基于多肽亲和介导定点偶联策略的抗体元素标签的合成及单细胞分析应用  

Synthesis and Single-cell Analysis Application of Antibody-Element-Tag Based on Peptide Affinity-mediated Site-specific Labeling Strategy

作　　者：王蕾 易立 李华敏 陈石[1] 杨利民[1] 严晓文[1,2] 王秋泉 WANG Lei;YI Li;LI Hua-Min;CHEN Shi;YANG Li-Min;YAN Xiao-Wen;WANG Qiu-Quan(MOE Key Laboratory of Spectrochemical Analysis&Instrumentation,Department of Chemistry,College of Chemistry and Chemical Engineering,Xiamen University,Xiamen 361005,China;Innovation Laboratory for Sciences and Technologies of Energy Materials of Fujian Province(IKKEM),Xiamen 361005,China)

机构地区：[1]厦门大学化学化工学院,化学系,谱学分析与仪器教育部重点实验室,厦门361005 [2]福建省能源材料科学与技术创新实验室(IKKEM),厦门361005

出　　处：《分析化学》2024年第10期1496-1507,共12页Chinese Journal of Analytical Chemistry

基　　金：国家重点研发计划项目(No.2022YFF0710200);国家自然科学基金项目(Nos.22074127,22193053);福建省能源材料科学与技术创新实验室项目(No.HRTP-[2022]-13);厦门大学校长基金项目(No.20720200073)资助。

摘　　要：电感耦合等离子体质谱(ICP-MS)因具有灵敏度高、准确度好、抗干扰能力强和多元素检测等优点,近年来在生物分子定量分析中显示出巨大的应用潜力。元素标签通过靶向基团与元素报告基团组装,实现对目标生物分子的特异性标记和信号报告,对基于ICP-MS的生物定量分析的性能具有至关重要的作用。抗体可与细胞表面的抗原高度特异性结合,是最常用的靶向递送载体之一。传统的随机化学偶联方法难以控制抗体的偶联位点和载荷数量,会增加抗体偶联物的异质性,影响抗体与抗原的特异性识别和结合能力,降低元素标签的靶向能力、定量分析的准确性和标签稳定性。针对这些问题,本研究对抗体的定点偶联策略进行了研究,采用多肽亲和标记技术,将一段短链DNA定点偶联到免疫球蛋白G(IgG)抗体K248位,通过DNA碱基互补配对,实现了单克隆抗体与稀土元素Eu修饰的噬菌体MS2衣壳蛋白的精准组装,并基于此制备了高灵敏度和高选择性的元素标签。本方法克服了随机偶联造成的元素标签的异质性问题,确保了元素标签的靶向特异性、亲和力和信号放大能力。研究结果表明,此元素标签与癌细胞表面的表皮生长因子受体(Epidermal growth factor receptor,EGFR)特异性结合,利用单细胞ICP-MS技术成功实现了癌细胞表面抗原的单细胞分析检测。Recently,inductively coupled plasma mass spectrometry(ICP-MS)has shown great potential in the quantitative analysis of biomolecules due to its high sensitivity,high accuracy,anti-interference ability and multielement detection capability.Elemental tags enable specific labeling of target biomolecules with element-reporter groups,playing a critical role in the performance of bioanalysis using ICP-MS.Antibodies are highly specific to antigens on cell surfaces and are one of the most commonly used targeted delivery vehicles.Traditional random chemical coupling methods are difficult to control the coupling sites and loading numbers of element reporters,which lead to heterogeneity of antibody conjugates,affecting the specific recognition and binding ability of antibodies and antigens,and thus reducing the targeting ability and quantitative accuracy.To solve these issues,site-specific coupling of antibodies with element reporter was studied in this work.By using peptide-mediated affinity labeling technology,a short chain DNA was specifically labeled to immunoglobulin G(IgG)antibody K248,and then DNA base complementary pairing was used to realize accurate assembly of monoclonal antibodies and Eu-modified phage MS2 capsid,resulting in the preparation of highly sensitive and selective element tags.This method overcame the heterogeneity problem of element tags caused by random coupling,and ensured the targeting specificity,affinity and signal amplification ability of element tags.The element tag prepared here showed a specificity to bind/label with epidermal growth factor receptor(EGFR)biomarker on the surface of cancer cells,realizing single cancer cell analysis using ICP-MS.

关 键 词：多肽亲和标记 定点偶联 抗体 元素标签 单细胞-电感耦合等离子体质谱 

分 类 号：Q503[生物学—生物化学] O657.63[理学—分析化学]