Antigen detection using fluorophore-modified antibodies and magnetic microparticles

In this submission, we detail a new method for detecting mouse Immunoglobulin G (IgG) utilizing fluorophore-modified antibodies and antibody-modified magnetic microparticles. This was accomplished by adding an excess amount of fluorescein isothiocyanate (FITC)-modified goat anti-mouse IgG (F(ab')2 fragment specific to mouse IgG), and allowing them to react for some time. After a given reaction time, the bound antibody could be isolated from the unbound, excess antibody via addition of goat anti-mouse IgG (Fc fragment specific)-modified magnetic microparticles. After application of a magnetic field, the free, unbound antibody could be isolated and the fluorescence intensity of the isolated solution detected. We show that the fluorescence intensity decreased linearly as antigen concentration increased, has a detection limit of 0.65 nM, and was species specific. In a subsequent experiment, we demonstrated that simple filtration could also be used to separate the bound from unbound antibodies, which enhances the simplicity and ultimate utility of the assay. We also show that the approach can be used to detect two different analytes in a single solution, which could be easily modified to detect multiple antigens. Finally, we demonstrate that simple observation by the naked eye could be used to detect specific antigens in a sample. This sensing approach could be easily modified to detect many other antigens, even biomarkers for disease. It is this versatility, and simplicity that makes this sensing approach potentially very impactful.