Feasibility study of enzyme-amplified sandwich immunoassay using protein G capillary affinity chromatography and laser induced fluorescence detection

Abstract

The feasibility of performing the enzyme-amplified sandwich immunoassay with protein G capillary affinity chromatography and laser induced fluorescence (LIF) detection was investigated using the determination of human immunoglobulin G (hIgG) as a model system. The incubated mixture of samples containing hIgG and alkaline phosphatase (ALP) conjugated goat anti-human IgG F(ab')2 fragment was loaded onto the capillary column packed with recombinant protein G bound perfusive support in neutral pH. After nonretained compounds were eluted, the fluorogenic ALP substrate, fluorescein diphosphate (FDP), was loaded onto the capillary column followed by stop-flow incubation. Finally, the product of ALP-mediated hydrolysis of FDP, fluorescein, was swept out of the capillary column and detected with a LIF detector using the 488 nm line of an argon ion laser as the excitation source. Chromatographic conditions were optimized. The calibration curve for hIgG was linear over the range of 0.5-50 pmol/L (r2=0.999) with the limit of detection of 0.2 pmol/L.