Rolling Circle Amplification Improves Sensitivity in Multiplex Immunoassays on Microspheres

Abstract

Microspheres are a proven solid-phase format for development of assays used in both research and clinical diagnostics. They can be manufactured in homogeneous batches incorporating very precise quantities of fluorescent dyes. Different amounts of fluorescent dyes in individual microspheres are easily distinguished by a flow cytometer. Quantitative immunoassays can be performed on the surface of the bead by use of a second fluorophore-based detection system compatible with the flow cytometer. The different bead types can be used to prepare separate immunoassays, which can later be combined in a multiplexed assay capable of simultaneously detecting multiple analytes (1)(2). The cytometric bead array products from Becton Dickinson and the Luminex bead format assays manufactured by Upstate Biotechnologies, Bio-Rad, and Biosource take advantage of this approach to multiplexing. These multiplex immunoassays are built on beads distinguishable in flow cytometers by their different fluorophore content. There are, however, numerous examples where important biological markers for cancer, infectious disease, or biochemical processes are present in body fluids or tissues at concentrations too low to be detected by conventional immunoassays (3)(4)(5). With these needs in mind, we have adapted the Rolling Circle Amplification (RCA) reporter system (6)(7)(8) to the detection of protein antigens (immunoRCA). In immunoRCA, the 5′ end of an RCA primer is attached to an antibody. Thus, in the presence of circular DNA, Phi29 DNA polymerase, and nucleotides, the rolling circle reaction produces a concatamer of the complement of the circular DNA sequence that extends from the end of the original primer remaining attached to the antibody. The amplified DNA can be detected by hybridization of complementary oligonucleotide probes or by antibodies specific for nucleotide analogs incorporated during the RCA reaction. RCA is well suited to flow cytometry bead assay formats because the amplification …