Enterovirus 71 (EV71) is responsible for the outbreaks of hand-foot-and-mouth disease that caused significant mortality in children, but no vaccine is available yet. EV71 virus-like particle (VLP) is the empty capsid consisting of viral structural proteins but can elicit potent immune responses, rendering VLP a promising EV71 vaccine candidate. To evaluate whether VLP remains stable after long-term storage, which is crucial for advancing the VLP vaccine to the clinical setting, we evaluated the effects of NaCl concentration, buffers and temperatures on the VLP stability. We first validated the use of dynamic light scattering (DLS) for measuring the hydrodynamic diameter (approximate to 30-35 nm) of VLP, which was close to the VLP diameter (approximate to 25-27 nm) as measured by transmission electron microscopy (TEM). Using these techniques, we found that EV71 VLP remained stable for 5 months in sodium phosphate (NaPi) buffers with various NaCl concentrations. EV71 VLP also remained morphologically stable in NaPi, citrate and TE+ buffers for 5 months, yet the enzyme-linked immunosorbent assay (ELISA) revealed that the VLP stored in citrate and TE+ buffers partially lost the immunogenicity after 5 months. In contrast, the VLP stored in the NaPi buffer at 4 degrees C remained stable macroscopically and microscopically for 5 months, as judged from the DLS, TEM and ELISA. The VLP stored at 25 degrees C and 37 degrees C also retained stability for 1 month, which would obviate the need of a cold chain during the shipping. These data altogether proved the stability of EV71 VLP and suggested that the VLP is amenable to bioprocessing and storage. (C) 2013, The Society for Biotechnology, Japan. All rights reserved.