Tunable affinity and heat stable antibody targeting of nanoparticles

描述（由申请人提供）：我们提出的研究将证明，我们可以通过纳米尺度工程的可变淋巴细胞受体（VLR）抗体，生产一种具有可调亲和力和热稳定性的靶向载体。我们将通过VLR功能化纳米/微粒的结合试验来证明这些靶向性。我们将创建多价VLR抗体，具有一系列的价，连接体长度，并具有合适的残基附着在纳米/微粒上。VLRs的多价重组形式将识别炭疽芽孢杆菌孢子外壳蛋白BclA。我们将用生物物理技术和单分子成像来验证合适的价和连接体排列。我们将利用表面等离子体共振（SPR）和基于原子力显微镜力谱的单分子结合测量来测量VLR抗体与BclA抗原的结合强度。我们将测量动力学速率和由此产生的多价抗体的亲和力，作为价和连接体长度的函数。我们还将验证即使在高温下抗原结合也会发生。我们将多价VLRs与几种微/纳米颗粒结合，并使用高通量流式细胞术验证其与炭疽杆菌的粘附性。我们将测量颗粒粘附和凝集作为a) VLR分子价，b) VLR连接体间距，c)颗粒大小和d)温度的函数。我们的目标是扩大纳米颗粒靶向病原体抗体的用途：1)确定设计原则，通过简单的修饰价来提供合理的方法来控制分子亲和力；2)利用热稳定到70°C或更高温度的抗体分子。这一建议的成功完成将开辟几条富有成效的发现之路，这将导致对病原体和生物战剂的传感器和对策的改进。我们期望创造具有可调抗原亲和力和热稳定性的纳米/微粒。这两个影响将极大地提高基于抗体的检测器、对策和诊断方法的可行性。

DESCRIPTION (provided by applicant): Our proposed study will demonstrate that we can produce a targeting vehicle capable of both tunable affinity and heat-stability through nanoscale engineering of Variable Lymphocyte Receptors (VLR) antibodies. We will demonstrate these targeting qualities through binding assays of VLR functionalized nano/microparticles. We will create multivalent VLR antibodies with a range of valencies, linker lengths, and that have suitable residues for attachment to nano/microparticles. The multivalent recombinant forms of VLRs will recognize the Bacillus anthracis spore coat protein called BclA. We will verify the proper valency and linker arrangement with biophysical techniques and single molecule imaging. We will measure the binding strength the VLR antibodies to the BclA antigen with surface plasmon resonance (SPR) and single-molecule binding measurements based upon atomic force microscopy force spectroscopy. We will measure the kinetic rates and the resulting affinity of the multivalent antibodies as a function of valency and linker length. We will also validate that antigen binding occurs even when raised to high temperatures. We will conjugate the multivalent VLRs to several micro/nanoparticles and validate adhesion to B. anthracis using high throughput flow cytometry. We will measure particle adhesion and agglutination as a function of a) VLR molecular valency, b) VLR linker spacing, c) particle size, and d) temperature. We aim to broaden the usefulness of nanoparticle targeting of pathogens with antibodies by 1) identifying design principles that offer a rational method for controlling the molecular affinity through simple modification of valency and 2) utilizing an antibody molecule that is heat-stable up to temperatures of 70¿C or greater. Successful completion of this proposal will open several fruitful paths of discovery which will result in improved sensors and countermeasures for pathogens and biowarfare agents. We anticipate the creation of nano/microparticles with tunable antigen affinity and heat stability. These two impacts will drastically improve the viability of antibody- based detectors, countermeasures, and diagnosis methods. PUBLIC HEALTH RELEVANCE: We propose to create nano/microparticles that are targeted by the newly discovered VLR antibody. We will create recombinant forms of VLRs that we anticipate will offer the following benefits to drug delivery, reagent development, and sensors, including rational adjustment of antibody affinity and temperature stability.