Min Thae Thae, Yamabhai Montarop
Molecular Biotechnology Laboratory, School of Biotechnology, Institute of Agricultural Technology, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand.
ACS Omega. 2020 Dec 22;6(1):762-774. doi: 10.1021/acsomega.0c05340. eCollection 2021 Jan 12.
The single-chain variable fragment (scFv) of monoclonal antibodies is a promising recombinant nanostructure for various medical applications, including bioimaging and targeted therapy. While numerous scFv antibodies against eukaryotic cell surface proteins (especially cancer biomarkers) have been generated and engineered to suit various purposes, only a few specific scFv against bacterial cell surfaces have been developed, especially those of human origin. Recent incidents of emerging multidrug-resistant pathogenic bacteria and the realization of the importance of a balanced microbiota on the health of the host has led to more interests in the development of recombinant antibacterial antibodies as a detection probe or targeted therapy for bacterial infections. This study reports the generation of two specific human antibacterial scFv using phage display antibody technology. The recombinant scFv fragments of about 30 kDa and a diameter of 5 nm were produced and purified from engineered that can enhance cytosolic disulfide bond formation. As a proof of principle, and were used as model Gram-positive and Gram-negative bacteria, respectively. Specificity at the strain and species level to both planktonic and biofilm forms of these bacteria were demonstrated in various assay formats, namely, ELISA, flow cytometry, western blot, immunofluorescence, and electron microscopy via the hexa-histidine tag. This recombinant scFv generation platform can be applied for other bacteria, and since the scFv obtained has a benefit of being a human origin, it could be conveniently engineered for various therapeutic or theranostic applications with minimized adverse immunoreaction.
单克隆抗体的单链可变片段（scFv）是一种很有前景的重组纳米结构，可用于包括生物成像和靶向治疗在内的各种医学应用。虽然已经产生并设计了许多针对真核细胞表面蛋白（尤其是癌症生物标志物）的scFv抗体以满足各种目的，但针对细菌细胞表面的特异性scFv却只开发了少数几种，尤其是源自人类的。近期出现的多重耐药病原菌事件以及认识到平衡微生物群对宿主健康的重要性，引发了人们对开发重组抗菌抗体作为细菌感染检测探针或靶向治疗手段的更多兴趣。本研究报告了利用噬菌体展示抗体技术产生的两种特异性人类抗菌scFv。从能够增强胞质二硫键形成的工程菌中制备并纯化了约30 kDa、直径为5 nm的重组scFv片段。作为原理验证，分别使用[具体革兰氏阳性菌名称]和[具体革兰氏阴性菌名称]作为革兰氏阳性菌和革兰氏阴性菌模型。通过六组氨酸标签，在酶联免疫吸附测定（ELISA）、流式细胞术、蛋白质免疫印迹、免疫荧光和电子显微镜等各种检测形式中，证明了对这些细菌的浮游和生物膜形式在菌株和物种水平上的特异性。这种重组scFv生成平台可应用于其他细菌，并且由于获得的scFv源自人类，因此可以方便地进行工程改造，用于各种治疗或诊疗应用，同时将不良免疫反应降至最低。
