目的 建立基于细胞表面特异性识别的荧光标记小分子抗体的鉴定方法并应用于食管癌外周血循环肿瘤细胞(circulating tumor cells,CTCs)检测。方法 本研究前期利用噬菌体技术高通量筛选富集到单链噬菌体抗体NFC 20b和NFC 70a,标记荧光后,观察在斑马鱼体内与食管癌细胞的聚集示踪,并用于检测食管癌患者的外周血CTCs;同时通过高通量蛋白组芯片对抗原蛋白表达谱进行分析。结果 荧光小分子抗体NFC 20b和NFC 70a于不同时间点,在斑马鱼的背主动脉、尾动脉和尾静脉中均能观察到抗体分布,与空白组和阴性抗体组相比,两抗体与移植瘤结合荧光更强;用蛋白芯片筛选抗原蛋白表达谱,初步确定NFC 20b结合的抗原蛋白为细胞周期蛋白依赖性激酶2(CDK2)。结论 荧光标记的小分子抗体可以通过血液循环分布到斑马鱼的卵黄囊,并能与移植瘤有明显的聚集结合效应,可以用于检测食管癌患者外周血CTCs;利用蛋白芯片技术可以辅助鉴定抗体所结合的抗原种类。
Abstract
Objective To establish a method of identification of fluorescent-labeled small molecule antibodies based on cell surface specific recognition and to verify their applicability in detection of peripheral blood circulating tumor cells (CTCs) of esophageal cancer.Methods Phage antibodies NFC 20b and NFC 70a were obtained via high-throughput screening and enrichment using phage display technology in the early stage of this study. After fluorescence labeling, the aggregation tracer between them and esophageal cancer cells was observed in zebrafish. Small molecule antibodies were also used to detect CTCs in peripheral blood of esophageal cancer patients. Meanwhile, the expression profile of antigen proteins was analyzed using the high-throughput protein chip.Results The distribution of fluorescent labeled small molecule antibodies NFC 20b and NFC 70a could be observed in the dorsal aorta, caudal artery and caudal vein of zebrafish, so was the binding of antibodies to transplanted tumors in yolk sac. The protein chip used to screen the expression profile of the antigen protein proved that the antigen protein bound by NFC 20b was cyclin dependent kinase 2(CDK2).Conclusions Small molecule antibodies labeled by fluorescence labeling technology can be distributed to yolk sac through blood circulation, and can aggregate with and bind to transplanted tumors of esophageal cancer in yolk sac. This approach can be used to detect CTCs in peripheral blood of ESCC. The protein chip can be used to assist in identifying the antigen binding to small antibodies.
关键词
循环肿瘤细胞 /
斑马鱼 /
荧光标记 /
抗体 /
蛋白芯片
Key words
circulating tumor cells /
zebrafish /
fluorescent labeled /
antibody /
protein chip
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基金
国家自然科学基金资助项目(31500756);军队创新培育工程资助项目(16QNP023)
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