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首页> 美国卫生研究院文献>Scientific Reports >An innovative flow cytometry method to screen human scFv-phages selected by in vivo phage-display in an animal model of atherosclerosis
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An innovative flow cytometry method to screen human scFv-phages selected by in vivo phage-display in an animal model of atherosclerosis

机译：一种创新的流式细胞术方法用于筛选在动脉粥样硬化动物模型中通过体内噬菌体展示选择的人scFv噬菌体

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Atherosclerosis is a chronic, progressive inflammatory disease that may develop into vulnerable lesions leading to thrombosis. This pathology is characterized by the deposition of lipids within the arterial wall and infiltration of immune cells leading to amplification of inflammation. Nowadays there is a rising interest to assess directly the molecular and cellular components that underlie the clinical condition of stroke and myocardial infarction. Single chain fragment variable (scFv)-phages issuing from a human combinatorial library were selected on the lesions induced in a rabbit model of atherosclerosis after three rounds of in vivo phage display. We further implemented a high-throughput flow cytometry method on rabbit protein extracts to individually test one thousand of scFv-phages. Two hundred and nine clones were retrieved on the basis of their specificity for atherosclerotic proteins. Immunohistochemistry assays confirmed the robustness of the designed cytometry protocol. Sequencing of candidates demonstrated their high diversity in VH and VL germline usage. The large number of candidates and their diversity open the way in the discovery of new biomarkers. Here, we successfully showed the capacity of combining in vivo phage display and high-throughput cytometry strategies to give new insights in in vivo targetable up-regulated biomarkers in atherosclerosis.
机译：动脉粥样硬化是一种慢性进行性炎性疾病，可能发展成脆弱的病变，导致血栓形成。这种病理学的特征在于脂质在动脉壁内的沉积和免疫细胞的渗透导致炎症的加剧。如今，越来越需要直接评估构成中风和心肌梗塞临床状况的分子和细胞成分。在三轮体内噬菌体展示后，从人组合文库中发出的单链片段可变（scFv）噬菌体选择在动脉粥样硬化兔模型中诱导的病变上。我们进一步对兔蛋白提取物实施了高通量流式细胞术，以单独测试一千个scFv噬菌体。根据其对动脉粥样硬化蛋白的特异性，检索了209个克隆。免疫组织化学测定证实了所设计的细胞计数方案的鲁棒性。候选者的测序证明了他们在VH和VL种系使用方面的高度多样性。大量的候选人及其多样性为发现新的生物标记打开了道路。在这里，我们成功地展示了结合体内噬菌体展示和高通量细胞术策略的能力，从而为动脉粥样硬化的体内靶向上调生物标志物提供了新的见解。

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