Sadler Christy J, Creamer Adam, Giang Kim Anh, Darmawan Kevion K, Shamsabadi André, Richards Daniel A, Nilvebrant Johan, Wojciechowski Jonathan P, Charchar Patrick, Burdis Ross, Smith Francesca, Yarovsky Irene, Nygren Per-Åke, Stevens Molly M
Department of Materials, Department of Bioengineering, Institute of Biomedical Engineering Imperial College London, London SW7 2AZ, U.K.
Department of Physiology, Anatomy and Genetics, Department of Engineering Science, Kavli Institute for Nanoscience Discovery, University of Oxford, Oxford OX1 3QU, U.K.
J Am Chem Soc. 2025 Apr 9;147(14):11925-11940. doi: 10.1021/jacs.4c17452. Epub 2025 Mar 26.
Immunoreagents, most commonly antibodies, are integral components of lateral flow immunoassays. However, the use of antibodies comes with limitations, particularly relating to their reproducible production, and poor thermal and chemical stability. Here, we employ phage display to develop affibodies, a class of nonimmunoglobulin affinity proteins based on a small three-helix bundle scaffold, against SARS-CoV-2 Spike protein. Subsequently, we demonstrate the utility and viability of affibodies to directly replace antibodies in lateral flow immunoassays. In addition, we highlight several physiochemical advantages of affibodies, including their ability to withstand exposure to high temperature and humidity while maintaining superior performance compared to their antibody counterparts. Furthermore, we investigate the adsorption mechanism of affibodies to the surface of gold nanoparticle probes via a His-tag, introduced to also facilitate recombinant purification. Through molecular dynamics simulations, we elucidate the structural and physical characteristics of affibody dimers which result in high-performing detection probes when immobilized on nanoparticle surfaces. This work demonstrates that affibodies can be used as direct replacements to antibodies in immunoassays and should be further considered as alternatives owing to their improved physiochemical properties and modular design.
免疫试剂，最常见的是抗体，是侧向流动免疫分析的重要组成部分。然而，抗体的使用存在局限性，特别是在其可重复生产以及热稳定性和化学稳定性较差方面。在此，我们利用噬菌体展示技术开发了亲和体，这是一类基于小三螺旋束支架的非免疫球蛋白亲和蛋白，用于针对严重急性呼吸综合征冠状病毒2（SARS-CoV-2）刺突蛋白。随后，我们证明了亲和体在侧向流动免疫分析中直接替代抗体的实用性和可行性。此外，我们强调了亲和体的几个物理化学优势，包括它们在暴露于高温和高湿度环境下仍能保持性能优于其抗体对应物的能力。此外，我们研究了亲和体通过引入His标签吸附到金纳米颗粒探针表面的机制，该标签也有助于重组纯化。通过分子动力学模拟，我们阐明了亲和体二聚体的结构和物理特性，这些特性在固定在纳米颗粒表面时会产生高性能的检测探针。这项工作表明，亲和体可以在免疫分析中直接替代抗体，并且由于其改善的物理化学性质和模块化设计，应进一步被视为替代方案。
