Real-time label-free detection of complement activation products in human serum by white light reflectance spectroscopy

Abstract

We present a label-free, real-time sensor based on white light reflectance spectroscopy for quantitating the complement activation product C3b and its metabolites as a biomarker in human serum. Our novel sensor allows real-time monitoring of biomolecular reactions (in this case, antigen–antibody reactions) taking place on a reflective surface within a flow cell. Detection was based on monitoring the increase in film thickness caused by its immunoreaction with a specific antibody; this reaction was seen as a shift in the wavelength at which constructive interference was observed . Quantitation of C3b was achieved by immobilizing a specific mouse monoclonal antibody onto the refractive surface and monitoring the rate of the signal changes occurring during the first 60 s of the immunoreaction between the antibody and known concentrations of purified C3b or dilutions of complement-activated human serum. The lowest detectable concentration of purified C3b was 20 ng/mL, and complement activation products in human serum samples could be detected at dilutions as high as 6000-fold. The advantages of the method include its relatively low cost, short analysis time, and high assay sensitivity and reliability. Thus, this novel assay method can be used to monitor serum C3b produced as a result of complement activation in a variety of normal and pathologic conditions.