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目的:对通过CDR3导向抗体库法筛选到的3株人源化膀胱癌噬菌体抗体进行进一步分析鉴定。方法:用ELISA方法对筛选特异性克隆进行特异性鉴定、抗原表位的核实;采用硫氰酸盐洗脱ELISA法评估其亲和力;选用活性较高的克隆进行膀胱癌组织的免疫组化实验。结果:3株阳性克隆可溶性表达后能够与膀胱癌EJ细胞特异性结合;与鼠源噬菌体抗体(BDI)相比,2株克隆的亲和指数比较低,均为0.25mol/L,1株克隆的亲和指数为0.7mol/L,略低于鼠源的亲和指数(0.9mol/L)的水平。选择活性较高的克隆进行膀胱癌组织的免疫组化实验,显示该克隆能够与癌组织特异性结合。结论:用CDR3导向库法获得的人源抗体片段基本保持了原鼠源单克隆抗体(mAb)BDI的特性。
OBJECTIVE: To further identify and identify the three humanized phage antibody against human bladder cancer selected by CDR3-directed antibody library. Methods: The specific clones were screened by ELISA for the identification of antigenic epitopes. The affinity of the screened clones was evaluated by thiocyanate elution ELISA. The more active clones were selected for immunohistochemistry of bladder cancer. Results: The three positive clones were able to specifically bind to bladder cancer EJ cells after soluble expression. Compared with the murine phage antibody (BDI), the positive clones of the two clones had relatively low affinity scores of 0.25 mol / L and one clone The affinity index was 0.7mol / L, slightly lower than the mouse affinity index (0.9mol / L) levels. The higher activity of the selected clones for immunohistochemistry of bladder cancer tissue showed that the clone can specifically bind with cancer tissue. Conclusion: The human antibody fragments obtained by CDR3-directed library method basically retain the characteristics of the original murine monoclonal antibody (mAb) BDI.