Enzyme-Mediated Site-Specific Conjugation of Antibodies to Nanoparticles

项目概要/摘要 尽管进行了广泛的努力，但抗体-金纳米颗粒（AuNP）缀合化学的当前策略是： 不普遍适用于所有抗体，是pH依赖性的，导致随机取向以降低活性， 和/或具有有限的稳定性。这些问题必须得到解决，以实现AuNP使能的全部潜力- 并促进诊断竞技场的广泛接受和实施。远景目标 本研究的目的是开发一种将抗体吸附在金纳米颗粒上以形成高活性， 定向的和稳定的缀合物，用于AuNP激活的免疫测定。我们的核心假设是- 酶介导的多肽与抗体的Fc片段的位点特异性缀合，以掺入多- 多个硫醇基团和高密度的局部正电荷将促进定向的和稳健的吸附， 金纳米颗粒我们进行这些研究的理由是，成功完成将推动新的设计， 高活性和稳定的抗体-金纳米颗粒缀合物是推进AuNP-使能的平台所急需的。 形式技术。在令人信服的初步数据的推动下，我们将测试我们的假设，并朝着我们的目标前进。 通过完成以下目标实现长期研究目标：1）酶介导的修饰和表征- 抗体的原位分析; 2）修饰的抗体吸附到AuNP上的原位分析;和3）定量分析 抗体-AuNP偶联物的抗原结合活性。在我们以前和正在进行的工作中，我们详细介绍了 局部蛋白质电荷和巯基官能团对取向和亲和力的作用，分别为， 将蛋白质吸附到金纳米颗粒上。在第一个目标下，将合成一种短的、富含半胱氨酸和赖氨酸的肽 使用微生物transglu-缀合至抗体Fc片段上保守的位点特异性Q295， 通过质谱法和蛋白质电荷确认结合。下 第二个目标是竞争性蛋白质结合测定和纳米颗粒追踪分析，已经在我们的实验室中得到验证， 将用于定量评价肽修饰的抗体与金纳米颗粒的结合亲和力。 在最后一个目标下，将使用先前证明的酶测定来定量抗原结合活性， 用未修饰的和肽修饰的抗体形成的生物缀合物。这一战略之所以具有创新性，是因为 多肽作为抗体和AuNP表面之间的交联剂的用途以及多肽作为抗体和AuNP表面之间的交联剂的用途， 用于肽与抗体Fc区的位点特异性缀合的酶。该项目意义重大- 因为它可以精确控制抗体吸附到金纳米颗粒上，而无需蛋白质工程 并且将导致提供优化固定化的流线型过程的通用设计原则 化学形成强大的和高功能的纳米粒子探针。

PROJECT SUMMARY/ABSTRACT Despite extensive efforts, current strategies of antibody-gold nanoparticle (AuNP) conjugation chemistries are not universally applicable to all antibodies, are pH dependent, result in random orientation for diminished activity, and/or have limited stability. These issues must be addressed to realize the full potential of AuNP-enabled as- says and promote wide-spread acceptance and implementation in the diagnostics arena. The long-term goal of this research is to develop a strategy to immobilize antibody onto gold nanoparticles to form highly active, oriented, and stable conjugates for use in AuNP-enabled immunoassays. Our central hypothesis is that en- zyme-mediated, site-specific conjugation of a polypeptide to the Fc fragment of an antibody to incorporate multi- ple thiol groups and a high density of localized positive charge will promote oriented and robust adsorption onto gold nanoparticles. Our rationale for these studies is that successful completion would drive the novel design of highly active and stable antibody-gold nanoparticle conjugates critically needed to advance AuNP-enabled plat- form technologies. Driven by compelling preliminary data, we will test our hypothesis and progress toward our long-term research goal by completing the following Aims: 1) Enzyme-mediated Modification and Characteriza- tion of Antibodies; 2) In situ Analysis of Modified Antibody Adsorption onto AuNPs; and 3) Quantitative Analysis of Antigen-binding Activity for Antibody-AuNP Conjugates. In our previous and ongoing work, we have detailed the role of localized protein charge and thiol functional groups on the orientation and affinity, respectively, for the adsorption of proteins to gold nanoparticles. Under the first aim, a short, cysteine- and lysine-rich peptide will be conjugated to the site-specific Q295 that is conserved on the Fc fragment of the antibody using microbial transglu- taminase (mTG) and the conjugation will be confirmed by mass spectrometry and protein charge. Under the second aim, a competitive protein binding assay and nanoparticle tracking analysis, already validated in our lab, will be used to quantitatively evaluate the binding affinity of the peptide-modified antibody to gold nanoparticles. Under the last aim, a previously proven enzyme assay will be used to quantify the antigen-binding activity for bioconjugates formed with unmodified and peptide-modified antibodies. This strategy is innovative because of the use of a polypeptide as a crosslinking agent between the antibody and the AuNP surface and the use of an enzyme for site-specific conjugation of the peptide to the Fc region the antibody. The project is significant be- cause it enables the precise control of antibody adsorption onto gold nanoparticles without protein engineering and will lead to generalized design principles that provide a streamlined process to optimize immobilization chemistry to form robust and highly functional nanoparticle probes.