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目的从大容量天然噬菌体抗体库中筛选抗银环蛇毒素的人源单链抗体(ScFv),并进行鉴定。方法以银环蛇毒素为靶抗原,通过“吸附-洗脱-扩增”过程从大容量天然噬菌体抗体库中筛选特异性抗体,采用ELISA法检测其结合活性;与抗蛇毒素马血清进行竞争抑制ELISA,检测其中和活性;DNA指纹图谱分析和序列测定确定其抗体类型。结果经4轮筛选,获得了5株对银环蛇毒素具有结合活性的阳性克隆;其中1株结合活性高的单克隆抗体中和活性抑制率可达8.5%;DNA指纹图谱显示为3个不同的ScFv基因,序列分析表明其重链可变区均属于VHⅠ型,轻链可变区分别为VκⅡ、VκⅢ和VλⅡ。结论利用噬菌体抗体库技术获得了具有中和活性的抗银环蛇毒素人源单链抗体。
Objective To screen and identify anti-bungarotoxin human scFv from large-capacity natural phage antibody library. Methods The specific antibodies were screened from the bulk natural phage antibody library by using the “adsorption-elution-amplification” process, and the binding activity of the antibody was tested by ELISA. The anti-snake venom horse serum Competitive inhibition ELISA, detection of its neutralization activity; DNA fingerprinting analysis and sequencing to determine the type of antibody. Results After four rounds of screening, five positive clones with binding activity to bungarotoxin were obtained. Among them, the inhibition rate of neutralizing activity of one monoclonal antibody with high binding activity was up to 8.5%; DNA fingerprinting showed three different Of the ScFv gene. Sequence analysis showed that the heavy chain variable region belongs to VH Ⅰ type, and the light chain variable region is Vκ Ⅱ, Vκ Ⅲ and Vλ Ⅱ. Conclusion The anti-bungarotoxin humanized single chain antibody with neutralizing activity was obtained by the phage antibody library technology.