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Generation of bioluminescent enzyme immunoassay for ferritin by single-chain variable fragment and its NanoLuc luciferase fusion

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Abstract

Ferritin, widely present in liver and spleen tissue, is considered as a serological biomarker for liver diseases and cancers. The detection of ferritin may be an important tool in health diagnosis. In this study, 14 non-immunized chicken spleens were utilized to construct a single-chain fragment (scFv) phage library. After 4 rounds of panning, 7 unique clones were obtained. The optimal clone was further screened and combined with NanoLuc luciferase (Nluc) as a dual functional immunoprobe to bioluminescent enzyme immunoassay (BLEIA), which was twice as sensitive as its parental scFv-based double-sandwich enzyme-linked immunoassay (ds-ELISA). The cross-reactivity analysis revealed that the proposed methods were highly selective and suitable for clinical detection. To further verify the performance of the immunoassays, serum samples were tested by the proposed methods and a commercial ELISA kit, and there was a good correlation between the results. These results suggested that scFv fused with Nluc might be a powerful dual functional tool for rapid, practically reliable, and highly sensitive ferritin detection.

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Funding

This work was financially supported by the Guangzhou Science and Technology Foundation (201903010034), the Natural Resources Science Foundation of Guangdong Province (2018A030313926), the Science and Technology Foundation Key R&D Program of Guangdong Province (2019B020209009; 2019B020218009), the R&D Program of Guangdong Province Drug Administration (2021TDZ09; 2021YDZ06), NIH-NIEHS (RIVER Award) R35 ES030443-01, and NIH-NIEHS (Superfund Award) P42 ES004699.

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Author notes

  1. Qiyi He and Li Yang contributed equally to this work.

Authors and Affiliations

  1. Department of Pharmaceutical Engineering, School of Biomedical and Pharmaceutical Sciences, Guangdong University of Technology, Guangzhou, 510006, People’s Republic of China

    Qiyi He, Mingxia Lin, Huiyi Yang, Xiping Cui & Suqing Zhao

  2. Faculty of Chemical Engineering and Light Industry, Guangdong University of Technology, Guangzhou, 510006, People’s Republic of China

    Qiyi He & Yanxiong Fang

  3. Biotherapy Center, Third Affiliated Hospital of Sun Yat-Sen University, Guangzhou, 510630, People’s Republic of China

    Li Yang

  4. Department of Entomology and Nematology and UCD Comprehensive Cancer Center, University of California, Davis, Davis, CA, 95616, USA

    Mark R. McCoy & Bruce D. Hammock

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  1. Qiyi He
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  2. Li Yang
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  3. Mingxia Lin
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  4. Huiyi Yang
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  8. Yanxiong Fang
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  9. Suqing Zhao
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Corresponding authors

Correspondence to Yanxiong Fang or Suqing Zhao.

Ethics declarations

All animal experimental procedures were carried out according to protocols approved by the Animal Care and Use Committee of Guangdong Province, People’s Republic of China.

Five serum samples from healthy volunteers included 2 males and 3 females aged 25–45 were obtained from the Third Affiliated Hospital of Sun Yat-sen University which complied with the Helsinki Declaration of 1975 and approved by the Guangdong University of Technology Ethics Committee.

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The authors declare no competing interests.

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He, Q., Yang, L., Lin, M. et al. Generation of bioluminescent enzyme immunoassay for ferritin by single-chain variable fragment and its NanoLuc luciferase fusion. Anal Bioanal Chem 414, 6939–6946 (2022). https://doi.org/10.1007/s00216-022-04261-7

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  • DOI: https://doi.org/10.1007/s00216-022-04261-7

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