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⁻¹ and 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.

具有位点特异性、定向和共价模式的固定化抗体对于提高固相免疫测定的灵敏度具有重要意义。在这里，我们开发了一种新的抗体偶联策略，可以以定向和共价方式固定抗体。在这项研究中，IgG-Fc 结合蛋白（Z 域）携带位点特异性光交联剂p-苯甲酰基-L-苯丙氨酸和单个 C 端半胱氨酸 (Cys) 手柄是基因工程设计的。在紫外线照射下，嵌合蛋白使 Cys 手柄以 Fc 特异性和共价结合模式与天然抗体偶联，从而产生新型硫醇化抗体。因此，基于巯基和马来酰亚胺基团之间的交联，开发了一种将抗体共价、定向固定到马来酰亚胺修饰的磁性纳米粒子 (MNP) 的方法。将抗体偶联的 MNP 应用于基于 MNP 的酶联免疫吸附试验 (ELISA) 以检测癌胚抗原。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，其灵敏度比传统微孔板 ELISA（2.0 ng mL ^-1）高约 100 倍。因此，所提出的抗体固定化方法可用于表面功能化以灵敏检测各种生物标志物。