Immunoassays with rolling circle DNA amplification: a versatile platform for ultrasensitive antigen detection.
Journal: 2000/October - Proceedings of the National Academy of Sciences of the United States of America
ISSN: 0027-8424
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.
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Proc Natl Acad Sci U S A 97(18): 10113-10119

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

Molecular Staging Incorporated, 66 High Street, Guilford, CT 06437; Department of Genetics, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06510; and Department of Pathology, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06510
To whom reprint requests should be addressed. E-mail: ude.elay@draW.divaD.
Contributed by David C. Ward
Contributed by David C. Ward
Accepted 2000 May 22.

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.

Keywords: prostate-specific antigen, human IgE, IgG, ELISA, immuno-microarrays
Abstract

Antibody-based detection systems for specific Ags are versatile and powerful tools for various molecular and cellular analyses, as well as clinical diagnostics (1). The power of such systems originates from the considerable specificity of Abs for particular antigenic epitopes. There are, however, numerous examples where important biological markers for cancer, infectious disease, or biochemical processes are present at too low a concentration in body fluids or tissues to be detected by using conventional immunoassays. Recent advances in the field of low-level Ag detection include the development of stronger fluorochromes and chemiluminescent substrates for use in ELISAs, immunofluorescence-based staining and immunoblotting (2), and the application of signal amplification methods such as tyramide deposition (3). Although these techniques can be quite powerful, greater sensitivity and specificity are often required, particularly when working with limited amounts of sample material or when Ag density is extremely low. With these needs in mind, we have adapted the recently described rolling circle amplification (RCA) reporter system (4) for the detection of protein Ags.

The use of DNA amplification for the detection of Abs bound to Ag has been documented previously (510). In immuno-PCR, a unique DNA sequence tag is associated with a specific Ab using streptavidin-biotin interactions, alternative bridging moieties, or covalent linkage. Abs bound to Ag are then detected by PCR amplification of the associated DNA tag. Multiple Abs and multiple DNA tags have been used (10) to analyze several Ags simultaneously. Although immuno-PCR was shown to be significantly more sensitive than ELISA, gel electrophoresis was required after DNA amplification in solution to separate and/or quantitate the different amplified DNA tags. The requirements for thermal cycling and product separation by gel electrophoresis have restricted the widespread adoption of immuno-PCR as an alternative to ELISA and have precluded its utility in immunohistochemical or array formats.

RCA driven by DNA polymerase can replicate circularized oligonucleotide probes with either linear or geometric kinetics under isothermal conditions (4). Using a single primer, RCA generates hundreds of tandemly linked copies of the circular template within a few minutes. In ImmunoRCA, the 5′ end of this primer is attached to an Ab. In the presence of circular DNA, DNA polymerase, and nucleotides, the rolling circle reaction results in a DNA molecule consisting of multiple copies of the circle DNA sequence that remains attached to the Ab (Fig. (Fig.1).1). The amplified DNA can be detected in a variety of ways, including direct incorporation of hapten-labeled or fluorescently labeled nucleotides, or by hybridization of fluor-labeled or enzymatically labeled complementary oligonucleotide probes. ImmunoRCA, therefore, represents a novel approach for signal amplification of Ab–Ag recognition events. Although RCA reactions can be carried out with either linear or geometric kinetics (4), the signal-generation paradigm we have used in this study is based exclusively on the linear RCA model. Here, we describe the construction of Ab–DNA conjugates and demonstrate the utility of using these conjugates to detect Ags in several different immunoRCA formats.

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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.

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Acknowledgments

We thank Dr. Linhua Fang for characterizing the performance of the conjugates in solution.

Acknowledgments

Abbreviations

CACHETcondensation of amplified circles after hybridization of encoding tags
RCArolling circle amplification
GMBSN-[γ-maleimidobutyryloxy]succinimide ester
PSAprostate-specific Ag
TBSTris-buffered saline
Abbreviations

Footnotes

This contribution is part of the special series of Inaugural Articles by members of the National Academy of Sciences elected on May 2, 2000.

Article published online before print: Proc. Natl. Acad. Sci. USA, 10.1073/pnas.170237197.

Article and publication date are at www.pnas.org/cgi/doi/10.1073/pnas.170237197

Footnotes

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