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<p>The latest global pandemic and the ongoing threat of emerging pathogens have highlighted the need for effective diagnostic strategies for infectious diseases. Current methods primarily depend on immunoassays, such as enzyme-linked immunosorbent assays and rapid diagnostic tests, which utilize antibodies to detect specific analytes. However, traditional mammalian cell systems for antibody production are expensive and carry a risk of viral contamination. To address these limitations, in this study, we developed an <i>Escherichia coli</i>-based antibody fragment platform, Fab<sub>2</sub>-Sav/Rassf/Hpo domain (SARAH), by fusing an anti-parallel coiled-coil structure derived from the SARA domain of human Mst1 kinase to the antigen-binding (Fab) fragment to enhance stability and binding affinity. Using bovine interferon-gamma (bIFN-γ) as a model antigen, Fab<sub>2</sub>-SARAH exhibited superior performance to traditional constructs. In addition, as Fab<sub>2</sub>-SARAH was optimized for <i>E. coli</i>, a 5-L fed-batch fermentation was successfully performed to produce Fab<sub>2</sub>-SARAH at a g/L scale. When incorporated into a lateral flow immunoassay (LFIA), Fab<sub>2</sub>-SARAH demonstrated a limit of detection of 7.91&#xa0;ng/mL for bIFN-γ. The LFIA also detected bIFN-γ in fetal bovine serum, indicating its applicability. These results suggest the potential of Fab<sub>2</sub>-SARAH for scalable, cost-effective, and rapid diagnostics to manage infectious diseases. While further studies are warranted to determine whether this technology is applicable to other biomarkers and diagnostic formats, this study serves as a foundation for potentially transforming global disease management and surveillance.</p>

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Fab2-SARAH: An Escherichia coli-Based Antibody Fragment Platform for Scalable, Cost-Effective In Vitro Diagnostics

  • 期刊论文

  • 发表日期：

    2025年7月11日

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  • Young Su Kim,
  • Seo Yeong Oh,
  • Hye-Jeong Lee,
  • Seong Uk Son,
  • Hwabong Jeong,
  • Hyeran Kim,
  • Juyeon Jung,
  • Young-Phil Kim,
  • Eun-Kyung Lim,
  • Jungoh Ahn

摘要

The latest global pandemic and the ongoing threat of emerging pathogens have highlighted the need for effective diagnostic strategies for infectious diseases. Current methods primarily depend on immunoassays, such as enzyme-linked immunosorbent assays and rapid diagnostic tests, which utilize antibodies to detect specific analytes. However, traditional mammalian cell systems for antibody production are expensive and carry a risk of viral contamination. To address these limitations, in this study, we developed an Escherichia coli-based antibody fragment platform, Fab2-Sav/Rassf/Hpo domain (SARAH), by fusing an anti-parallel coiled-coil structure derived from the SARA domain of human Mst1 kinase to the antigen-binding (Fab) fragment to enhance stability and binding affinity. Using bovine interferon-gamma (bIFN-γ) as a model antigen, Fab2-SARAH exhibited superior performance to traditional constructs. In addition, as Fab2-SARAH was optimized for E. coli, a 5-L fed-batch fermentation was successfully performed to produce Fab2-SARAH at a g/L scale. When incorporated into a lateral flow immunoassay (LFIA), Fab2-SARAH demonstrated a limit of detection of 7.91 ng/mL for bIFN-γ. The LFIA also detected bIFN-γ in fetal bovine serum, indicating its applicability. These results suggest the potential of Fab2-SARAH for scalable, cost-effective, and rapid diagnostics to manage infectious diseases. While further studies are warranted to determine whether this technology is applicable to other biomarkers and diagnostic formats, this study serves as a foundation for potentially transforming global disease management and surveillance.