title: A simple and rapid immunoassay system using green fluorescent protein tag.
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.
authors.affiliation: Department of Biochemistry, Faculty of Pharmaceutical Sciences, Health Sciences, University of Hokkaido, Japan.
authors.forename: T
authors.initials: T
authors.lastname: Aoki
authors.forename: M
authors.initials: M
authors.lastname: Kaneta
authors.forename: H
authors.initials: H
authors.lastname: Onagi
authors.forename: Y
authors.initials: Y
authors.lastname: Takahashi
authors.forename: K S
authors.initials: KS
authors.lastname: Koch
authors.forename: H L
authors.initials: HL
authors.lastname: Leffert
authors.forename: H
authors.initials: H
authors.lastname: Watabe
mesh_terms: D000818:Animals; D004926:Escherichia coli; D019084:Fluorescent Antibody Technique, Indirect; D049452:Green Fluorescent Proteins; D006801:Humans; D007202:Indicators and Reagents; D008164:Luminescent Proteins; D008970:Molecular Weight; D010751:Phosphopyruvate Hydratase / Q000032:analysis; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000032:analysis; D012756:Sheep; D013050:Spectrometry, Fluorescence
chemical_list: D007202:Indicators and Reagents; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D010751:Phosphopyruvate Hydratase