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Development of a phage display-mediated immunoassay for the detection of vascular endothelial growth factor

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Abstract

Because of the critical role of vascular endothelial growth factor (VEGF) in angiogenesis and its significantly increased serum levels in early stages of cancer, VEGF is considered an important prognostic biomarker in different cancers. Herein, the amplification power of PCR combined with phage displaying anti-VEGF VHH, a sensitive real-time immunoassay, was precisely designed based on phage display-mediated immuno-PCR (PD-IPCR) for the detection of VEGF. This system benefits from strong and specific binding of antigen and antibody in a sandwich immunosorbent assay platform using avastin (anti-VEGF monoclonal antibody) as the capture antibody. The anti-VEGF phage particles were used as both anti-VEGF agent and DNA template in the PD-IPCR. Anti-VEGF phage ELISA showed a linear range of 3–250 ng/ml and a limit of detection (LOD) of 1.1 ng/ml. Using the PD-IPCR method, the linear range of VEGF detection was found to be 0.06–700 ng/ml, with a detection limit of 3 pg/ml. The recovery rate in serum ranged from 83% to 99%, with a relative standard deviation of 1.2–4.9%. These values indicate that the method has good sensitivity for use in clinical analysis. The proposed method was successfully applied to the clinical determination of VEGF in human serum samples, and the results showed excellent correlation with conventional ELISA (R2 = 0.995). The novel immunoassay provides a specific and sensitive immunoassay protocol for VEGF detection at very low levels.

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Acknowledgements

The authors express their gratitude to the research council of Tarbiat Modares University and Ministry of Sciences, Research, and Technology for financial support. We also thank Dr. Mehrdad Behmanesh for his advice and for kindly providing equipment.

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Authors and Affiliations

  1. Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, Jalal Ale Ahmad Highway, Tehran, 14115-154, Iran

    Zahra S. Rezaei, Sadegh Hasannia & Reza H. Sajedi

  2. Department of Biology, Faculty of Sciences, University of Guilan, Rasht, Guilan, 4199613776, Iran

    S. Shirin Shahangian

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  1. Zahra S. Rezaei
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  2. S. Shirin Shahangian
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Contributions

ZSR performed all the experiments, analysed the data, and contributed to manuscript writing. SSS was involved in the discussions and revised the manuscript. SH contributed to manuscript revision and analysis of the data. RHS proposed the original idea, designed and supervised the experiments, formed the experimental concept and contributed to analysis of the data, provided intellectual input, and edited the manuscript.

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Correspondence to Reza H. Sajedi.

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The authors declare that they have no conflict of interest.

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Human serum samples for real sample analysis were provided from Laleh hospital, Tehran, Iran (Ethical Committee approval ID: IR.MODARES.REC.1399.034, awarded by Medical Sciences of Tarbiat Modares University, Tehran, Iran). Human participants were not directly involved in the present study.

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Rezaei, Z.S., Shahangian, S.S., Hasannia, S. et al. Development of a phage display-mediated immunoassay for the detection of vascular endothelial growth factor. Anal Bioanal Chem 412, 7639–7648 (2020). https://doi.org/10.1007/s00216-020-02901-4

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  • DOI: https://doi.org/10.1007/s00216-020-02901-4

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