摘要
目的运用生物膜干涉(BLI)技术,建立高通量定量检测单克隆抗体浓度的方法。方法使用Octet Red 96分子相互作用分析仪,根据样品与protein A传感器结合后光相对干涉位移变化,检测抗体浓度,并对这种检测方法进行方法学考察。结果protein A传感器能特异性地与单克隆抗体结合,抗体工作标准品浓度在7.81~500μg/mL与结合速率呈良好的回归关系,相关系数R 2为0.9999;检测限为0.6μg/mL,定量限为2μg/mL;回收率为98.90%~100.40%;重复性的相对标准偏差(RSD)为2.17%,中间精密度的RSD为2.52%;45 d内protein A传感器重复使用60次,阳性对照值的RSD为3.7%;再生液pH变化±0.2、平衡液吐温-20比例变化±5%的RSD值为0.99%。检测温度与转盘速度的变化是影响样品定量检测的因素,检测结果与高效液相数据高度一致(R^2=0.90)。结论运用BLI技术能实现高通量定量检测单克隆抗体浓度,该方法具有样品适应性强、分析速度快、准确度高的优势,可用于单克隆抗体细胞株的筛选、排序及蛋白表达的检测与监控。
Objective To establisha high-throughput quantitative method to determine the concentration of monoclonal antibody based on bio-layer interference(BLI)technique.Methods To determine the antibody concentration based on the spectral shift after the sample was reacted with the protein A sensor,we used Octet Red 96 platform,and performed a methodological investigation.Results Protein A biosensor could react with antibody specifically without matrix effection,and the increase binding rate was proportional to the concentration of monoclonal antibody in the range of 7.81-500μg/mL,with correlation coefficient R^2=0.90.The LOQ was 0.6μg/mL and DOQ was 2μg/mL.The recovery was 98.90%-100.40%.The RSD of precision was 2.17%and intermediate precision was 2.52%.Protein A sensor could be reused 60 times,and RSD of positive control value was 3.7%in 45 days.The RSD of sample concentration was 0.99%when regeneration liquid pH varied±0.2 and tween-20 percentage varied±5%in neutralization liquid,the test temperature and shake speed were main factors which affected test.The quantitative method had comparable data correlated to UPLC-Protein A method.Conclusion The rapid concentration measurement method can be used for clone selection,ranking and monoclonal antibody expression monitoring with advantage of high throughput and high speed.
作者
胡妍
罗安
车耀健
刘全裕
HU Yan;LUO An;CHE Yaojian;LIU Quanyu(Immunotherapy Institute,Fujian Medical University,Fuzhou 350122,China;Department of Pharmacy,Fujian Health College,Fuzhou 350101,China;Scholl of Pharmacy,Fujian Medical University,Fuzhou 350122,China)
出处
《福建医科大学学报》
2020年第3期139-143,共5页
Journal of Fujian Medical University
基金
福建省教育厅中青年教师教育科研项目(JT180169)
福建省卫生计生委青年科研课题(2016-2-38)。
关键词
生物膜
抗体
单克隆
biofilms
antibodies
monoclonal
