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Isolation and characterization of a novel nanobody for detection of GRP78 expressing cancer cells
Biotechnology and Applied Biochemistry ( IF 3.2 ) Pub Date : 2020-04-09 , DOI: 10.1002/bab.1916 Hossein Aghamollaei 1 , Mostafa Ghanei 1 , Mohammad Javad Rasaee 2 , Ali Mohammad Latifi 3 , Hamid Bakherad 4 , Mahdi Fasihi-Ramandi 5 , Ramezan Ali Taheri 6 , Seyed Latif Mousavi Gargari 7
Biotechnology and Applied Biochemistry ( IF 3.2 ) Pub Date : 2020-04-09 , DOI: 10.1002/bab.1916 Hossein Aghamollaei 1 , Mostafa Ghanei 1 , Mohammad Javad Rasaee 2 , Ali Mohammad Latifi 3 , Hamid Bakherad 4 , Mahdi Fasihi-Ramandi 5 , Ramezan Ali Taheri 6 , Seyed Latif Mousavi Gargari 7
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Glucose‐regulated protein 78 (GRP78) is an endoplasmic reticulum (ER) chaperone that has been shown that is overexpressed in cancer cells. Overexpression of GRP78 on cancer cells makes this molecule a suitable candidate for cancer detection and targeted therapy. VHH is the binding fragment of camelid heavy‐chain antibodies also known as “nanobody.” The aim of this study is to isolate and produce a new recombinant nanobody using phage display technique to detect cancer cells. Using the c‐terminal domain of GRP78 (CGRP) as an antigen, four rounds of biopanning were performed, and high‐affinity binders were selected by ELISA. Their affinity and functionality were characterized by surface plasmon resonance (SPR) cell ELISA and immunocytochemistry. A unique nanobody named V80 was purified. ELISA and SPR showed that this antibody had high specificity and affinity to the GRP78. Immunofluorescence analysis showed that V80 could specifically bind to the HepG2 and A549 cancer cell lines. This novel recombinant nanobody could bind to the cell surface of different cancer cells. After further evaluation, this nanobody can be used as a new tool for cancer detection and tumor therapy.
中文翻译：
新型纳米抗体的分离和表征，用于检测表达GRP78的癌细胞
葡萄糖调节蛋白78（GRP78）是一种内质网（ER）分子伴侣，已显示在癌细胞中过表达。GRP78在癌细胞上的过表达使该分子成为癌症检测和靶向治疗的合适候选者。VHH是骆驼科重链抗体的结合片段，也称为“纳米抗体”。这项研究的目的是使用噬菌体展示技术分离和生产一种新的重组纳米抗体，以检测癌细胞。使用GRP78（CGRP）的c末端结构域作为抗原，进行了四轮生物淘选，并通过ELISA选择了高亲和力的结合物。它们的亲和力和功能通过表面等离子体共振（SPR）细胞ELISA和免疫细胞化学来表征。纯化了一个独特的名为V80的纳米抗体。ELISA和SPR表明该抗体对GRP78具有高特异性和亲和力。免疫荧光分析表明，V80可以特异性结合HepG2和A549癌细胞系。这种新颖的重组纳米抗体可以与不同癌细胞的细胞表面结合。经过进一步评估，该纳米抗体可以用作癌症检测和肿瘤治疗的新工具。
更新日期：2020-04-09
中文翻译：
新型纳米抗体的分离和表征，用于检测表达GRP78的癌细胞
葡萄糖调节蛋白78（GRP78）是一种内质网（ER）分子伴侣，已显示在癌细胞中过表达。GRP78在癌细胞上的过表达使该分子成为癌症检测和靶向治疗的合适候选者。VHH是骆驼科重链抗体的结合片段，也称为“纳米抗体”。这项研究的目的是使用噬菌体展示技术分离和生产一种新的重组纳米抗体，以检测癌细胞。使用GRP78（CGRP）的c末端结构域作为抗原，进行了四轮生物淘选，并通过ELISA选择了高亲和力的结合物。它们的亲和力和功能通过表面等离子体共振（SPR）细胞ELISA和免疫细胞化学来表征。纯化了一个独特的名为V80的纳米抗体。ELISA和SPR表明该抗体对GRP78具有高特异性和亲和力。免疫荧光分析表明，V80可以特异性结合HepG2和A549癌细胞系。这种新颖的重组纳米抗体可以与不同癌细胞的细胞表面结合。经过进一步评估，该纳米抗体可以用作癌症检测和肿瘤治疗的新工具。
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