分泌型ICOS-mIg重组融合蛋白定量检测方法的建立和应用

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。

Development and application of a method for rapid quantification of recombinant ICOS-mIg fusion protein

AIM: To set up a double antibody sandwich ELISA for rapid quantification of recombinant ICOS-mIg fusion protein and evaluate its sensitivity, specificity and linear detection range. METHODS: Recombinant ICOS-mIg fusion protein was prepared by molecular biology technique. Sheep anti-mouse IgG and horse anti-mouse IgG-HRP conjugate were selected as coating antibody and detection antibody, respectively for sandwich ELISA by pair-matching experiments. A standard curve for determining the recombinant ICOS-mIg fusion protein was plotted and applied for quantification of ICOS-mIg fusion protein. RESULTS: A sandwich ELISA for quantification of the recombinant ICOS-mIg fusion protein was developed. Its linear detection was 7.8 to 500 microg/L(R(2)=0.9911, P<0.0001). CONCLUSION: The recombinant ICOS-mIg fusion protein can be determined by this sandwich ELISA rapidly and precisely.