一种使用绿色荧光蛋白标签的简单快速免疫分析系统。

A simple and rapid immunoassay system using green fluorescent protein tag.

作者信息

Aoki T, Kaneta M, Onagi H, Takahashi Y, Koch K S, Leffert H L, Watabe H

机构信息

Department of Biochemistry, Faculty of Pharmaceutical Sciences, Health Sciences, University of Hokkaido, Japan.

出版信息

J Immunoassay. 1997 Nov;18(4):321-33. doi: 10.1080/01971529708005825.

DOI:10.1080/01971529708005825

PMID:9358339

Abstract

A fusion protein between green fluorescent protein (GFP) and neuron-specific enolase (NSE) was expressed in Escherichia coli. The GFP-NSE fusion protein migrated at 62 kDa in SDS-PAGE and retained the fluorescence under non-heating conditions. However, heat-denatured GFP-NSE was non-fluorescent and migrated at 74 kDa corresponding to the theoretical value. This suggests that the special structure of GFP, which is not denatured by SDS, influences its mobility in SDS-PAGE under non-heating conditions. The fluorescence intensity of GFP-NSE was measurable over a wide range by spectrophotometry or densitometry. The competitive immunoassay for NSE was performed using GFP-NSE as labeled antigen. Under our assay conditions, the working range of this system was about 2 -60 ng. This simple and rapid fluorescence immunoassay (FIA) using GFP-tagged antigen may be applicable to many protein markers.

摘要

绿色荧光蛋白（GFP）与神经元特异性烯醇化酶（NSE）之间的融合蛋白在大肠杆菌中表达。GFP-NSE融合蛋白在SDS-PAGE中迁移至62 kDa，并在非加热条件下保持荧光。然而，热变性的GFP-NSE无荧光，且迁移至74 kDa，与理论值相符。这表明GFP的特殊结构不会被SDS变性，在非加热条件下会影响其在SDS-PAGE中的迁移率。GFP-NSE的荧光强度可通过分光光度法或光密度法在很宽的范围内进行测量。使用GFP-NSE作为标记抗原进行NSE的竞争性免疫测定。在我们的测定条件下，该系统的工作范围约为2 - 60 ng。这种使用GFP标记抗原的简单快速荧光免疫测定（FIA）可能适用于许多蛋白质标志物。