分泌型ICOS-mIg重组融合蛋白定量检测方法的建立和应用  

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

作　　者：王健[1] 沈茜[1] 

机构地区：[1]第二军医大学长海医院中心实验室,上海200433

出　　处：《细胞与分子免疫学杂志》2005年第5期646-649,共4页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家高技术研究发展计划(863)资助项目(No.2002AA214091)

摘　　要：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7.8～500 μg/L,标准曲线的回归方程为: y=-0.7864+1.1635 log(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.AIM： To set up a double antibody sandwich ELISA for rapid quantification of recombinant ICOS-mlg 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 μg/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.

关 键 词：重组融合蛋白 ELISA 可诱导共刺激分子 免疫球蛋白恒定片段 

分 类 号：R392.11[医药卫生—免疫学]