目的 应用微量致细胞病变（micro-cytopathic effect，CPE）法建立抗柯萨奇病毒A组6型（coxsackievirus A6，CV-A6）中和抗体检测方法并进行方法学验证。方法 用人横纹肌瘤细胞和CV-A6检测毒株建立基于CPE的中和抗体检测方法，并对相对准确度、精密度、线性、专属性和耐用性等进行验证。结果 建立了针对CV-A6免疫血清的中和抗体效价测定方法。3份不同稀释度（1×、16×、256×）的血清参考品各测定3次，相对偏倚分别为﹣2%、﹣4%、﹣16%，拟合线性回归斜率为1.065，相对准确度良好；几何变异系数（geometric coefficient of variation, GCV）为19%～50%，重复性良好。取10份小鼠免疫血清于不同天测定3次中和抗体效价，GCV为0%～75%，中间精密度良好。将抗CV-A6中和抗体血清参考品稀释9个稀释度，独立测定3次，拟合直线的回归系数为0.93，直线回归具有统计学意义（F=340.99，P＜0.000 1）。阴性血清中和效价均＜8，表明专属性良好。不同细胞代次、不同细胞密度、不同病毒载量、不同培养时间、待检血清反复冻融不同次数及短暂储存时间范围的GCV为23%～101%，表明该方法耐用性良好。结论 成功建立了抗CV-A6中和抗体检测方法，该方法具有较好的相对准确度、精密度、线性、专属性及耐用性，可用于评价CV-A6免疫血清的中和抗体水平。

Objective To establish and validate the detection method of coxsackievirus A6 (CV-A6) neutralizing antibody by micro-cytopathic effect (CPE) method.Methods A neutralizing antibody detection method based on CPE was established using human rhabdomyosarcoma (RD) cells and CV-A6 standard detection strains. It was verified for relative accuracy, precision, linearity, specificity, and durability.Results A determination method of neutralizing antibody titer for CV-A6 immune serum was established. Three serum reference samples of different dilutions (1×, 16×, and 256×) were measured 3 times with relative biases of ﹣2%, ﹣4%, and ﹣16%, respectively. The slope of the fitted linear regression was 1.065, indicating good relative accuracy. The geometric coefficients of variation (GCVs) were 19%-50%, and the repeatability was good. The GCVs of 10 mouse immune sera neutralizing antibody titers measured for 3 times on different days were 0%-75%, indicating good intermediate precision. The regression coefficient of best fit line for the neutralizing antibody titers of CV-A6 neutralizing antibody serum reference at 9 dilution levels measured 3 times was 0.93, and the linear regression was statistically significant (F=340.99, P<0.000 1). The neutralizing titers of negative sera were all less than 8, showing good specificity. The GCVs of different RD cell passages, different RD cell densities, different viral loads, different culture days, different number of repeated freeze-thaw times and short-term storage time range of serum to be tested were 23%-101%, indicating good durability.Conclusions The CV-A6 neutralizing antibody detection method has been successfully established with good relative accuracy, precision, linearity, specificity, and durability. It can be used to evaluate the neutralizing antibody levels of immune sera against CV-A6.