Thanongsaksrikul Jeeraphong, Srimanote Potjanee, Tongtawe Pongsri, Glab-Ampai Kittirat, Malik Aijaz Ahmad, Supasorn Oratai, Chiawwit Phatcharaporn, Poovorawan Yong, Chaicumpa Wanpen
Graduate Programme in Biomedical Science, Faculty of Allied Health Sciences, Thammasat University, 99 Moo 18 Paholyothin Road, Klong Luang, Rangsit, Pathum Thani, 12120, Thailand.
Faculty of Medicine Siriraj Hospital, Center of Research Excellence on Therapeutic Proteins and Antibody Engineering, Mahidol University, Bangkok, Thailand.
Arch Virol. 2018 May;163(5):1141-1152. doi: 10.1007/s00705-018-3731-z. Epub 2018 Jan 22.
Enterovirus-71 (EV71) and coxsackievirus-A16 (CA16) frequently cause hand-foot-mouth disease (HFMD) epidemics among infants and young children. CA16 infections are usually mild, while EV71 disease may be fatal due to neurologic complications. As such, the ability to rapidly and specifically recognize EV71 is needed to facilitate proper case management and epidemic control. Accordingly, the aim of this study was to generate antibodies to EV71-virion protein-2 (VP2) by phage display technology for further use in specific detection of EV71. A recombinant peptide sequence of EV71-VP2, carrying a predicted conserved B cell epitope fused to glutathione-S-transferase (GST) (designated GST-EV71-VP2/131-160), was produced. The fusion protein was used as bait in in-solution biopanning to separate protein-bound phages from a murine scFv (MuscFv) phage display library constructed from an immunoglobulin gene repertoire from naïve ICR mice. Three phage-transformed E. coli clones (clones 63, 82, and 83) produced MuscFvs that bound to the GST-EV71-VP2/131-160 peptide. The MuscFv of clone 83 (MuscFv83), which produced the highest ELISA signal to the target antigen, was further tested. MuscFv83 also bound to full-length EV71-VP2 and EV71 particles, but did not bind to GST, full-length EV71-VP1, or the antigenically related CA16. MuscFv83 could be a suitable reagent for rapid antigen-based immunoassay, such as immunochromatography (ICT), for the specific detection and/or diagnosis of EV71 infection as well as epidemic surveillance.
肠道病毒71型（EV71）和柯萨奇病毒A16型（CA16）经常在婴幼儿中引发手足口病（HFMD）疫情。CA16感染通常症状较轻，而EV71疾病可能因神经系统并发症而致命。因此，需要具备快速、特异性识别EV71的能力，以促进妥善的病例管理和疫情控制。相应地，本研究的目的是通过噬菌体展示技术产生针对EV71病毒体蛋白2（VP2）的抗体，以便进一步用于EV71的特异性检测。制备了一段携带预测保守B细胞表位并与谷胱甘肽-S-转移酶（GST）融合的EV71-VP2重组肽序列（命名为GST-EV71-VP2/131-160）。该融合蛋白用作溶液内生物淘选的诱饵，以从由未经免疫的ICR小鼠免疫球蛋白基因库构建的鼠单链抗体（MuscFv）噬菌体展示文库中分离与蛋白结合的噬菌体。三个经噬菌体转化的大肠杆菌克隆（克隆63、82和83）产生了与GST-EV71-VP2/131-160肽结合的MuscFv。对产生针对靶抗原最高ELISA信号的克隆83的MuscFv（MuscFv83）进行了进一步测试。MuscFv83也与全长EV71-VP2和EV71颗粒结合，但不与GST,全长EV71-VP1或抗原相关的CA16结合。MuscFv83可能是用于基于抗原的快速免疫测定（如免疫层析法（ICT））的合适试剂，用于EV71感染的特异性检测和/或诊断以及疫情监测。
