人CD48亚型特异性抗体的鉴定及其表征分析

Identification and Characterization Analysis of Isoform Specific Antibodies to Human CD48

CD48是淋巴细胞活化信号分子家族成员,是免疫细胞发挥功能的重要分子,在癌症、自身免疫和过敏反应等过程中发挥重要作用。人(Homo sapiens) CD48蛋白有两种亚型,其中CD48亚型1(CD48-1)含有243个氨基酸,CD48亚型2(CD48-2)含有252个氨基酸,它们N端有217个氨基酸完全一致。本研究从合成的单链抗体(single-chain variable fragment, scFv)噬菌体展示文库中筛选到靶向CD48蛋白的scFv 1C2和2C1, scFv 1C2能结合CD48-1与CD48-2两种亚型,而scFv 2C1仅靶向CD48-2。ELISA实验测得1C2和2C1对CD48蛋白的亲和常数分别为120 nmol/L和66 nmol/L。稳定性检测结果表明,scFv对温度敏感,但对pH变化不敏感。1C2和2C1可作为Western blot和免疫组织化学实验的一抗,用于检测分析细胞和胃癌组织样本中的两种CD48亚型。综上所述,筛选获得的两种抗体可用于肿瘤中CD48不同亚型的表达与功能分析,在肿瘤的诊断和治疗方面有着很大的潜力。

CD48,a member of signaling lymphocyte activation molecule family,is identified as an important molecule in immune cell functions,with roles in cancer,autoimmunity and allergy.Two isoforms of human(Homo sapiens)CD48 protein existed,CD48-1(CD48 antigen isoform 1,length of 243 aa)and CD48-2(CD48 antigen isoform 2,length of 252 aa).They have 217 identical amino acids on the N-terminus.In this study,single-chain variable fragment(scFv)1C2 and 2CI targeting CD48 protein were obtained from a synthetic scFv phage display library,the scFv 1C2 that binds to CD48-1 and CD48-2,and the scFv 2C1 that only recognizes CD48-2.ELISA analysis showed that the affinity constants of 1C2 and 2C1 to CD48 protein were 120 nmol/L and 66 nmol/L,respec-tively.Stability test result showed that the scFv antibodies are sensitive to temperature change,but not sensitive to pH change.Immu-nohistochemical assay showed that 1C2 and 2C1 can be used to detect CD48 protein isoforms in gastric cancer samples as primary anti-bodies.Taken together,these results showed that the two antibodies developed in the study can be used to analyze the protein levels and function of CD48 isoforms and have potentials to be used for diagnostic and treatment of tumors.