Immunoassays with rolling circle DNA amplification: A versatile platform for ultrasensitive antigen detection

We describe an adaptation of the rolling circle amplification (RCA) reporte r system for the detection of protein Ags, termed "immunoRCA." In immunoRCA , an oligonucleotide primer is covalently attached to an Ab; thus, in the p resence of circular DNA, DNA polymerase, and nucleotides, amplification res ults in a long DNA molecule containing hundreds of copies of the circular D NA sequence that remain attached to the Ab and that can be detected in a va riety of ways. Using immunoRCA, analytes were detected at sensitivities exc eeding those of conventional enzyme immunoassays in ELISA and microparticle formats. The signal amplification afforded by immunoRCA also enabled immun oassays to be carried out in microspot and microarray formats with exquisit e sensitivity. When Ags are present at concentrations down to fM levels, sp ecifically bound Abs can be scored by counting discrete fluorescent signals arising from individual Ag-Ab complexes. Multiplex immunoRCA also was demo nstrated by accurately quantifying Ags mixed in different ratios in a two-c olor, single-molecule-counting assay on a glass slide, ImmunoRCA thus combi nes high sensitivity and a very wide dynamic range with an unprecedented ca pability for single molecule detection. This Ag-detection method is of gene ral applicability and is extendable to multiplexed immunoassays that employ a battery of different Ags, each labeled with a unique oligonucleotide pri mer, that can be discriminated by a color-coded visualization system. Immun oRCA-profiling based on the simultaneous quantitation of multiple Ags shoul d expand the power of immunoassays by exploiting the increased information content of ratio-based expression analysis.