噬菌体抗体库固相筛选条件的初步研究

目的:探讨噬菌体抗体库的固相筛选条件,为筛选方案的设计提供实验依据。方法:利用多种针对HEVNE2蛋白的特异性噬菌体人源抗体和非特异性噬菌体人源抗体,对噬菌体抗体与抗原的结合时间、抗原包被的浓度、洗涤强度和洗脱方式等多种筛选的条件进行初步探索。结果:阳性噬菌体抗体与抗原反应1min,就可较好结合,洗涤次数为20～30次、洗涤液的pH为5时,筛选得到的阳性率最高。包被抗原的浓度对筛选的阳性率没有明显影响,用10mg/L抗原竞争洗脱60min,可得到较高的阳性率。结论:噬菌体抗体库的筛选是一个非常复杂的过程,其中的各个条件之间有着密切的联系,应该根据具体情况进行调整。

Preliminary study on the conditions of solid-phase screening phage antibody library

AIM: To explore the conditions of solid-phase screening phage antibody library and to provide the experimental basis for the design of screening project. METHODS: Diverse antibodies including HEV NE2-specific and non-specific humanized phage antibodies were used to study the screening conditions, such as the binding time of phage antibodies to antigen, the concentration of coating antigen, the washing times and elution method. RESULTS: The best binding time of positive phage antibody to antigen was 1 min. The highest positive rate of screening was obtained under the conditions of washing for 20 to 30 times and pH value of the washing solution being 5. The concentration of the coating antigen had no obvious influence on the positive rate of screening. Higher positive rate was obtained by using 10 mg/L antigen to competitively elute for 1 h. CONCLUSION: Solid-phase screening of the phage antibody library is a very complex process, in which there are close relationship between the conditions, therefore, appropriate readjustment should be made for screening conditions according to concrete conditions.