基于重组荧光病毒的流式细胞术检测抗CV-A16中和抗体的研究

Development of a Coxsackievirus A16 Neutralization Assay Using Flow Cytometry Based on Reconstructed Fluorescent Virus

建立基于重组荧光病毒CV-A16-EGFP的流式快速测定CV-A16感染或免疫动物血清中和抗体效价的方法。在Vero细胞上对不同感染时间点,不同病毒接种量情况下的CV-A16-EGFP病毒的增殖特性进行分析;随后,利用流式检测不同病毒滴度下的EGFP阳性细胞数量、EGFP阳性细胞百分比(%)、及EGFP阳性细胞的平均荧光密度值,并换算出不同病毒滴度与三者之间分别的数值对应关系。最后,以平均荧光密度值作为替代判断病毒病变效应的指标,并利用流式荧光检测及传统微量中和方法对CV-A16感染恒河猴血清及CV-A16免疫的羊血清中和抗体效价进行检测,并对两种方法的检测结果进行比较。在感染后24h,不同的病毒滴度与对应的平均荧光密度值呈良好的线性关系。利用流式荧光检测的CV-A16中和抗体效价与传统的微量中和实验相比,两者检测结果具有较好的线性相关性,线性回归系数为R^2=0.8026;传统的微量中和实验检测的平均中和抗体效价略高于流式荧光检测的中和抗体效价,但是差异无统计学意义(P>0.05)。利用流式荧光检测可以迅速检测CV-A16中和抗体效价,并且实验结果与传统的微量中和实验具有较好的一致性。相比与传统方法,该方法更简单、快速、灵敏,为CV-A16抗血清的中和抗体检测提供参考。

To develop a method for rapid measurement of neutralizing antibody titer in coxsackievirus(CV)A16-immunized and CV-A16-infected animals based on flow cytometry. Proliferation of the CV-A16-EGFP virus was measured at different time points after infection and different virus-inoculation doses in Vero cells.Subsequently,the number,percentage and mean fluorescence intensity(MFI) of EGFP-positive cells for different virus titers were identified by flow cytometry. Also,the corresponding relationships between different virus titers and the three parameters mentioned above were calculated. The MFI was regarded as an alternative indicator to represent cytopathic effects. Then,the flow cytometry fluorescent-based neutralizing antibody titer against CV-A16 was validated by comparison with the conventional microneutralization assay by testing the CV-A16-infected serum of rhesus monkeys and CV-A16-immunized goat serum. At 24 h after infection,there was a good linear relationship between different viral titers and MFI. The neutralizing antibody titers determined by the two methods mentioned above were well correlated,and the linear regression coefficient was R^2= 0.8026.The mean neutralization antibody titer determined by the conventional microneutralization assay was slightly higher than that measured by flow cytometry fluorescence-based detection,however,the difference was not significant(P>0.01). The experimental results of our CV-A16 neutralization assay based on flow cytometry using fluorescence-based detection were in good accordance with those of the traditional microneutralization assay. Compared with the latter,our method was simpler,faster and more sensitive for measurement of neutralizing antibody titers against CV-A16 in vitro,which could provide a reference for determination of neutralizing antibody titers in CV-A16 antiserum.