Quantitative measurement of bitagged recombinant proteins using an immunometric assay:: Application to an anti-substance P recombinant antibody

作     者：Boquet, D Créminon, C Clément, G Frobert, Y Nevers, MC Essono, S Grassi, J 

作者机构：CEA Saclay Serv Pharmacol & Immunol DRM DSV F-91191 Gif Sur Yvette France CEA Saclay Lab Associe CEA INRA F-91191 Gif Sur Yvette France 

出 版 物：《ANALYTICAL BIOCHEMISTRY》 (分析生物化学)

年 卷 期：2000年第284卷第2期

页      面：221-230页

核心收录：

学科分类：0710[理学-生物学] 071010[理学-生物化学与分子生物学] 07[理学] 0703[理学-化学] 

主　　题：氨基酸序列 抗体 单克隆/分析 抗体 单克隆/遗传学 抗体 单克隆/免疫学 抗体亲和力 抗体特异性 碱基序列 克隆 分子 DNA引物 DNA 互补 免疫测定/方法 免疫球蛋白可变区/分析 免疫球蛋白可变区/遗传学 免疫球蛋白可变区/免疫学 分子序列数据 神经肽类 重组蛋白质类/分析 重组蛋白质类/免疫学 可重复性 结果 P物质/免疫学 动物 小鼠 

摘      要：We have developed two different immunometric assays to directly quantify both the total and the active fractions of a recombinant antibody (single chain fragment variable, or ScFv) as obtained in a crude extract from an Escherichia coli expression system. For total determination, the assay is based on the simultaneous recognition of two different peptide Tag sequences (Ha-Tag and Myc-Tag) at each of the N- and C-terminal extremities of the recombinant protein. A monoclonal antibody (mAb 12CA5, directed against Ha-Tag), coated on microtiter plates, is used for capture, and the mAb 9E10 (directed against Myc-Tag), labeled with acetylcholinesterase (AChE, EC 3.1.1.7), acts as tracer. In parallel, for the determination of the active fraction, the capture is performed using microtiter plates coated with the antigen, while solid-phase-immobilized ScFv is measured using the same 9E10 tracer mAb, A synthetic peptide in which the two Tag sequences were joined was used as a standard, thus avoiding the laborious purification of a recombinant protein as reference. The method was applied to the direct measurement, in periplasmic extracts, of the total and active;fractions of an ScFv produced at different induction temperatures. (C) 2000 Academic Press.