Fluorophor-linked immunosorbent assay: a time- and cost-saving method for the characterization of antibody fragments using a fusion protein of a single-chain antibody fragment and enhanced green fluorescent protein
Fluorophor-linked immunosorbent assay: a time- and cost-saving method for the characterization of antibody fragments using a fusion protein of a single-chain antibody fragment and enhanced green fluorescent protein
复制标题
DOI：
10.1016/s0003-2697(02)00290-7
复制
发表时间：
2002-10-01
影响因子：
2.9
通讯作者：
Schmid, RD
中科院分区：
文献类型：
--
作者：
Oelschlaeger, P;Srikant-Iyer, S;Schmid, RD
描述了一种用于表征单链抗体片段（scFv）的新型测定法，称为荧光团连接免疫吸附测定法（弗里萨）。该方法的原理是scFv与增强型绿色荧光蛋白（EGFP）的融合。可通过测量EGFP结构域的荧光来检测与固定的半抗原结合的scFv结构域。不需要酶联免疫吸附测定（ELISA）所必需的二抗和酶反应的耗时结合。因此，完成弗里萨所需的1.5小时测定时间远短于可比ELISA，后者需要约5小时。这使得弗里萨适用于其中短测定时间是必需的应用，例如在定向进化实验中筛选scFv的突变体文库或在生产过程中监测功能性表达的重组蛋白的量。与可比较的ELISA相反，当确定功能性scFv的相对量时，弗里萨显示不饱和。来自表达融合蛋白的大肠杆菌的细胞提取物的可溶性级分的量和归一化荧光信号显示出线性相关性，R-2 > 0.99。通过用s-三嗪特异性scFv K411 B检测s-三嗪示例性地显示了竞争性弗里萨用于检测分析物的有用性。(C)2002 Elsevier Science（美国）。All rights reserved.
A novel assay, referred to as fluorophor-linked immunosorbent assay (FLISA), for the characterization of single-chain antibody fragments (scFvs) is described. The principle of the method is the fusion of an scFv to enhanced green fluorescent protein (EGFP). The scFv domain, which binds to the immobilized hapten, can be detected by measuring the fluorescence of the EGFP domain. The time-consuming binding of secondary antibodies and enzyme reaction, necessary for enzyme-linked immunosorbent assays (ELISAs) are not required. Consequently, the assay time of 1.5 h needed to complete the FLISA is much shorter than that of comparable ELISAs, which require about 5 h. This renders the FLISA suitable for applications where a short assay time is essential, such as screening of mutant libraries of scFvs in directed evolution experiments or monitoring of the amount of functionally expressed recombinant protein during production processes. In contrast to a comparable ELISA, the FLISA showed no saturation when determining the relative amount of functional scFv. The amount of the soluble fraction of cell extracts from Escherichia coli expressing the fusion protein and the normalized fluorescence signal showed a linear correlation with R-2 > 0.99. The usefulness of a competitive FLISA for the detection of analytes is shown exemplarily by the detection of s-triazines with the s-triazine-specific scFv K411B. (C) 2002 Elsevier Science (USA). All rights reserved.