Development of a sensitive and specific assay to detect the presence of immune complex in human serum 2253742

Abstract

Abstract Introduction In several autoimmune diseases, such as systemic lupus erythematosus (SLE), glomerulonephritis or vasculitis, autoantibodies recognize and bind self-protein, forming immune complexes (IC). IC deposition or improper clearance can lead to inflammation and tissue damage. Here, we describe the development of a plate-based assay to detect presence of IC in serum samples. Methods Employing a FcgRIIb reporter cell line, we observed that IC formed in vitro in assay media induced signal over antigen or antibody alone. However, in vitro formed IC spiked in serum completely abrogate luciferase signal. This effect was caused by an unknown serum component and was independent of IgG levels, since similar results were obtained by spiking IC into IgG depleted serum. To optimize this assay, we formed IC, spiked it into neat or multiple serum dilutions in assay media and conclude that a dilution factor of 1000 is required to overcome matrix interference. Due to the matrix interference, we developed a more sensitive assay that could detect IC formation in a more concentrated serum. Results Therefore, we developed a plate-based assay using streptavidin coated plates with biotinylated recombinant FcgRIIb to capture IC and a sulfo-tagged anti-light chain antibody to detect. We observed in vitro formed IC spiked into healthy serum, IgG depleted serum or assay buffer is detected with no matrix interference and the signal to background (antibody or antigen alone) of 13. Conclusion All together, we describe an assay that specifically detects immune complexed IgG that can be used to screen the presence of IC in serum from patients. Funding Source n/a Topic Categories Basic Autoimmunity (BA)