抗人心脏型脂肪酸结合蛋白单克隆抗体的制备、表位鉴定及其应用  

Preparation,epitope analysis and clinical application of monoclonal antibodies against heart-type fatty acid binding protein

作　　者：韩起[1] 丛珊[1] 易维京[1] 陈莎[1] 梁文斌[1] 陈安[1] 胡川闽[1] 李淑慧[1] 

机构地区：[1]第三军医大学医学检验系临床生物化学教研室,重庆400038

出　　处：《细胞与分子免疫学杂志》2013年第6期620-624,共5页Chinese Journal of Cellular and Molecular Immunology

基　　金：第三军医大学科技成果转化基金(2010xzh08)

摘　　要：目的制备抗人心脏型脂肪酸结合蛋白(H-FABP)单克隆抗体(mAb),鉴定其基本特性并通过配对检测临床样本血清,对已配对的mAb进行表位鉴定。方法通过已制备的H-FABP抗原和合成肽免疫BALB/c小鼠,常规融合筛选后进行抗体亚型鉴定、效价和亲和力检测,纯化mAb后通过间接ELISA、Western blot法对mAb进行特异性检测;将获得的mAb分别作为捕获和检测抗体,经过配对建立检测重组H-FABP的ELISA体系,并初步用于临床血清检测;采用生物信息学分析设计2段H-FABP表位,通过原核表达获得其短肽并纯化,通过Western blot法对配对的mAb进行表位鉴定。结果成功获得4株抗H-FABP特异性mAb,亚类分别为IgG2a和IgG2b,抗体效价为1∶51 200～1∶1024 000,亲和力最高可达到9.02×109mol/L;经Western blot法和间接ELISA鉴定均能够特异检测重组H-FABP蛋白,经过配对发现mAb 3-H5和1-F10能够检测重组H-FABP,并在其临床血清样本检测中发现正常组和急性心梗组之间H-FABP水平具有显著性统计学差异;通过表位鉴定发现未知表位的mAb 1-F10能够特异识别位点为H-FABP的第86到第133位氨基酸。结论制备了4株高特异性和高亲和力的抗H-FABPmAb,成功建立了检测临床样本血清中的ELISA体系,并对其表位进行了分析鉴定。Objective To prepare and characterize the monoclonal antibody （mAb） against recombinant human heart- type fatty acid binding protein （H-FABP） and apply it to the clinical analysis. Methods BALB/c mice were immunized with recombinant H-FABP （rH-FABP） from prokaryotic expression or synthesized peptide fragment. After common fusion and screening, the subtypes, titer and affinity of mAbs were detected respectively. After purification, the specificity of mAbs was tested by indirect ELISA and Western blotting. ELISA system was established by pair mapping and applied in the detection of clinical samples. Two epitope peptides were designed by bioinformatics and used to detect the epitope of 1-F10 through Western blotting. Results Four different hybridoma clones secreting anti-H-FABP antibodies were developed, with high titres of 1：51 200-1：1024 000, Immunoglobulin types of these mAbs were found to be IgG2a or IgG2b, respectively. The affinity of the mAb 1-F10 even reached 9.02 ×10^9 mol/L. ELISA and Western blotting showed that these mAbs could identify H-FABP specifically. In addition, the pair mapping of monoclonal antibodies 3-H5-1-F10 could recognize H-FABP in human serum samples. Furthermore, the specific target recognized by 1-F10 mAb was located within amino acid 86-133 of H-FABP. Conclusion Four highly specific mAbs against H-FABP were successfully obtained and the established ELISA system could be used to detect the H-FABP in clinical serum samples, and the epitope of 1-F10 mAb was also verified.

关 键 词：H-FABP 单克隆抗体 ELSIA体系检测 表位鉴定 

分 类 号：Q811.4[生物学—生物工程] R446.62[医药卫生—诊断学]