A simple and rapid immunoassay system using green fluorescent protein tag

Area of Science:

• Biochemistry
• Molecular Biology
• Immunotechnology

Background:

• Neuron-specific enolase (NSE) is a biomarker.
• Green fluorescent protein (GFP) is a useful reporter molecule.
• Developing novel detection methods for biomarkers is crucial.

Purpose of the Study:

• To create a fusion protein of GFP and NSE.
• To characterize the fusion protein's properties.
• To develop a fluorescence immunoassay (FIA) for NSE detection.

Main Methods:

• Expression of GFP-NSE fusion protein in Escherichia coli.
• SDS-PAGE analysis under non-heating and heat-denatured conditions.
• Spectrophotometry and densitometry for fluorescence measurement.
• Development of a competitive immunoassay using GFP-NSE as labeled antigen.

Main Results:

• The GFP-NSE fusion protein showed retained fluorescence under non-heating SDS-PAGE.
• Heat-denatured GFP-NSE lost fluorescence and migrated at its theoretical molecular weight.
• The fluorescence intensity was measurable over a wide range.
• The developed FIA system demonstrated a working range of 2-60 ng for NSE detection.

Conclusions:

• The unique structure of GFP influences its mobility in SDS-PAGE.
• The GFP-NSE fusion protein is suitable for quantitative fluorescence measurements.
• The developed fluorescence immunoassay is a simple, rapid, and potentially broadly applicable method for detecting protein markers like NSE.