甘精胰岛素生物学活性定量免疫荧光测定法和胰岛素生物测定法的等效性研究  

Study on equivalence of biological activity of insulin glargine by quantitative immunofluorescence assay and insulin bioassay

作　　者：高益敏 张红梅 何开勇 尹登科 孙备 许雷鸣 GAO Yi-min;ZHANG Hong-mei;HE Kai-yong;YIN Deng-ke;SUN Bei;XU Lei-ming(Anhui Institute for Food and Drug Control,Hefei 230051,China;Recombinant Vaccine Research and Development Joint Key Laboratory of Anhui Province,Hefei 230051,China;School of Pharmacy,Anhui University of Chinese Medicine,Hefei 230012,China;Hubei Institute for Drug Control,Wuhan 430064,China)

机构地区：[1]安徽省食品药品检验研究院,安徽合肥230051 [2]重组疫苗研究开发安徽省联合共建学科重点实验室,安徽合肥230051 [3]安徽中医药大学药学院,安徽合肥230012 [4]湖北省药品监督检验研究院,湖北武汉430064

出　　处：《中国药理学通报》2024年第11期2193-2199,共7页Chinese Pharmacological Bulletin

基　　金：国家药典委员会国家药品标准制修订研究课题(No BZ2024046);安徽省药品监督管理局监管科学研究重点项目(No AHYJ-KJ-202306);安徽省药品监督管理局科技创新项目(No AHYJ-KJ-202004)。

摘　　要：目的建立甘精胰岛素基于转基因细胞CHO-INSRB 1284的定量免疫荧光生物学活性测定法(In-Cell Western,ICW),并将其与《中国药典》胰岛素生物测定法进行等效性研究。方法CHO-INSRB 1284细胞以1.5×10^(8) L^(-1)细胞密度铺板,1500μmol·L^(-1)甘精胰岛素稀释25倍后再进行3倍系列梯度稀释,在微孔板刺激细胞20 min,经固定、透化及抗体孵育步骤,利用odyssey双色红外荧光成像系统,进行定量免疫荧光生物学活性检测。结果ICW中甘精胰岛素的浓度与相对效价具有良好的量效关系;方法专属性好,相对准确度、中间精密度及线性均符合要求;7批甘精胰岛素样品采用两种方法测定的生物学活性结果相对偏差低于10%,用SPSS软件和SAS软件分析,表明方法具有相关性及等效性。结论建立的甘精胰岛素生物学活性定量免疫荧光测定法,方法专属性好,准确度及精密度高,与体内胰岛素生物测定法具有相关性和等效性,可用于甘精胰岛素的体外药效评价和质量控制。Aim To establish a quantitative immunofluorescent bioactivity assay(ICW)for insulin glargine based on CHO-INSRB 1284 transgenic cells,and to study its equivalence with insulin bioassay of Ch.P.Methods The cells were diluted 25 times with 1.5×10^(8) L^(-1) cell density plates and 1500μmol·L^(-1) insulin glargine,and then diluted with a 3-fold gradient series.The cells were stimulated in microporous plates for 20 min.After fixation,permeation and antibody incubation.Quantitative immunofluorescence biological activity was detected by odyssey two-color infrared fluorescence imaging system.Results There was a good dose-effect relationship between the concentration of insulin glargine in ICW and its relative potency.The method had good specificity,and the relative accuracy,intermediate precision and linearity met the requirements.The relative deviation of biological activity results of 7 batches of insulin glargine samples measured by the two methods was less than 10%.The results were analyzed by SPSS and SAS software,which showed that the methods were correlated and equivalent.Conclusions The quantitative immunofluorescence assay for the biological activity of insulin glargine can be established.The method has good specificity,high accuracy and precision,and has correlation and equivalent with biotiter assay,which can be applied to in vitro efficacy evaluation and quality control of insulin glargine.

关 键 词：生物学活性 甘精胰岛素 定量免疫荧光测定法 转基因细胞 方法学验证 等效性评价 

分 类 号：R587.1[医药卫生—内分泌]