Enzyme-Mediated Site-Specific Conjugation of Antibodies to Nanoparticles

酶介导的抗体与纳米颗粒的位点特异性缀合

• 批准号: 10436681
• 负责人: Jeremy D. Driskell
• 金额: $ 36.99万
• 依托单位: ILLINOIS STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-01 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10436681
• 关键词: Address; Adsorption; Affinity; Antibodies; Binding; Binding Proteins; Biological; Biological Assay; Charge; Chemicals; Chemistry; Coupling; Cysteine; Data; Detection; Development; Diagnostic; Diagnostic Imaging; Disease; Drug Delivery Systems; Elements; Environment; Enzymes; Exhibits; Fc Immunoglobulins; Goals; Imaging technology; Immobilization; Immunoassay; Immunoglobulin Fragments; Immunoglobulin G; In Situ; Intervention; Laboratories; Learning; Lysine; Mass Spectrum Analysis; Measures; Mediating; Medical; Mission; Modification; Motivation; Nanostructures; Outcome; Peptide antibodies; Peptides; Process; Property; Protein Engineering; Proteins; Public Health; Publications; Reagent; Research; Resolution; Role; Site; Students; Sulfhydryl Compounds; Surface; Technology; Testing; Therapeutic; Transglutaminases; United States National Institutes of Health; Work; antigen binding; crosslink; density; design; functional group; innovation; insight; microbial; nanoGold; nanobiotechnology; nanomaterials; nanoparticle; novel; novel strategies; polypeptide; skills; small molecule; student participation; symposium; undergraduate student; zeta potential

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.

项目摘要/摘要 尽管做出了广泛的努力，但当前的抗体金纳米颗粒（AUNP）结合化学策略是 并非普遍适用于所有抗体，均取决于pH，导致活性减少的随机取向， 和/或稳定性有限。必须解决这些问题，以实现启用AUNP的全部潜力。 在诊断领域说并促进广泛接受和实施。长期目标 这项研究的是制定一种将抗体固定在金纳米颗粒上的策略，以形成高度活跃的， 定向且稳定的共轭物，可用于启用AUNP的免疫测定。我们的中心假设是 Zyme介导的多肽与抗体的FC片段的位点特异性共轭，以结合多种多肽 PLE硫醇基团和高密度的局部正电荷将促进定向和可靠的吸附到 金纳米颗粒。我们对这些研究的理由是，成功完成将推动新颖的设计 高度活跃和稳定的抗体 - 纳米粒子偶联物需要促进启用AUNP的平台 形式技术。在引人入胜的初步数据的驱动下，我们将检验我们的假设并朝着我们 长期研究目标通过完成以下目的：1）酶介导的修饰和特征 - 抗体的影响； 2）原位分析改性抗体吸附到AuNP上； 3）定量分析 抗体抗体 - unp结合物的抗原结合活性的在我们以前正在进行的工作中，我们已经详细介绍了 局部蛋白电荷和硫醇官能团在方向和亲和力中的作用 蛋白质吸附到金纳米颗粒上。在第一个目的下，短的半胱氨酸和赖氨酸富含肽将是 通过微生物转lu-结合在抗体的FC片段上与位点特异性Q295共轭 坦明酶（MTG）和结合将通过质谱和蛋白质电荷确认。在 第二个目标是一种竞争性蛋白质结合测定和纳米颗粒跟踪分析，已在我们的实验室中进行了验证， 将用于定量评估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，先前经过证实的酶测定将用于量化抗原结合活性的 由未修饰和肽修饰抗体形成的生物缀合物。该策略是创新的，因为 将多肽用作抗体和AUNP表面之间的交联剂 将肽与FC区域的抗体特异性结合的酶。该项目很重要 - 因为它可以在没有蛋白质工程的情况下精确控制抗体吸附到金纳米颗粒上 并将导致一般的设计原则，该原理提供了简化的过程以优化固定化 化学形成强大且高度功能的纳米粒子探针。