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目的:建立双抗体夹心ELISA法,定量检测重组可诱导共刺激分子(ICOS)-mIg融合蛋白的分泌型表达,并对其灵敏度、特异性及线性检测范围进行评价。方法:用分子生物学技术制备重组ICOS-mIg融合蛋白。以羊抗小鼠IgG为包被抗体,HRP标记的马抗小鼠IgG为检测抗体,通过配对试验、方阵滴定试验及绘制mIgG浓度与A450值的标准曲线,建立定量检测重组ICOS-mIg融合蛋白的双抗体夹心ELISA法。结果:建立了双抗体夹心ELISA法,检测的线性范围为7.8~500μg/L,标准曲线的回归方程为:y=-0.7864+1.1635log(x),R2=0.9911,P<0.0001。应用该方法可快速检测哺乳动物细胞表达的重组ICOS-mIg融合蛋白的分泌量。结论:建立了一种可快速定量检测重组ICOS-mIg融合蛋白分泌表达的双抗体夹心ELISA法。该法灵敏、准确、快速、实用性好,对优化ICOS-mIg融合蛋白表达细胞的筛选和大规模制备具有重要价值。
Objective: To establish a double-antibody sandwich ELISA for the quantitative detection of the secretory expression of recombinant inducible co-stimulatory molecules (ICOS) -mIg fusion protein, and to evaluate its sensitivity, specificity and linearity detection range. Methods: Recombinant ICOS-mIg fusion protein was prepared by molecular biology techniques. Using goat anti-mouse IgG as coating antibody and HRP-labeled horse anti-mouse IgG as detection antibody, a standard curve of mIgG concentration and A450 value was drawn by paired test, square titration test and quantitative analysis of recombinant ICOS-mIg fusion Double antibody sandwich ELISA. Results: A sandwich ELISA was established. The linear range was 7.8-500 μg / L. The regression equation of the standard curve was y = -0.7864 + 1.1635 log (x), R2 = 0.9911, P <0.0001. This method allows rapid detection of the secretion of recombinant ICOS-mIg fusion protein expressed in mammalian cells. Conclusion: A double-antibody sandwich ELISA was developed to detect the secretion of recombinant ICOS-mIg fusion protein rapidly and quantitatively. The method is sensitive, accurate, rapid and practical. It is of great value to optimize the screening and large-scale preparation of ICOS-mIg fusion protein expressing cells.