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.

Graphical abstract

中文翻译：

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用于检测血管内皮生长因子的噬菌体展示介导的免疫测定的开发。

由于血管内皮生长因子（VEGF）在血管生成中的关键作用及其在癌症早期阶段的血清水平显着升高，因此VEGF被认为是不同癌症中重要的预后生物标志物。在此，基于噬菌体展示介导的免疫PCR（PD-IPCR），精确设计了PCR结合噬菌体展示抗VEGF VHH（一种灵敏的实时免疫测定法）的扩增能力以检测VEGF。该系统得益于在夹心免疫吸附测定平台中使用阿瓦斯汀（抗-VEGF单克隆抗体）作为捕获抗体的抗原与抗体的强而特异性结合。抗VEGF噬菌体颗粒在PD-IPCR中既用作抗VEGF剂又用作DNA模板。抗VEGF噬菌体ELISA的线性范围为3–250 ng / ml，检出限（LOD）为1.1 ng / ml。使用PD-IPCR方法，发现VEGF检测的线性范围为0.06-700 ng / ml，检测极限为3 pg / ml。血清的回收率从83％到99％不等，相对标准偏差为1.2–4.9％。这些值表明该方法在临床分析中具有良好的敏感性。该方法成功地用于人血清中VEGF的临床测定，结果与常规ELISA（R ²  ＝ 0.995）。新颖的免疫测定为非常低水平的VEGF检测提供了特异性和灵敏的免疫测定方案。

图形概要