Selective and sensitive fluorescence detection method for pig IgG based on competitive immunosensing strategy and...

A novel fluorescence detection method based on competitive immunoassay and magnetic bioseparation technique was developed and applied to the determination of pig immunoglobulin G (IgG) in serum samples. Core-shell structured Fe₃O₄@SiO₂ nanoparticles were synthesized by chemical coprecipitation, followed by functionalization with amino groups and immobilization of pig IgG antibodies. The synthesized Fe₃O₄@SiO₂-antibody nanoparticles were employed as the probe for the competitive immune recognition of the target antigens in samples and the antigens labeled with fluorescein isothiocyanate (FITC). After the magnetic separation of probes binding with these two types of antigens, fluorescence of the free FITC-labeled antigens was measured for the quantification of the target antigens, since the ratio of the FITC-labeled antigens in supernatant before and after the competitive immune recognition depends on the amount of the target antigens in sample, due to the competitive nature of the binding of the antibody for these two types of antigens. Under the optimal conditions, a linear relationship was obtained between the change of fluorescence intensity and the concentration of pig IgG in a range from 0.75 to 23.50 µg L^-1, with a detection limit (LOD) of 0.031 µg L^-1. With the facile-prepared probes, this fluorescence competitive method can provide a rapid, specific and highly sensitive immunoassay protocol for the determination of target proteins in complex matrix samples.