Adding a Twist to LateralFlow Immunoassays: A DirectReplacement of Antibodies with Helical Affibodies, from Selectionto Application

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 His₆-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.