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

Abstract

We describe an adaptation of the rolling circle amplification (RCA) reporter system for the detection of protein Ags, termed "immunoRCA. " In immunoRCA, an oligonucleotide primer is covalently attached to an Ab; thus, in the presence of circular DNA, DNA polymerase, and nucleotides, amplification results in a long DNA molecule containing hundreds of copies of the circular DNA sequence that remain attached to the Ab and that can be detected in a variety of ways. Using immunoRCA, analytes were detected at sensitivities exceeding those of conventional enzyme immunoassays in ELISA and microparticle formats. The signal amplification afforded by immunoRCA also enabled immunoassays to be carried out in microspot and microarray formats with exquisite sensitivity. When Ags are present at concentrations down to fM levels, specifically bound Abs can be scored by counting discrete fluorescent signals arising from individual Ag-Ab complexes. Multiplex immunoRCA also was demonstrated by accurately quantifying Ags mixed in different ratios in a two-color, single-molecule-counting assay on a glass slide. ImmunoRCA thus combines high sensitivity and a very wide dynamic range with an unprecedented capability for single molecule detection. This Ag-detection method is of general applicability and is extendable to multiplexed immunoassays that employ a battery of different Abs, each labeled with a unique oligonucleotide primer, that can be discriminated by a color-coded visualization system. ImmunoRCA-profiling based on the simultaneous quantitation of multiple Ags should expand the power of immunoassays by exploiting the increased information content of ratio-based expression analysis.

Figure 1

Schematic of immunoRCA assay. (Top Left) A reporter Ab conjugated to an oligonucleotide binds to a test analyte that is captured on a solid surface by covalent attachment or by a capture Ab. (Top Right) A DNA circle hybridizes to a complementary sequence in the oligonucleotide. (Bottom Left) The resulting complex is washed to remove excess reagents, and the DNA tag is amplified by RCA. (Bottom Right) The amplified product is labeled in situ by hybridization with fluor-labeled oligonucleotides.

Figure 2

Comparison of immunoRCA and conventional immunoassay in an ELISA format. Filled circles, ELISA of human IgE with immunoRCA, using an anti-human IgE-DNA conjugate; open squares, ELISA of human IgE with an anti-human IgE-alkaline phosphatase conjugate.

Figure 3

Detection of PSA by immunoRCA in a microspot assay. (A) Fluorescent microscope images of immunoRCA signals obtained with different concentrations of human PSA captured on microspots of anti-human PSA Ab. Concentrations: (Upper Left) 5 pg/ml PSA; (Lower Left) 0.5 pg/ml PSA; (Upper Right) 0.1 pg/ml PSA; (Lower Right) 0 pg/ml PSA. (B) Fluorescence in microscope images was quantitated as described in Methods and plotted versus PSA concentrations incubated on microspots.

Figure 4

ImmunoRCA anti-human IgE microarray dose-response for purified IgE. Signals from six microarray spots were averaged for each point, and the background (no IgE) signal was subtracted. (Inset) Microarray scanner image of anti-human IgE array incubated with 1 ng/ml IgE.

Figure 5

Dual Ag detection using immunoRCA-CACHET. (A) The RCA products from the anti-avidin and the anti-sheep Ab conjugates were decorated with fluorescein- and Cy3-labeled detector oligonucleotides, respectively. Each image shown is the superimposition of two separate images, with fluorescein and Cy3 signals pseudocolored in green and red, respectively. (Upper Left) Avidin:anti-digoxigenin (anti-dig) = 1:1; (Upper Right) avidin:anti-dig = 1:4; (Lower Left) avidin:anti-dig = 1:20; (Lower Right) avidin:anti-dig = 0:1. (B) Quantitation of avidin (FITC) and anti-digoxigenin (Cy3) signals.