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，我们可以通过可变淋巴细胞受体（VLR）抗体的纳米级工程化来生产具有可调亲和力和热稳定性的靶向载体。我们将通过VLR功能化的纳米/微粒的结合测定来证明这些靶向品质。我们将创建多价VLR抗体，其具有一系列化合价、接头长度，并且具有用于连接至纳米/微米颗粒的合适残基。VLR的多价重组形式将识别称为BclA的炭疽芽孢杆菌孢子外壳蛋白。我们将用生物物理技术和单分子成像来验证正确的化合价和接头排列。我们将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。我们将测量多价抗体的动力学速率和所得亲和力作为化合价和接头长度的函数。我们还将验证即使在高温下也会发生抗原结合。我们将多价VLR与几种微米/纳米颗粒缀合并验证与B的粘附。炭疽杆菌的高通量流式细胞术。我们将测量颗粒粘附和凝集，作为a）VLR分子价、B）VLR接头间距、c）颗粒尺寸和d）温度的函数。我们的目标是通过1）确定设计原则，提供一种合理的方法来控制分子亲和力，通过简单的修饰化合价和2）利用抗体分子，是热稳定的温度高达70 ℃或更高，以扩大纳米粒子靶向病原体的抗体的有用性。该提案的成功完成将开辟几条富有成效的发现途径，从而改进传感器和病原体和生物战剂的对策。我们预期产生具有可调抗原亲和力和热稳定性的纳米/微粒。这两个影响将大大提高基于抗体的检测器、对策和诊断方法的可行性。 公共卫生相关性：我们建议创建新发现的VLR抗体靶向的纳米/微粒。我们将创建重组形式的VLRs，我们预计将为药物递送、试剂开发和传感器提供以下益处，包括合理调整抗体亲和力和温度稳定性。

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.