EV71特免血浆筛选方法的建立及其应用

Establishment and application of method to screen anti-EV71 positive plasma

目的考察献血员血浆中抗EV71中和抗体的状况，同时建立适宜的高通量筛选EV7l特免血浆的方法，为制备EV71特异性免疫球蛋白提供技术支持。方法以经典微量细胞病变中和方法测定血浆样品及市售普通静注丙种球蛋白中EV71抗体滴度，探索用于制备EV71特异性免疫球蛋白血浆的参考指标。据此，建立筛选血浆的高通量阈值中和方法，并与传统方法进行比较。应用筛选的EV71特免血浆制备EV71特免球蛋白，并应用细胞模型和动物模型评价其中和抗体活性。结果以经典法检测，约20％的原料血浆中抗体滴度达1：50或以上，混合后的效价为1：180，达到市售普通静注丙种球蛋白的抗体水平。以480份样品检测，所建立的阈值法与经典方法符合率为88．1％；制备的EV71特免球蛋白质量符合国家标准，EV71中和活性比静注免疫球蛋白提高约16倍，对动物的保护作用显著提高。结论成功建立了EV71特免血浆的筛选方法及血浆筛选参考指标；所建立的高通量筛选方法操作简便，可用于研制EV71特异性免疫球蛋白的血浆筛选；制备的EV71特免球蛋白中和效价显著提高，对动物的保护作用明显增强，对于重症手足口病的防治可能具有重要意义。

Objective To evaluate the titers of EV71 neutralizing antibody in donators＇ plasma, and to establish a high throughput screening method to screening anti-EV71 positive plasma for the produc- tion of EV71-specifie IVIG in practice. Methods The EV71 neutralizing antibody titers in plasma samples and in commercial IVIGs （intravenous immunoglobulins ） were evaluated by micro-cytopathic effect neutral- ization test （MCPENT）, and a selecting criteria was determined for screening anti-EV71 positive plasma. Then, a high throughput neutralizing method for screening plasma was established and a comparison with MCPENT was conducted. EV71-specific IVIG was prepared from screened anti-EV71 source plasma, and its neutralizing activity was evaluated in cell model and mouse model. Results The results of MCPENT showed that the titers of EV71 neutralizing antibody were 1 ： 50 or above in 20% of the source plasma, and reached 1 ： 180 after being pooled, approaching to the titer of commercial IVIG. In 480 samples, the coincidence rate between the established high throughput neutralizing method and MCPENT was 88.1%. The quality of prepared EV71-specific IVIG was complied with the national standard, and its neutralizing potency was 16 times of that of IVIG, suggesting EV71-specific IVIG had a significantly improved protecting effect on experi- mental neonatal mice. Conclusion The screening method and the screening criteria for EV71 specific plas- ma were successfully established. The established high throughput neutralizing method that was operated eas- ily could be used for plasma screening in the development of EV71-specific IVIG. The prepared EVTl-spe- eific IVIG, whose neutralizing potency was significantly improved, showed a markedly protective effect on experimental neonatal mice, suggesting it might be very important for the prevention and treatment of hand- foot-and-mouth disease.