一种从抗狂犬病病毒噬菌体抗体库中筛选和验证中和抗体的方法

Method for screening and verifying neutralizing antibodies from anti-rabies virus phage antibody display library

目的建立一种从噬菌体库抗体库中高效筛选和验证抗狂犬病病毒中和抗体的方法。方法①定性分析:从经过灭活的CVS-11筛选噬菌体抗体库中挑取单克隆于96孔板中培养,制备噬菌体抗体,取培养上清进行快速荧光灶抑制试验(rapid fluorescent focus inhibition test,RFFIT),选择可明显抑制病毒感染的克隆测序,获得具有中和活性的抗体可变区序列;②定量分析:挑选有中和活性的克隆,重新制备噬菌体抗体颗粒,纯化后进行RFFIT分析;扩增抗体可变区基因,构建真核瞬时表达质粒。瞬转HEK-293 EBNA1细胞,培养上清经Mabselect SuRe亲和纯化后测定抗体比活。结果定性分析获得的噬菌体抗体颗粒与全分子抗体体外中和活性显著相关;剔除低活性序列后,活性高于0.5 IU/mL的噬菌体抗体颗粒与其全分子抗体体外中和活性之间无显著相关;所有纯化后噬菌体抗体颗粒活性>0.5 IU/mL的序列其全分子抗体体外中和活性均>500 IU/mg。结论构建了一种从抗狂犬病病毒噬菌体库中高效筛选、验证中和抗体的方法。

Objective To develop a method for highly efficient screening and verifying of anti-rabies virus neutralizing antibodies from phage antibody display library.Methods①Qualitative analysis:Phage antibody library was screened by using inactivated CVS-11 monoclones and cultured in 96-well plates.Culture supernatant was analyzed by fluorescence focus inhibition test(rapid fluorescent focus inhibition test,RFFIT).The clones that significantly inhibited viral infection were sequenced;②Quantitative analysis:neutralizing activity of purified phage that significantly inhibited viral infection was analyzed by RFFIT,and the genes for antibody variable region were obtained by PCR amplification of the clones.Eukaryotic transient expression plasmids of full-length antibodies were constructed and transfected into HEK-293 EBNA1 cells.Full-length antibodies were purified from culture supernatants by Mabselect SuRe affinity chromatography.The neutralizing activity of antibody was analyzed by RFFIT.Results The neutralization activity from the obtained phage antibody particles was significantly correlated with that of full-length antibody in vitro by qualitative analysis.On the other hand,after removed off sequence with a low activity,there was no a significant correlation between the neutralization activity higher than 0.5 IU/mL from phage antibody particles and the neutralization activity from a full-length antibody.It is greater than 500 IU/mg for neutralization activity from the full-length antibodies that is corresponding to higher than 0.5 IU/mL from the purified phage antibody particles.Conclusion One efficient method was developed for screening and verifying antibodies with active neutralizing activity from anti-rabies virus phages display library.