Fluorophore–antibody conjugates with high photobleaching resistance, high chemical stability, and Fc-specific attachment is a great advantage for immunofluorescence imaging. Here, an Fc-binding protein (Z-domain) carrying a photo-cross-linker (p-benzoylphenylalanine, Bpa) fused with enhanced green fluorescent protein (EGFP), namely photoactivatable Z_Bpa–EGFP recombinant, was directly generated using the aminoacyl-tRNA synthetase/suppressor tRNA technique without any further modification. By employing the photoactivatable Z_Bpa–EGFP, an optimal approach was successfully developed which enabled EGFP to site-selectively and covalently attach to native antibody (IgG) with approximately 90% conjugation efficiency. After characterizing the Fc-specific and covalent manner of the EGFP-photoconjugated antibody, its excellent photobleaching resistance for immunofluorescence imaging was demonstrated in a model study by monitoring the toll-like receptor 4 (TLR4) expression in HepG2 cells. The proposed approach here for the preparation of a novel fluorescent antibody is available and reliable, which would play an important role in fluorescence immunoassay, and is expected to be extended to the generation of other biomolecule-photoconjugated antibodies, such as other fluorescent proteins for multiplex immunofluorescence imaging or reporter enzymes for highly sensitive enzyme immunoassays.

Graphical abstract

具有高抗光漂白性，高化学稳定性和Fc特异性结合的荧光团-抗体结合物是免疫荧光成像的一大优势。在此，使用氨基酰基直接生成带有光交联剂（对苯甲酰基苯基丙氨酸，Bpa）与增强型绿色荧光蛋白（EGFP）融合的Fc结合蛋白（Z结构域），即可光活化的Z _Bpa –EGFP重组体。 -tRNA合成酶/抑制剂tRNA技术，无需任何进一步修饰。通过使用可光活化的Z _Bpa–EGFP成功开发出了一种最佳方法，该方法使EGFP能够以约90％的结合效率与天然抗体（IgG）进行位点选择和共价结合。在表征了EGFP-光偶联抗体的Fc特异性和共价方式后，通过监测HepG2细胞中的Toll样受体4（TLR4）表达，在模型研究中证明了其对免疫荧光成像的出色的抗光漂白性。这里提出的用于制备新型荧光抗体的方法是可行且可靠的，它将在荧光免疫测定中发挥重要作用，并有望扩展到其他生物分子-光偶联抗体的产生，

图形概要