Directional immobilization of antibody onto magnetic nanoparticles by Fc-binding protein-assisted photo-conjugation for high sensitivity detection of antigen.
Directional immobilization of antibody onto magnetic nanoparticles by Fc-binding protein-assisted photo-conjugation for high sensitivity detection of antigen.
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DOI：
10.1016/j.aca.2021.339054
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发表时间：
2021-11
影响因子：
6.2
通讯作者：
Jin-Bao Tang;Hong-ming Yang;Xiao-Yi Gao;Xianghua Zeng;Feng-Shan Wang
中科院分区：
文献类型：
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作者：
Jin-Bao Tang;Hong-ming Yang;Xiao-Yi Gao;Xianghua Zeng;Feng-Shan Wang
具有位点特异性、定向性和共价性的固定化抗体对提高固相免疫分析的灵敏度具有重要意义。在这里，我们开发了一种新的抗体缀合策略，可以以定向和共价的方式缀合抗体。在这项研究中，IgG-Fc结合蛋白（Z结构域）携带位点特异性光交联剂，对苯甲酰基-L-苯丙氨酸，和一个单一的C-末端半胱氨酸（Cys）处理的基因工程。在UV照射后，嵌合蛋白使得Cys柄能够以Fc特异性和共价缀合模式与天然抗体偶联，从而产生新型巯基化抗体。因此，基于巯基和马来酰亚胺基团之间的交联，开发了用于将抗体共价、定向固定到马来酰亚胺修饰的磁性纳米颗粒（MNP）的方法。将抗体偶联的MNP应用于MNP酶联免疫吸附试验（ELISA）检测癌胚抗原。基于MNP的ELISA的定量线性范围为0.1-100 ng mL− 1，检测限为0.02 ng mL−1，比传统的微孔板ELISA（2.0 ng mL−1）灵敏约100倍。因此，所提出的抗体固定方法可用于表面功能化，用于各种生物标志物的灵敏检测。
Immobilized antibodies with site-specific, oriented, and covalent pattern are of great significance to improve the sensitivity of solid-phase immunoassay. Here, we developed a novel antibody conjugation strategy that can immobilize antibodies in a directional and covalent manner. In this study, an IgG-Fc binding protein (Z domain) carrying a site-specific photo-crosslinker,p-benzoyl-L-phenylalanine, and a single C-terminal cysteine (Cys) handle was genetically engineered. Upon UV irradiation, the chimeric protein enables the Cys handle to couple with the native antibody in Fc-specific and covalent conjugation pattern, resulting in a novel thiolated antibody. Thus, an approach for the covalent, directional immobilization of antibodies to maleimide-modified magnetic nanoparticles (MNPs) was developed on the basis of the crosslinking between sulfhydryl and maleimide groups. The antibody-conjugated MNPs were applied in MNP-based enzyme-linked immunosorbent assay (ELISA) for the detection of carcinoembryonic antigen. The MNP-based ELISA presented a quantification linear range of 0.1–100 ng mL−1and detection limit of 0.02 ng mL−1, which was approximately 100 times more sensitive than the traditional microplate ELISA (2.0 ng mL−1). Thus, the proposed antibody immobilization approach can be used in surface functionalization for the sensitive detection of various biomarkers.