抗人神经肽Y单克隆抗体的制备及鉴定  

Preparation and Identification of Monoclonal Antibody Against Human Neuropeptide Y

作　　者：丁克祥[1] 董萍 杨永鹏 孙乐 孙哲 尹晴[1] 韩晋云[1] 左夏林[1] 丁宇[4] 丁振华[5] 

机构地区：[1]南方医科大学（原第一军医大学）,中国广州510515 [2]大连市董萍医疗美容整形医院,中国大连116011 [3]京天成生物技术（北京）有限公司,中国北京100085 [4]华中科技大学同济医学院同济医院,中国武汉430030 [5]南方医科大学公共卫生学院及热带医学学院,中国广州510515

出　　处：《国际老年医学杂志》2011年第4期145-150,共6页International Journal of Geriatrics

摘　　要：目的：建立抗人神经肽Y（NPY）的单克隆抗体细胞株。方法：用合成的hNPY多肽偶联KLH免疫BALB／c小鼠，取其脾细胞与骨髓瘤细胞SP2／0融合，用ELISA法筛选出阳性克隆，经克隆化后建立抗人NPY杂交瘤细胞株，并对得到的单抗进行亚类、效价、纯度、亲和常数以及结合位点的测定和分析。结果：获得了3株稳定分泌抗hNPY单抗杂交瘤细胞株，IgG亚类鉴定分别为IgG3、IgG2b、IgG1，腹水效价为1：20万～1：200万，纯化抗体效价为0．05μg／ml-0．0005μg／ml，4A8抗体纯度达到95％以上，抗体亲和常数分别为5．27×10^6L／mol、3．39×10^6L／mol、1．05×10^7L／mol，不同单抗配对经时间分辨免疫荧光法检测结果提示3株单抗是针对不同抗原决定簇。结论：成功制备了3株抗hNPY单抗，为研究NPY及其抗体在脂肪移植及肥胖治疗中的机制和应用奠定基础。OBJECTIVE： To produce monoclonal antibodies （McAb） against human neuropeptide Y （hNPY）. METHODS： The BALB/c mouse were immunized with synthetic peptide hNPY coupled to KLH. The immunized mouse speleen cells were fused with SP2/0 cells to raise monoelonal hybridoma cell lines, and the culture supematants of hybridomas were screened by ELISA method against bNPY. The monoclonal antibodies were further characterized by examinations of their immunoglobulin class or subclass, valence, purity, affinity constants and binding sites. RESULTS： Three hybridomas producing antibodies against hNPY were obtained. IgG isotypes of the three McAb were IgG3, IgG2b and IgG1 respectively. The titers of the ascites were between 1： 2×10^5 - 1： 2×10^6dilutions and the titers of the purified antibodies were between 0.05 μg,/ml - 0.0005 μg/ml. The purity of Portein - G purified monoclonal antibody 4A8 was 〉 95%. The affinity constants were 5.27×10^6L/ tool, 3.39×10^6L/mol and 1.05×10^7L/mol respectively for those three McAbs. It has also shown that all three McAbs recognize different epitopes on hNPY by McAb pairings in the time -re-solved fluoroimmunoassay. CONCLUSION： Three hNPY - specific McAbs are successfully developed, which lays the foundation of further studies of NPY - mediated mechanisms and future applications of NPY and/or NPY antibody in fat transplantation and obesity treatment

关 键 词：人神经肽Y 多肽合成 BALB/C小鼠 单克隆抗体 制备 鉴定 

分 类 号：R339.21[医药卫生—人体生理学]