摘要
本研究采用均相时间分辨荧光技术,在LHCGR(-Ex10)-CRE-luc-HEK293细胞中通过竞争性免疫法检测细胞中cAMP的量,建立促黄体生成素(LH)体外生物学活性测定方法。对方法的关键参数进行优化,并对方法的相对准确度、中间精密度、线性和范围进行了验证。优化后确定实验条件为:起始浓度200 nmol·mL^(-1)、7倍梯度稀释、细胞密度24000 cells·well^(-1)、孵育时间30 min。结果表明,方法在64%~156%的相对效价水平范围内线性良好,相对准确度高,且中间精密度良好。本研究成功建立并验证了检测LH生物学活性的均相时间分辨荧光法,可用于重组促黄体生成素类药物生物活性评价和质量控制,并可用于鉴别重组促黄体生成素。
This study employs homogeneous time-resolved fluoroimmunoassay to quantitatively determine the concentration of cyclic adenosine monophosphate(cAMP)within LHCGR(-Ex10)-CRE-luc-HEK293 cells,utilizing a competitive immunoassay method.The research sets forth an in vitro biological activity assessment methodology for luteinizing hormone(LH).Key parameters of the methodology are optimized,and the relative accuracy,intermediate precision,linearity,and range are validated.The optimized experimental conditions are defined as follows:an initial concentration of 200 nmol·mL^(-1),a seven-fold gradient dilution,a cell density of 24000 cells·well^(-1),and a duration of incubation of 30 min.The findings indicate that the method exhibits excellent linearity across a relative potency range of 64%to 156%,high relative accuracy,and satisfactory intermediate precision.The successful establishment and validation of a homogeneous time-resolved fluoroimmunoassay for the evaluation of LH biological activity is reported.This methodology is applicable for the assessment of biological activity and quality control of recombinant LH-like drugs,as well as for the differentiation of recombinant LH.
作者
邓哲
张孝明
孙悦
李晶
吕萍
相宏宇
梁成罡
DENG Zhe;ZHANG Xiao-ming;SUN Yue;LI Jing;LÜPing;XIANG Hong-yu;LIANG Cheng-gang(School of Life Sciences,Jilin University,Changchun 130012,China;National Institutes for Food and Drug Control,State Key Laboratory of Drug Regulatory Science,NHC Key Laboratory of Research on Quality and Standardization of Biotech Products,NMPA Key Laboratory for Quality Research and Evaluation of Biological Products,Beijing 102629,China;Heilongjiang Institute for Drug Control,Harbin 150088,China)
出处
《药学学报》
北大核心
2026年第1期266-272,共7页
Acta Pharmaceutica Sinica
关键词
促黄体生成素
生物学活性
均相时间分辨荧光
方法学验证
绒毛膜促性腺激素
质量控制
luteinizing hormone
biological activity
homogeneous time-resolved fluorescence
methodological validation
chorionic gonadotropin
quality control
