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Nanobody based immunoassay for alpha fetal protein detection using streptavidin-conjugated polymerized horseradish peroxidase for signal amplification

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Abstract

The enzyme-linked immunosorbent assay (ELISA) offers several advantages, including simple operation, high throughput, and low cost, making it an ideal immunoassay method for efficient screening of disease-related biomarkers in clinical samples. However, the traditional colorimetric ELISA has relatively low sensitivity, which promotes the continuous emergence of various novel signal amplification technologies. In this work, we fused the AFP-specific nanobody (A1) with the streptavidin-binding peptide (SBP) to develop a fusion protein (A1-SBP) as biorecognition element in a colorimetric ELISA for detecting AFP. Besides, to further improve the sensitivity of the traditional colorimetric ELISA, the streptavidin-conjugated polymerized horseradish peroxidase (SA-PolyHRP) were selected as a detection probe for signal amplification. The proposed signal enhancement strategy demonstrated a limit of detection (LOD) of 0.597 ng/mL for the SA-polyHRP-based ELISA, which is 7.67-fold lower than that of the traditional SA-HRP-based ELISA without additional steps. Furthermore, the proposed SA-polyHRP-based ELISA showed a good correlation with the detection of clinical samples using the Roche E601 chemiluminescence immunoassay analyzer. Therefore, the proposed signal enhancement strategy is an attractive approach for improving the sensitivity of immunoassay without requiring additional steps.

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Acknowledgements

This research was funded by the Natural Science Foundation of Hainan Province (No. 322MS016) and the National Natural Science Foundation of China (No. 31901800, No. 32260623 and No.31801156), the Specific Research Fund of the Innovation Platform for the Academinicians of Hainan Province (No. YSPTZX202152) and Key Laboratory of Tropical Fruits and Vegetables Quality and Safety for State Market Regulation (No. ZX-2023001).

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Author notes

  1. Danyang Sun, Xing Liu have contributed equally to this work.

Authors and Affiliations

  1. National Engineering Research Center for Bioengineering Drugs and the Technologies, Institute of Translational Medicine, Nanchang University, Nanchang, 330031, China

    Qi Chen

  2. Key Laboratory of Tropical and Vegetables Quality and Safety for State Market Regulation, School of Food Science and Engineering, Hainan University, Haikou, 570228, China

    Danyang Sun, Xing Liu, Kunlu Bao & Long Wu

  3. State Key Laboratory of Military Stomatology & National Clinical Research Center for Oral Diseases & Shaanxi International Joint Research Center for Oral Diseases, Center for Tissue Engineering, School of Stomatology, The Fourth Military Medical University, Xian, 710299, China

    Huijuan Kuang

  4. Department of Clinical Laboratory, The Second Affiliated Hospital of Hainan Medical University, Haikou, 570311, China

    Hua Pei

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  1. Danyang Sun
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Correspondence to Qi Chen.

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Sun, D., Liu, X., Bao, K. et al. Nanobody based immunoassay for alpha fetal protein detection using streptavidin-conjugated polymerized horseradish peroxidase for signal amplification. ANAL. SCI. 39, 2059–2065 (2023). https://doi.org/10.1007/s44211-023-00423-4

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  • DOI: https://doi.org/10.1007/s44211-023-00423-4

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