Vaccine Research and Development Center, National Health Research Institutes, Taiwan.
J Virol Methods. 2010 Apr;165(1):42-5. doi: 10.1016/j.jviromet.2009.12.015. Epub 2009 Dec 29.
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.
肠道病毒 71 型（EV71）是手足口病（HFMD）的主要病原体，可导致幼儿频繁出现严重的神经并发症和死亡率。血清中和抗体反应是 EV71 感染和保护性免疫的主要指标。目前基于细胞病变抑制（CPE）的血清中和试验（Nt-CPE）需要进行人工显微镜检查，既费时又费力。在这项研究中，我们开发了一种高通量中和测定法，该测定法采用酶免疫测定法（EIA）检测横纹肌肉瘤（RD）细胞中 EV71 的生长，并测量针对 EV71 的血清中和抗体（Nt-EIA）。RD 细胞感染 100 TCID（50）的 EV71 36-42h 后具有最佳性能，并被选择用于 Nt-EIA。用 Nt-CPE 和 Nt-EIA 测量了 120 份人血清（59 份阴性血清，61 份阳性血清）的 EV71 中和抗体滴度。通过与 Nt-CPE 的定性比较，Nt-EIA 测定的 EV71 中和抗体滴度具有 100%的灵敏度（100%）、94.9%的特异性和 97.5%的一致性。在定量比较中，Nt-EIA 和 Nt-CPE 之间的相关系数在对数转换后为 0.91。总的来说，Nt-EIA 是一种替代定量测定 EV71 中和抗体对 EV71 的合适方法。
