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.prostate-specific antigen ͉ human IgE ͉ IgG ͉ ELISA ͉ immuno-microarrays

AFM fishing nanotechnology is the way to reverse the Avogadro number in proteomics

“…As soon as we gain the capacity to register single protein molecules and their complexes, we shall be able to address the problem of compiling proteomic maps in any biological material on a single-molecule level. The alternative approach in this research area, which will probably find wide application in the future, lies in the development of methods enabling replication of protein molecules [27] or the use of PCR for monitoring the antigen/antibody interaction reactions (immuno-PCR [28], immuno-RCA [29]), and the method for proximal coupling of aptamers [30]). However, insufficient development of these methods does not yet allow us to consider them as a basis for highly productive proteomic investigations.…”

Microarray methods for protein biomarker detection

“…RCA has been more commonly applied to DNA quantitation and mutation detection, 83,84 and in order to adapt it for protein detection, antibodies covalently conjugated to a DNA primer sequence were designed. [85][86][87] Fig. 3(a) outlines the RCA mechanism for the amplified detection of target proteins that are first directly labeled with biotin in serum solution before incubating on the antibody array.…”

Genomics, Molecular Epidemiology and Diagnostics of Infectious hypodermal and hematopoietic necrosis virus

“…By using specific primer pairs, the closed circular C probe is amplified under isothermal condition. After its initial discovery [98], it became widely used for diagnosis and single nucleotide polymorphism analysis [5,53,73,87,88,91]. In 2006, a simple and sensitive RAM assay was developed for detection of IHHNV by Teng et al [86].…”