Development of a high-throughput assay for measuring serum neutralizing antibody against enterovirus 71

Enterovirus 71 (EV71) is the main etiologic agent of hand, foot, and mouth disease (HFMD) and causes frequently severe neurological complications and mortality in young children. The serum neutralizing antibody response is the major indicator of EV71 infection and protective immunity. The current serum neutralization test based on inhibition of cytopathic effect (Nt-CPE) requires manual microscopic examination, which is time-consuming and labor-intensive. In this study, a high-throughput neutralization assay which employs enzyme immunoassay for detecting growth of EV71 in Rhabdomyosarcoma (RD) cells and measuring serum neutralizing antibody (Nt-EIA) against EV71 was developed. RD cells infected with 100 TCID(50) of EV71 for 36-42h had the best performance and were selected for Nt-EIA. One hundred and twenty human sera (59 negative sera, 61 positive sera) were measured for EV71 neutralization antibody titers by Nt-CPE and Nt-EIA. Neutralization antibody titers against EV71 determined by Nt-EIA had a high sensitivity (100%), specificity (94.9%) and agreement (97.5%) by a qualitative comparison with Nt-CPE. In the quantitative comparison, the correlation coefficient between Nt-EIA and Nt-CPE was 0.91 after log transformation. Overall, the Nt-EIA is a suitable alternative assay for the quantitation of EV71 neutralizing antibody to EV71.