In vitro selection of anti-gliadin single-domain antibodies from a naive library for cDNA-display mediated immuno-PCR
In vitro selection of anti-gliadin single-domain antibodies from a naive library for cDNA-display mediated immuno-PCR
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DOI：
10.1016/j.ab.2019.113490
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发表时间：
2020-01-15
影响因子：
2.9
通讯作者：
Nemoto, Naoto
中科院分区：
文献类型：
--
作者：
Jayathilake, Chathuni;Kumachi, Shigefumi;Nemoto, Naoto
麸质不耐受，或对麸质的不良肠道反应，是某些人群中相当常见的问题。乳糜泻是最严重的麸质不耐受形式，可导致消化系统永久性损伤。由于终身避免麸质是唯一可用的治疗方法，因此开发可靠的技术来识别食品中的麸质污染是很重要的。麦醇溶蛋白是谷蛋白的一种组分，已知在谷蛋白毒性中起主要作用。本研究利用cDNA展示技术，从羊驼源天然VHH文库中筛选抗毒麦醇溶蛋白的特异性单域抗体。cDNA展示方法是一种很有前途的体外展示技术，其使用精心设计的嘌呤霉素接头将不稳定的mRNA-蛋白质融合分子独特地转化为稳定的mRNA/cDNA-蛋白质融合分子。通过pulldown和间接ELISA法检测3个候选VHH的亲和力。此外，一种新的cDNA展示介导的免疫PCR方法（cD-IPCR）已成功地应用于检测食品中的醇溶蛋白。我们相信，这项工作表明了潜在的应用cDNA展示方法在选择结合剂对有毒和异质性的目标，如麦醇溶蛋白与免免疫制备方式。
Gluten intolerance, or adverse intestinal reactions to gluten, is a fairly common problem among certain groups of people. Celiac disease is the most severe form of gluten intolerance, which can lead to permanent damage in the digestive system. Since lifelong avoidance of gluten is the only available treatment, development of reliable techniques to identify gluten contamination in food is important. Gliadin, a component of gluten, is known to play a major role in gluten toxicity. In this study, cDNA display method was used to select specific single-domain antibodies against toxic gliadin from an alpaca-derived naive VHH library. The cDNA display method is a promising in vitro display technique, which uniquely converts an unstable mRNA-protein fusion molecule to a stable mRNA/cDNA-protein fusion molecule using a well-designed puromycin linker. Three candidate VHHs were selected and the affinities of the VHHs were observed by pulldown assay and indirect ELISA method. In addition, a novel cDNA display mediated immuno-PCR method (cD-IPCR) was successfully applied to detect gliadin in food. We believe this work demonstrates the potential application of the cDNA display method in selecting binders against toxic and heterogeneous targets such as gliadin with an immunization-free preparation manner.