Enzyme-Mediated Site-Specific Conjugation of Antibodies to Nanoparticles

项目摘要/摘要 尽管进行了广泛的努力，目前抗体-金纳米颗粒(AuNP)偶联化学的策略是 并不是普遍适用于所有抗体，是pH相关的，导致活性降低的随机取向， 和/或具有有限的稳定性。必须解决这些问题，以充分发挥启用AuNP的AS的潜力- 说并促进诊断领域的广泛接受和实施。长期目标 这项研究的目的是开发一种策略，将抗体固定在金纳米颗粒上，形成高活性的， 定向、稳定的结合物，用于AuNP启用的免疫分析。我们的中心假设是恩- 酶介导的多肽与抗体Fc片段的定点结合以整合多... 多个硫醇基团和高密度的定域正电荷将促进定向和强劲的吸附 金纳米粒子。我们进行这些研究的理由是，成功完成这些研究将推动 高活性和稳定的抗体-金纳米颗粒结合物是推进AuNP激活的平台至关重要的 形式技术。在令人信服的初步数据的推动下，我们将检验我们的假设，并朝着我们的 远期研究目标：1)酶介导的修饰和表征-- 抗体的检测；2)修饰抗体在AuNPs上吸附的原位分析；3)定量分析 抗体-AuNP结合物的抗原结合活性。在我们以前和正在进行的工作中，我们有详细的 局域蛋白质电荷和硫醇官能团分别对蛋白质的取向和亲和力的影响 蛋白质在金纳米颗粒上的吸附。在第一个目标下，一个短的、富含半胱氨酸和赖氨酸的多肽将是 利用微生物转录酶与抗体Fc片段上保守的位点特异性Q295结合。 三聚氰胺酶(MTG)和偶联产物将通过质谱学和蛋白质电荷法进行确认。在.之下 第二个目标是竞争性蛋白质结合分析和纳米颗粒跟踪分析，已经在我们的实验室得到验证， 将用于定量评价多肽修饰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分析来量化抗原结合活性 由未经修饰和多肽修饰的抗体形成的生物偶联物。这一战略是创新的，因为 使用多肽作为抗体和AuNP表面之间的交联剂以及使用 用于将多肽与抗体的Fc区进行定点结合的酶。这个项目意义重大，因为- 因为它可以在没有蛋白质工程的情况下精确控制抗体在金纳米颗粒上的吸附 并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，以形成坚固和高功能的纳米颗粒探测器。

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.