The plates were washed thrice and developed with TMB solution (Tiangen Biotech, Beijing, China) in a dark room for 15 min, and the enzyme reaction was stopped by adding 2 M H2SO4. SNX-5422 ic50 The absorbance at 450 nm was measured using a microplate reader (Bio-Rad, USA). Epitope mapping Enzyme-linked immunosorbent assay (ELISA) protocols were used for all the

epitope mapping experiments. Peptides LEE011 manufacturer truncated at the carboxyl end or the amino terminus were purchased from Scilight-Peptide (Beijing, China). The peptides were conjugated with Bull Serum Albumin (BSA). The purity was higher than 90%. In vitro neutralization assay EV71 BJ08 (genogroup C4) and BrCr-TR (genogroup A), were propagated in RD cells. Virus titers were determined using RD cells by the microtitration method and expressed as the 50% tissue culture infective dose (TCID50) according to the Reed–Muench method. Two-fold serial dilutions of sera were prepared using Minimum Essential Medium (MEM,Gibco®) containing 2% FBS. The EV71 stock was diluted to a working concentration of 100 TCID50/50 μl. The neutralization assay was conducted using 96-well

plates. In each well, 50 μl of diluted serum sample was mixed with 50 μl RAD001 in vitro of EV71 at 100 TCID50, and incubated overnight at 37°C. Next, 100 μl of cell suspension containing 10,000 RD cells was added to wells containing the virus/antiserum mixtures and incubated at 37°C. After 7 days, the cells were observed to evaluate the appearance of cytopathic effects. Neutralization titer was defined as the highest serum dilution that could completely protect cells from developing cytopathic effects. Mouse protection assay To evaluate

protective efficacy of the immunized sera against EV71 infection, in vivo infection experiments were performed. Briefly, 50 μl of sera or PBS were incubated with 10 LD50 of EV71 BrCr-TR (50 μl in sterile RD cell supernatant) at 37°C for 2 hour. Groups of one-day-old BALB/c suckling mice (n = 10 per group) for were inoculated intraperitoneally (i.p.) with the virus-sera mixture or virus-PBS mixture. All mice were monitored daily for clinical symptoms and death for up to 16 days after inoculation. Acknowledgements This work was supported by grants from International Science & Technology Cooperation (No. 2011DFG33200), National High Technology Research and Development Program of China (863 Program, No. 2012AA02A400), Program for New Century Excellent Talents in University (No. JU015001201001), Jilin Program for Development of Science and Technology (No.20106043) and Beijing Municipal Education Committee Foundation (JJ015001201301). References 1. Schmidt NJ, Lennette EH, Ho HH: An Apparently New Enterovirus Isolated from Patients with Disease of the Central Nervous System. J Infect Dis 1974,129(3):304–309.PubMedCrossRef 2. Brown BA, Pallansch MA: Complete nucleotide sequence of enterovirus 71 is distinct from poliovirus. Virus Res 1995,39(2–3):195–205.PubMedCrossRef 3.