酶标法检测促胰岛素分泌肽融合蛋白生物学活性  

Immunoassay for bioactivity determination of Exendin-4-Fc

作　　者：付艳丽 马亚茹 辛芳 孔涛 张艳丽 陈勇 FU Yan-li;MA Ya-ru;XIN Fang;KONG Tao;ZHANG Yan-li;CHEN Yong(The Fourth Research Laboratory,Lanzhou Institute of Biological Products Co.,Ltd,Vaccine Engineering Technology Center of Gansu,Lanzhou 730046,China)

机构地区：[1]兰州生物制品研究所有限责任公司第四研究室,甘肃省疫苗工程技术研究中心,甘肃兰州730046

出　　处：《生物技术》2022年第6期699-703,共5页Biotechnology

基　　金：中国生物重大专项(CNBG-LZ-043)。

摘　　要：[目的]建立促胰岛素分泌肽融合蛋白(Exendin-4-Fc fusion protein,简称E4F4)生物学活性的检测方法,并对新建方法进行优化及验证。[方法]采用U2OS/GLP-1R转基因细胞系,通过酶标法检测胞内cAMP含量的改变来测定E4F4生物学活性。[结果]U2OS/GLP-1R转基因细胞系在30代以内均可用于E4F4生物学活性的检测,细胞接种密度为1.2×10^(4)/孔,药物作用时间30 min为该方法的最佳检测条件;在0.00305~50μg/mL范围内,剂量效应曲线呈典型的S型半对数曲线;3批E4F4成品分别进行加标回收试验,回收率均在80%~120%之间;对同一批E4F4成品独立测定8次,CV%值为12.81%;3批E4F4成品在3个工作日内进行生物学活性检测,每批分别测定3次,9次试验的CV%为16.73%。[结论]经过细胞接种密度、细胞代次和药物作用时间的优化,建立的U2OS/GLP-1R细胞酶标法专属性强,精密度好,准确性高,可作为E4F4生物学活性的常规检测方法。[Objective]To establish the method for detection of Exendin-4-Fc fusion protein biological activity and optimize and verify the new approach.[Method]The U2 OS/GLP-1 R transduction cell line was used to determine the biological activity of E4 F4 by changes in intracellular cAMP content.[Result]The U2 OS/GLP-1 R transgenic cell line can be used for detection of E4 F4 biological activity in 30 generations,and the cellular vaccination density is 1.2×10^(4),drug action time 30 min is the best detection condition for this method;at 0.00305-50μg/mL,the dose reaction curve is typically S-type half-parallel curve;the 3 batch of E4 F4 finished products is applied to the recovery test,the recovery is between 80%-120%;the same batch of E4 F4 finished products were determined 8 times,the CV%is 12.81%;3 batches of E4 F4 finished products were active within 3 working days,each batch was measured 3 times,and the CV%of 9 tests was 16.73%.[Conclusion]Extracted by the cell vaccination density,the operation of cells and drug action time,the established U2 OS/GLP-1 R cell enzyme standard is strong,high precision,high accuracy,which can be used as the convention for E4 F4 biologically active detection method.

关 键 词：促胰岛素分泌肽融合蛋白 生物学活性 酶标法 方法优化 方法验证 

分 类 号：R392-33[医药卫生—免疫学]