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

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.