RA患者血清IgG抗体特异结合的噬菌体模拟肽的筛选

Screening of phage mimetic peptides recognized by serum IgG of RA patients

目的从噬菌体随机12肽库中,筛选能与RA患者血清IgG抗体特异结合的RA相关抗原表位的噬菌体模拟肽,并寻找对RA敏感性和特异性均高的模拟肽。方法提纯30例RA患者血清IgG抗体,以此作为筛选配基,对噬菌体12肽库进行4轮免疫筛选,通过ELISA方法对随机挑取噬菌体克隆进行初步鉴定,取混合阳性克隆利用Dot-ELISA方法分析15例RA患者和15例正常人血清的敏感性和特异性。结果经4轮免疫淘洗后,特异结合的噬菌体富集增加近100倍。随机挑取的11个噬菌体克隆,经鉴定均能与IgG抗体特异性结合,阳性率100%。混合的阳性克隆分别与15例RA患者和15例正常人血清反应,15例RA患者血清全部为阳性,正常人血清只有1例为阳性,敏感性为100%,特异性为93.3%。结论利用RA患者血清IgG抗体筛选噬菌体随机12肽库,能获得对RA敏感性和特异性均高的噬菌体多肽,故有望利用该技术开发新的RA特异性实验室诊断指标,为制备RA特异性诊断试剂盒提供实验基础。

Objective To screen peptides of RA related antigens that could be recognized by serum IgG of RA patients from phage displayed random display 12 peptides library, and to provide useful information for further study of the interaction between antigen and antibody in RA patients. Methods lgG was purified from serum of 30 patients with RA and was used as the ligand for 3iopanning of a phage displayed random display 12 peptides library. The positive phage clones were obtained through four rounds of biopanning. Double-antibody sandwich ELISA and Dot-ELISA were used to evaluate the characteristics of binding between phage borne peptides and IgG of RA patients mixed positive phage clones were used to detect 15 RA patients＇ serum and 15 healthy persons＇ serum by Dot-ELISA. Results The eluted phages were clinched nearly to 100 fold through four rounds of biopanning. In 11 phage clones selected randomly from the fourth round biopanning, all clones were positive. And Dot-ELISA tests proved that 11 phage clones could bind to IgG. The binding rate of mixed positive phage clones with IgG of RA patients was significantly higher than that of healthy persons. Conclusion By the use of serum IgG antibodies to screen the phage random polypeptide library, one can obtain multiple phage peptides which have high sensitivity and specificity for RA, this technique could be used to develop a new specific maker, and provide a new approach for developing ELISA kit to detect RA specific antibodies.