抗肿瘤坏死因子-α单克隆抗体对噬菌体随机12肽库的免疫筛选

目的 从噬菌体随机多肽库中筛选出能与肿瘤坏死因子-α特异性结合的短肽分子。方法 采用肿瘤坏死因子-α作为配基,免疫亲和筛选以融合蛋白形式在丝状噬菌体M13外壳蛋白Ⅲ表达的随机12肽文库,按“吸附-洗脱-扩增”的淘洗过程,经过3轮免疫筛选后,随机挑取噬菌体克隆用双抗体夹心ELISA及斑点ELISA测其特异性,并用混合的阳性噬菌体克隆分别与SLE患者和正常人血清反应,以斑点ELISA法分析其诊断系统性红斑狼疮的价值。结果 经过3轮免疫筛选,特异性结合的阳性噬菌体克隆富集增加近100倍,第3轮筛选后随机挑选7个阳性噬菌体克隆,经双抗体夹心ELISA和斑点ELISA检测,有5个克隆能与肿瘤坏死因子α特异性相结合,其中A值最高的5个克隆混合后与SLE患者血清结合率明显高于正常人,差异有显著性。结论 噬菌体随机肽库技术可以应用于分析、鉴定肿瘤坏死因子α的表位,继而为获得亚单位表位水平的诊断试剂和抗肿瘤坏死因子α的短肽疫苗提供依据。

Identifying the Epitope of Monoclonal Antibody with Phage-displayed Random Peptide Library

Objective To obtain the short peptides from phage-displayed random peptide library through screening the epitope of monoclonal antibody against tumor necrosis factor(TNF-α). Methods Anti-TNF-α was used to immunoscreen a phage random peptide library of 12 amino-acidresidues displayed as a fusion to protein Ⅲ of filamentous phage M13. The positive clones were obtained by three rounds of biopanning, and the reactivity of each clone binding to anti-TNF-α was examined by double-antibody sandwich ELISA and Dot-ELISA. Mixed positive phage clones were used to detect the serum from SLE patients and healthy persons by Dot-ELISA. Results The eluted phages were enriched nearly 100 fold through three rounds of biopanning, 7 phage clones from the third round biopanning were randomly selected and 5 clones of them could bind to the anti-TNF-α. The binding rate of mixed clones with SLE patients was significantly higher than that of healthy persons. Conclusion The phage display technique can be applied to study the anti-TNF-α antigenic peptides, and these epitopes provide the potential for developing immunodiagnostic reagents of vaccines.