重组人表皮生长因子生物活性检测方法的建立及与其他显色方法的比较  

Development of a method for determination of bioactivity of recombinant human epidermal growth factor and comparison of different chromogenic methods

作　　者：于雷[1] 史新昌[1] 刘兰[1] 李响[1] 裴德宁[1] 饶春明[1] YU Lei;SHI Xin-chang;LIU Lan;LI Xiang;PEI De-ning;RAO Chun-ming(National Institutes for Food and Drug Control,Beijing 100050,China)

机构地区：[1]中国食品药品检定研究院,北京100050

出　　处：《中国生物制品学杂志》2018年第9期985-989,共5页Chinese Journal of Biologicals

基　　金：中国食品药品检定研究院中青年发展研究基金(2017B3)

摘　　要：目的建立重组人表皮生长因子(recombinant human epidermal growth factor,rh EGF)生物学活性的检测方法,并与其他4种常见显色方法进行比较。方法参考《中国药典》三部(2015版)附录3528中3T3细胞/MTT显色法,建立rh EGF生物学活性的检测方法,去除细胞饥饿的步骤,并用10%SDS代替DMSO,同时对培养3T3细胞的PRIM1640培养液的胎牛血清浓度(0、2%、5%、10%)进行优化。采用优化方法及药典方法同时检测rh EGF标准品,对灵敏度、信噪比、线性及孔间变异度进行比较。分别采用优化方法、MTS显色法、CCK-8显色法、结晶紫显色法、Cell Titer-Glo显色法检测同批rh EGF供试品,比较各方法的检测结果。结果优化方法的灵敏度(半数有效浓度为2.84 IU/m L)、信噪比(1.40)、线性(R2>0.99)和孔间变异度(95%可信区间为1.576%~3.334%)均优于药典方法,且实验周期由6 d缩短为5 d。5种显色方法对rh EGF供试品的检测结果差异无统计学意义(P>0.05),CCK-8显色法检测结果的变异系数(CV)最小(16.8%),结晶紫显色法的CV值最大(23.3%)。结论优化方法更适用于rh EGF制品的常规生物学活性检测,且与其他4种显色方法等效。Objective To develop a method for determination of bioactivity of recombinant human epidermal growth factor（rh EGF） and with other four chromogenic methods. Methods A method for determination of bioactivity of rh EGF was developed according to the 3 T3 cells/MTT chromogenic assay in Annex 3528 of Chinese Pharmacopoeia（Volume Ⅲ,2015 edition）,in which the step of cell starvation was deleted,and DMSO was substituted with 10% SDS,while the fetal bovine serum concentration（0,2%,5% and 10%） in PRIM1640 medium was optimized. The methods before and after optimization were used for determination of standard rh EGF,of which the sensitivity,signal/noise ratio,linearity and variation between wells were compared. The rh EGF samples of the same batch was determined by the optimized method,MTS chromogenic method,CCK-8 chromogenic method,crystal violet chromogenic method and Cell Titer-Glo chromogenic method,and the results were compared. Results The sensitivity（median effective dose 2. 84 IU/m L）,signal/noise ratio（1. 40）,linearity（R^2 〉0. 99） and variation between wells（95% CI：1. 576% ~ 3. 334%） of the optimized method were superior to those of method in Chinese Pharmacopoeia（Volume Ⅲ,2015 edition）,while the test cycle was shortened from 6 d to 5 d. However,the determination results of test samples of rh EGF by the five chromogenic methods showed no significant difference（P 〉0. 05）. The CV of determination results by CCK-8 chromogenic method was the lowest（16. 8%）,while that by crystal violet chromogenic method（23. 3%）was the highest. Conclusion The optimized method was more suitable for the routine determination of bioactivity of rh EGF,which was equivalent to the other four chromogenic methods.

关 键 词：重组人表皮生长因子 生物学活性测定 3T3细胞 显色方法 

分 类 号：R392-33[医药卫生—免疫学]