Development of a phage display-mediated immunoassay for the detection of vascular endothelial growth factor
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RESEARCH PAPER
Development of a phage display-mediated immunoassay for the detection of vascular endothelial growth factor Zahra S. Rezaei 1 & S. Shirin Shahangian 2 & Sadegh Hasannia 1 & Reza H. Sajedi 1 Received: 16 March 2020 / Revised: 15 August 2020 / Accepted: 19 August 2020 # Springer-Verlag GmbH Germany, part of Springer Nature 2020
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. Keywords VEGF . Single-domain antibody . Phage ELISA . Phage display-mediated immuno-PCR (PD-IPCR)
Introduction Angiogenesis is the process involving the formation of new blood vessels from previous arteries. Physiological angiogenesis occurs during fetal development, wound healing, and menstruation, under precise temporal and spatial regulation [1]. Vascular endothelial growth factor (VEGF) is the key angiogenic factor in developmental, physiological, and pathological conditions [2]. VEGF dependence has been demonstrated in pathophysiological processes such as rheumatoid arthritis, psoriasis, arteriosclerosis, amyotrophic lateral sclerosis (ALS), age-related macular degeneration (AMD), diabetic
* Reza H. Sajedi [email protected] 1
Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, Jalal Ale Ahmad Highway, Tehran 14115-154, Iran
2
Department of Biology, Faculty of Sciences, University of Guilan, Rasht, Guilan 4199613776, Iran
retinopathy, sepsis, and tumor angiogenesis [3–9]. Abnormal and leaky tumor blood vessels reduce blood flow and increase interstitial fluid pressure in tumors, eventually causing hypoxia. Accordingly, in order to supply oxygen and nutrients for tumor gr
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