Magnetic Bead-Sensing-Platform-Based Chemiluminescence Resonance Energy Transfer and Its Immunoassay Application

A competitive immunoassay based on chemiluminescence resonance energy transfer (CRET) on the magnetic beads (MBs) is developed for the detection of human immunoglobulin G (IgG). In this protocol, carboxyl-modified MBs were conjugated with horseradish peroxidase (HRP)-labeled goat antihuman IgG (HRP-anti-IgG) and incubated with a limited amount of fluorescein isothiocyanate (FITC)-labeled human IgG to immobilize the antibody–antigen immune complex on the surface of the MBs, which was further incubated with the target analyte (human IgG) for competitive immunoreaction and separated magnetically to remove the supernatant. The chemiluminescence (CL) buffer (containing luminol and H₂O₂) was then added, and the CRET from donor luminol to acceptor FITC in the immunocomplex on the surface of MBs occured immediately. The present protocol was evaluated for the competitive immunoassay of human IgG, and a linear relationship between CL intensity ratio (<i><i>R</i> = I</i>₄₂₅/<i>I</i>₅₂₅) and human IgG concentration in the range of 0.2–4.0 nM was obtained with a correlation coefficient of 0.9965. The regression equation was expressed as <i>R</i> = 1.9871<i>C</i> + 2.4616, and a detection limit of 2.9 × 10^–11 M was obtained. The present method was successfully applied for the detection of IgG in human serum. The results indicate that the present protocol is quite promising for the application of CRET in immunoassays. It could also be developed for detection of other antigen–antibody immune complexes by using the corresponding antigens and respective antibodies