Identification and production of mouse scFv to specific epitope of enterovirus-71 virion protein-2 (VP2)
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.
Funding was provided by grants from the following sources: Thailand Research Fund (MRG5680105); Thammasat University (Project IDs 218028, 180630, and 151632); Center of Excellence in Clinical Virology (GCE 58-014-30-004); Research Chair Grant (P-15-50004); and, NSTDA Chair Professor Grant (P-1450624). The authors gratefully acknowledge Ms Jiratchaya Puenpa of Chulalongkorn University for EV71 propagation.
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All authors declare no personal or professional conflicts of interest, and no financial support from the companies that produce and/or distribute the drugs, devices, or materials described in this report.
Institutional guidelines for the care and use of animals were followed.
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This article does not contain any studies with human participants performed by any of the authors.
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