抗重组人白细胞介素-12单克隆抗体的制备及初步应用  

Preparation and Preliminary Application of McAbs against Recombinant Human IL-12

作　　者：李俐[1] 马颖哲[1] 张家颖[1] 张桂荣[1] 

机构地区：[1]吉林大学白求恩医学院生物化学与分子生物学中心,长春130021

出　　处：《中国生物制品学杂志》2008年第8期705-707,共3页Chinese Journal of Biologicals

基　　金：国家自然科学基金资助课题(30570688)

摘　　要：目的制备抗重组人白细胞介素-12(rhIL-12)单克隆抗体(McAb),并建立检测IL-12的双抗体夹心ELISA方法。方法以纯化的rhIL-12(p70)免疫BALB/c小鼠,制备单克隆抗体,建立双抗体夹心ELISA法,检测胃癌患者术前、术后血清IL-12水平的变化。结果筛选出3株稳定分泌抗rhIL-12的单抗杂交瘤细胞株,纯化后单抗纯度达95%以上。免疫球蛋白亚类2株为IgG1,1株为IgG2a,轻链属!型,相对亲和力依次为EM5>EM6>EV9。Western blot及ELISA交叉试验表明,单抗具有良好的特异性。建立的双抗体夹心ELISA方法检测rhIL-12的最低检出限为20pg/ml,线性范围为25～3200pg/ml。应用此法检测胃癌患者术前血清IL-12水平较正常对照组明显降低,术后10d明显升高。术后给予免疫增强型NE组IL-12水平较未给予组明显升高。结论制备的抗rhIL-12单克隆抗体,能够用于IL-12的体内外免疫学检测,为IL-12的研究、检测及临床应用奠定了基础。Objective To prepare the McAbs against recombinant human IL-12 （rhIL-12） and develop a double antibody sandwich ELISA method for determination of IL-12. Methods Immunize BALB/c mice with purified rhIL-12 （p70） to prepare McAbs. Determine the serum IL-12 levels of patients with gastric carcinoma before and after surgery by the double antibody sandwich ELISA kit prepared with the McAbs. Results Three hybridoma cell strains stably secreting McAb against rhIL-12 were screened, of which 2 secreted IgG1 and the other one secreted IgG2a, each with a κ chain. The secreted McAbs reached a purity of more than 95% after purification. In the order of relative affinity, the secreted McAbs were EMS, EM6 and EV9. Both Western blot and ELISA showed good specificity of secreted McAbs. The minimum detection limit and linear range of rhIL-12 determined by the prepared double antibody sandwich ELISA kit were 20 and 25 ～ 3200 pg/ml respectively. The serum IL-12 level of patients before surgery determined by the kit were significantly lower than those of normal control, but increased significantly 10 d after surgery. The IL-12 levels of patients treated with immunopotent NE were significantly higher than those untreated. Conclusion The McAbs prepared using rhIL-12 as antigen may be used for the in vitro and in vivo immunological determination of IL-12, which lays a foundation of study, detection and clinical application of IL-12.

关 键 词：重组人白细胞介素-12 单克隆抗体 双抗体夹心ELISA 胃癌 

分 类 号：R392-3[医药卫生—免疫学]