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Simple Bulk Readout of Digital Nucleic Acid Quantification Assays
Published on: September 24, 2015
Immunoassays with rolling circle DNA amplification: a versatile platform for ultrasensitive antigen detection
B Schweitzer1, S Wiltshire, J Lambert
1Molecular Staging Incorporated, 66 High Street, Guilford, CT 06437; Department of Genetics, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06510, USA.
Summary
We developed immunoRCA, a novel protein antigen detection method. This assay offers high sensitivity and single-molecule detection capabilities, significantly improving upon traditional immunoassays for advanced diagnostics.
Area of Science:
- Biotechnology
- Molecular Biology
- Immunology
Background:
- Conventional enzyme immunoassays (ELISA) have limitations in sensitivity and dynamic range.
- Protein antigen detection is crucial for diagnostics and research.
- Existing methods struggle with single-molecule detection and multiplexing.
Purpose of the Study:
- To adapt the rolling circle amplification (RCA) reporter system for protein antigen detection, termed immunoRCA.
- To achieve enhanced sensitivity and single-molecule detection capabilities for immunoassays.
- To demonstrate the applicability of immunoRCA in microspot, microarray, and multiplex formats.
Main Methods:
- Covalently attaching an oligonucleotide primer to an antibody (Ab).
- Utilizing circular DNA, DNA polymerase, and nucleotides for amplification.
- Detecting amplified DNA molecules, which remain attached to the Ab, via various methods including fluorescence.
Main Results:
- ImmunoRCA detected analytes with sensitivity exceeding conventional ELISA and microparticle assays.
- Achieved femtomolar (fM) level detection of antigens by counting discrete fluorescent signals from individual Ag-Ab complexes.
- Demonstrated multiplex immunoRCA for accurate quantification of mixed antigens in a two-color, single-molecule-counting assay.
Conclusions:
- ImmunoRCA combines high sensitivity, a wide dynamic range, and single-molecule detection capabilities.
- The method is broadly applicable and extendable to multiplexed immunoassays using color-coded visualization.
- ImmunoRCA-profiling offers expanded power for immunoassays through ratio-based expression analysis.
