A simple and rapid immunoassay system using green fluorescent protein tag.
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.
Aoki
T
T
Department of Biochemistry, Faculty of Pharmaceutical Sciences, Health Sciences, University of Hokkaido, Japan.
Kaneta
M
M
Onagi
H
H
Takahashi
Y
Y
Koch
K S
KS
Leffert
H L
HL
Watabe
H
H
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
D007202:Indicators and Reagents; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D010751:Phosphopyruvate Hydratase