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《基于报告基因的抗CD20单克隆抗_省略_ADCC生物学活性测定方法的建立_刘春雨》由会员上传分享，免费在线阅读，更多相关内容在行业资料-天天文库。
1、·94·药学学报ActaPharmaceuticaSinica2015,50(1):94−98基于报告基因的抗CD20单克隆抗体ADCC生物学活性测定方法的建立11*刘春雨,王兰,郭玮,于传飞,张峰,王文波,李萌,高凯(中国食品药品检定研究院,北京100050)摘要:利用Jurkat/NFAT-luc+FcγRIIIa转基因细胞系作为效应细胞,WIL2-S细胞系作为靶细胞,通过荧光素TM酶检测系统(BioGloLuciferaseAssaySystem)进行抗CD20单抗的ADCC生物学活性检测,并对实验条
2、件进行优化及方法学验证。结果显示抗CD20单抗在该方法中存在量效关系,且符合四参数方程式:y=(A−D)/[1+B−1(X/C)]+D。方法经优化确定靶细胞为WIL2-S细胞,抗体稀释浓度为18000ng·mL,1∶5倍的稀释倍数,效靶比为6∶1,诱导时间为6h。该方法具有良好的专属性,8次独立实验的回归分析、线性及平行性均通过统计学检验;4个不同稀释组回收率样本经3次测定,相对效价分别为(44.39±3.93)%、(72.74±2.78)%、(128.28±7.01)%和(168.19±2.70)%,变异
3、系数均小于10%,对应回收率分别为(88.78±7.85)%、(96.99±3.70)%、(102.63±5.61)%和(112.12±1.80)%。本研究利用转基因细胞法成功建立抗CD20单抗ADCC生物学活性检测方法,该方法专属性强、重复性好,准确性高,可作为抗CD20单抗ADCC生物学活性的常规检测方法。关键词:抗CD20单克隆抗体;ADCC;生物学活性中图分类号:R917文献标识码:A文章编号:0513-4870(2015)01-0094-05Developmentofanovelreporterg
4、enemethodfordeterminationofADCCpotencyofanti-CD20monoclonalantibody11LIUChun-yu,WANGLan,GUOWei,YUChuan-fei,ZHANGFeng,WANGWen-bo,*LIMeng,GAOKai(NationalInstitutesforFoodandDrugControl,Beijing100050,China)TMAbstract:ThebiologicalactivityofADCCbyanti-CD20mon
5、oclonalantibodywasdeterminedbyBioGloLuciferaseAssaySystemusingJurkat/NFAT-luc+FcγRIIIacelllineaseffectorcellandWIL2-Scelllineastargetcell.Thedevelopedmethodwasverifiedforspecificity,precisionandaccuracy.Anti-CD20monoclonalantibodyshowedadose-responsemodeby
6、thedevelopedmethod,andthedeterminationresultcompliedwithB]thefollowingfour-parameterequation:y=(A–D)/[1+(X/C)+D.Theoptimizedparametersofthemethod−1weredeterminedincludingtheantibodiesdilutedconcentration(18000ng·mL),dilutionrate(1∶5),theratioofeffectorcell
7、andtargetcell(6∶1),andinductiontime(6h).Thevaluesofeightindependenttestshavepassedastatisticaltestforcurveregressionanalysis,linearorparallelism,whichshowedthemethodpossessedgoodspecificity.Fourdifferentdilutegroupsofrecoveryratessampleweredeterminedfor3ti
8、mes,andtheresultshowedmeanrelativepotenciesof(44.39±3.93)%,(72.74±2.78)%,(128.28±7.01)%and(168.19±2.70)%respectively,withavariationcoefficientoflessthan10%,andtherecoveriesof(88.78±7.85)%,(96.99±3.70)%,收稿日期:2
