重组人EGF-IL-18融合蛋白直接竞争ELISA方法的建立  

Establishment of a direct competitive ELISA for recombinant human EGF-IL-18 fusion protein

作　　者：洪德芳[1] 艾冬冬[1] 丁麟超 吕建新[1] 钟连进[1] 厉保秋 高丽昌 金丽琴[1] 

机构地区：[1]温州医科大学检验医学院,生命科学学院,浙江温州325035 [2]山东弘立医学动物实验研究有限公司,山东济南250101

出　　处：《温州医学院学报》2014年第7期485-488,共4页Journal of Wenzhou Medical College

基　　金：浙江省重大科技专项(2009C13038);浙江省临床检验诊断学重中之重学科和浙江省重点科技创新团队资助项目(2010R50048)

摘　　要：目的：建立检测给药食蟹猴血清中重组人表皮生长因子-白介素-18 （rhEGF-IL-18）融合蛋白含量的直接竞争ELISA方法,为该融合蛋白的动力学研究提供方法学.方法：用棋盘法确定包被抗体和酶标抗原工作浓度,建立直接竞争ELISA方法,并对其进行方法学评价.结果：包被抗体和酶标抗原的最适工作浓度为4 μg/mL和1∶1 000.建立的标准曲线的曲线范围为100～ 900 ng/mL,R2为0.9898;日内精密度和日间精密度的最大变异系数分别为2.42％和2.20％;回收率95.3％ ～103.0％;样品在室温放置1 h、冻融2次和冻存20 d三种条件下检测结果均较稳定,相对标准差均小于15％;定量下限为20.0 ng/mL.结论：本研究建立的ELISA方法稳定且可行.Objective： To establish a direct competitive ELISA for determining recombinant human EGF-IL-18 （rhEGF-IL-18） fusion protein levels in serum samples of macaca faseicularis and perform a validation for the assay which would be used in pharmacokinetics. Methods： The optimal concentrations of coating antibody and enzyme-labelled antigen were determined by chessboard titration crossing test. The direct competitive ELISA for determining rhEGF-IL-18 was established and the methodology performance was evaluated. Results： The optimal concentrations of coating antibody and enzyme-labelled antigen were 4 μg/mL and 1：1 000, respectively. The range of the standard curve was 100-900 ng/mL （R2=0.9898）. The RSD of intra-day and inter-day were lower than 2.42% and 2.20%, respectively. The recovery was 95.3%-103.0%. The RSD of the samples standing at room temperature 1 h, freezing and thawing twice, cryopreserved for 20 days were less than 15%. The lower limit of quantification was 20.0 ng/mL. Conclusion： ELISA established in this study is stable and feasible.

关 键 词：重组人表皮生长因子-白介素-18融合蛋白 方法学评价 

分 类 号：R331[医药卫生—人体生理学]