Development of a Bead-Based Multiplexed Immunoassay Using Image Cytometric Analysis

Abstract

Keywords: multiplexed immunoassay image cytometry, bead-based assayIn this paper, we present the demonstration of a size-encoded bead-based multiplexed immunoassay with image cytometric decoding and readout. Antibodies were immobilized on polystyrene microspheres by physical adsorption and employed for the demonstration of this duplexed immunoassay. First, we carried out a duplexed immunoassay with a mouse and rat IgG detection system and discussed the problem of cross-reactivity inherent to these immunoreaction systems. We then carried out a duplexed immunoassay with a mouse IgG and human albumin detection system, which was less cross-reactive. We subsequently demonstrated the application of size-encoded image cytometric decoding and the detection of mouse IgG and human albumin using this duplexed immunoassay system. BackgroundImmunoassays are one type of analytical method, which are widely applied for the detection and quantitation of proteins, owing to their simplicity and high sensitivity.(1-4) Recent progress in proteomics has revealed correlations among disease, bioactivity, and molecular information, and this has strongly motivated the clinical application of multiplexed protein assay methods.(5-7)Protein microarrays, which are based on specific antibodies, are the ideal tool for multiplexed immunoassays. (8,9) A spot of antibody is printed locally using microprinting technology for the application of conventional, but multiplexed, immunoreactions. Therefore, antibody printing is becoming the main technical difficulty in carrying out these assays. Moreover, the reaction kinetics are relatively slow owing to the lengthy diffusion of molecules, because the reaction is carried out on a planar surface. (10,11) As an alternative technique, bead-based multiplexed immunoassays have been intensively researched to overcome the drawbacks of microarray technology. (12)(13)(14)(15) In the case of beadbased immunoassays, multiplexing is achieved by preparing multiple antibody-immobilized beads corresponding to each analyte, and thus multiplexed immunoreactions can be implemented