选和定量方法来测量细胞膜与单抗之间的结合亲和力。商用Biacore“仪器--目前是测量结合动力学的黄金标准--适用于固定在金膜衬底上的纯化分子，主要是蛋白质。然而，该仪器不适合定量单抗与天然膜插入状态的细胞表面抗原之间的相互作用。我们建议使用一种新的仪器，纳米灯(激光照射的金属孔)阵列，以高空间分辨率定量单抗与锚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造膜验证了这个平台，用于结合动力学测量。本文提出的工作将通过在金属纳米孔上重组少突胶质细胞和神经元细胞膜来进一步优化平台，以测量和定量它们与人治疗性IgM的结合亲和力，以确定候选抗原。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传递的信号方面发挥重要作用。作为未来的发展方向，我们还提出了重组悬挂在独立金属纳米孔底物上的独立脂膜的可能性，整合了跨膜蛋白如GPCRs，并展示了利用人造膜系统整合与两侧缓冲溶液接触的跨膜蛋白的可行性。

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.