Adding a Twist to Lateral Flow Immunoassays: A Direct Replacement of Antibodies with Helical Affibodies, from Selection to 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 His<sub>6</sub>-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.

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Publication Details

Journal
Journal of the American Chemical Society
Published
2025-03-26
DOI
https://doi.org/10.1021/jacs.4c17452
Citations
11
Primary Topic
Monoclonal and Polyclonal Antibodies Research
Type
article
Field-Weighted Citation Impact
14.96

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article

Adding a Twist to Lateral Flow Immunoassays: A Direct Replacement of Antibodies with Helical Affibodies, from Selection to Application

Kevion K. Darmawan, Kim Anh Giang, Patrick Charchar, Francesca Smith et al.
11 citations
Journal of the American Chemical Society
Monoclonal and Polyclonal Antibodies Research
14.96
article

Adding a Twist to Lateral Flow Immunoassays: A Direct Replacement of Antibodies with Helical Affibodies, from Selection to Application

Kevion K. Darmawan, Kim Anh Giang, Patrick Charchar, Francesca Smith, Daniel A. Richards, Molly M. Stevens, André Shamsabadi, Ross Burdis, Per‐Åke Nygren, Irene Yarovsky, Adam Creamer, Johan Nilvebrant, Jonathan P. Wojciechowski, Chris Sadler
article en
11 citations

Abstract

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<sub>6</sub>-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.

Journal of the American Chemical SocietyVol. 147(14)
ETH Zurich (CH), University of Oxford (GB), Bioengineering (Switzerland) (CH), MIT University (MK), NIHR Imperial Biomedical Research Centre (GB), AlbaNova (SE), Bioengineering Center (RU), Imperial College London (GB), RMIT University (AU), KTH Royal Institute of Technology (SE)
Wellcome Trust, National Computational Infrastructure, Royal Academy of Engineering, University of Oxford, Rosetrees Trust, Eidgenössische Technische Hochschule Zürich, Kungliga Tekniska Högskolan, Horizon 2020 Framework Programme, Engineering and Physical Sciences Research Council, Australian Research Council
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Openalex Percentile: Top 1%
Monoclonal and Polyclonal Antibodies Research
14.96
DOIOpen Access PDFOpenAlex
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