双抗体夹心ELISA定量检测IL-2-HSA融合蛋白

以抗人IL-2多克隆抗体为包被抗体，以IL-2-HSA融合蛋白为夹心抗原，以辣根过氧化物酶（HRP）标记的抗HSA单克隆抗体为检测抗体，建立了一种定量测定完整的IL-2-HSA融合蛋白的双抗体夹心ELISA方法。包被抗体和检测抗体的最佳工作浓度分别为为2 μg/mL和0.5 μg/mL，方法的线性检测范围39.06~1250 ng/mL，标准曲线回归方程为y=0.442 9 x-1.143 3，r=0.996 6，灵敏度可达10.25 ng/mL，批内、批间变异系数分别为6.40%和7.81%，发酵上清液中回收率为98.13%~102.94 %，与IL-2、HSA基本无交叉反应，健全性分析表明发酵液及小鼠血清组分及稀释倍数对该方法无影响。该方法可用于该融合蛋白的发酵过程优化、分离纯化工艺、后期药动学、临床研究等的定量检测。

A new sandwich ELISA method for quantitative analysis of fusion protein IL-2-HSA

A double antibody sandwich ELISA for quantitative analysis of recombinant fusion protein IL-2-HSA was constructed using a polyclonal antibody to human IL-2 for capture and a monoclonal antibody to HSA with HRP-labeled conjugate for detection.The optimal concentration of the first coating antibody and detection antibody were 2 μg/mL and 0.5 μg/mL，respectively.Regression equation of the linear calibration curve was：y=0.442 9 x-1.143 3 with a correlation coefficient of 0.996 6，and the linear detection ranged from 39.06 ng/mL to 1 250 ng/mL.Recovery from the supernatant of fermentation broth was 98.13% to 102.94%.The specificity assay indicated that it had little cross-reactions with IL-2 and HSA.The soundness analysis suggested that fermentation broth，mouse serum and dilution had no influence on the method.The present method can be used in the studies on fermentation，purification and clinical diagnosis.