检测重组人鼠嵌合抗VEGFR2单克隆抗体的ELISA方法的建立  

Development of ELISA Method of Determining Recombinant Human Chimeric Mouse of Anti-VEGFR2 Monoclonal Antibody

作　　者：鲁杰[1,2] 李慧[1] 张茂桃[2] 刘运龙[2] 陈知航[2] 李丽[2] 刘学龙[1] 程远国[2] 

机构地区：[1]延边大学,吉林延吉133000 [2]军事医学科学院微生物流行病研究所,北京100071

出　　处：《生物技术通讯》2016年第3期427-431,共5页Letters in Biotechnology

摘　　要：目的：建立一种新型、快速定量检测食蟹猴血清中抗血管内皮生长因子受体2（VEGFR2）单克隆抗体浓度的方法.方法：将特异性抗原VEGFR2-His包被在固相载体上,加稀释的受试药品血清,然后加入HRP标记的羊抗人IgG-（h＋1）,再加入TMB显色液,最后加入1 mol/L硫酸终止液,在酶标仪上用双波长读取D4s450/560nm值.结果：建立了定量检测食蟹猴血清中抗VEGFR2单克隆抗体浓度的ELISA法,方法的线性范围为400～6.25 ng/mL,定量下限为6.25ng/mL,板内精密度介于-13.6％～8.3％,板间精密度介于-6.1％～6.3％,与Avastin、Actemra、Cetuximab均无交叉,室温稳定性及冻融稳定性良好,无稀释效应.结论：通过方法学的确证,本实验建立的方法可满足抗VEGFR2单克隆抗体在食蟹猴体内的药代动力学研究要求,可用于抗VEGFR2单克隆抗体的检测.Objective：To establish a new and rapid ELISA method for quantitive determination of anti-vascular endothelial growth factor receptor 2 （VEGFR2）monoclonal antibody in the blood serum of Macacafascicularis.Methods：The specific antigen VEGFR2-His bag was coated on the solid phase carrier,and diluted serum was added,followed by goat anti-human IgG-HRP as detecting antibody,TMB color liquid and sulfuric acid as termination liquid.Finally,the absorbance of each well was measured by a microplate reader under 450/560 nm.Results：The linearity range of the new ELISA method was 400～6.25 ng/mL,the lower limit of quantification was 6.25 ng/mL,the within-plate precision ranged from-13.6％ to 8.3％,the between-plates precision was-6.1％ to 6.3％.There was no cross-reaction with Avastin,Actemra,and Cetuximab.Room temperature and freeze-thaw stability were validated,and no dilution effet was found.Conclusion：This ELISA method met the requirements of pharmacokinetic study,and could be used to determine anti-VEGFR2 monoclonal antibody in cynomolgus monkeys.

关 键 词：血管内皮生长因子受体2 酶联免疫吸附法 药代动力学 

分 类 号：Q503[生物学—生物化学] R392.1[医药卫生—免疫学]