Nucleic acid linked immune-sandwich assay (NULISA)
Inventors
Luo, Yuling • Feng, Wei • Grzybowski, Adrian • YIN, Yiyuan • Chen, Shiping
Assignees
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Publication Number
US-12188929-B2
Publication Date
2025-01-07
Expiration Date
Abstract
Disclosed herein are highly sensitive immunoassays that utilize a capture/release mechanism to reduce non-specific binding and achieve detection with attomolar-level sensitivity. Kits that can be used for carrying out these highly sensitive immunoassays are also disclosed herein.
Core Innovation
The disclosed system detects an analyte across at least two samples using two antibody or antibody-fragment binding moieties that bind distinct epitopes on the analyte to form an immunocomplex. Nucleic acid target labels carry analyte-specific identity barcodes, while a double-stranded nucleic acid sample label carries a sample-specific identity barcode. Additionally, the system incorporates paramagnetic beads coupled to receiving groups that bind corresponding presenting groups on the binding moieties.
The platform utilizes barcode-based, proximity-dependent analyte detection where target and sample identifiers link into nucleic acid reporters for pooled multiplex readout. Bead-captured immunocomplexes release into solution before signal generation, which enriches complexes, reduces nonspecific background, and enhances assay sensitivity. Furthermore, co-detection of both target identity barcodes distinguishes intended binder pairs and diminishes false signals during multiplexed analysis.
Claims Coverage
The claims identify one independent claim directed to a system for detecting an analyte in at least two samples, comprising four inventive features.
Paired antibody binding to distinct analyte epitopes
A first antibody or antibody fragment and a second antibody or antibody fragment bind to different epitopes on the analyte to form an immunocomplex.
Analyte-specific nucleic acid target labels and presenting groups
The first binding moiety comprises a first presenting group with a nucleic acid tag hybridized to a first nucleic acid target label, and the second binding moiety comprises a second presenting group with a nucleic acid capture probe hybridized to a second nucleic acid target label, wherein each target label comprises an analyte-specific identity barcode.
Paramagnetic beads coupled with receiving groups
The system comprises first paramagnetic beads coupled with a first receiving group and second paramagnetic beads coupled with a second receiving group, which bind to the first and second presenting groups, respectively.
Sample-specific double-stranded nucleic acid label
A double-stranded nucleic acid sample label comprises a longer strand with 5′ and 3′ overhangs that hybridize with the first and second nucleic acid target labels, and a shorter strand comprising a sample-specific identity barcode.
The independent claim combines distinct-epitope antibody binding, analyte-specific target labels, bead-associated receiving groups, and a sample-specific nucleic acid label to detect an analyte across multiple samples.
Stated Advantages
The approach enables multiplex analysis across analytes and samples.
Co-detection of both target identity barcodes distinguishes intended binder pairs, reduces false signals, and improves specificity.
The capture-and-release mechanism of bead-captured immunocomplexes into solution before signal generation enriches complexes, reduces nonspecific background, and improves signal-to-noise ratio and assay sensitivity.
Controlled partial capture of abundant analytes manages detector bandwidth.
The platform achieves attomolar protein detection limits, reaching 97.6 aM by sequencing and 36.0 aM by qPCR, with three of five five-plex assays showing detection limits below 500 aM and average coefficients of variation below 30%.
Documented Applications
Detection of an analyte in at least two samples using analyte-specific target identifiers and a sample-specific label.
Multiplex analyte detection across multiple samples, with reporters detected by qPCR, digital PCR, or next-generation sequencing.
Five-analyte multiplex NULISAseq detection of IL-4, IL-6, IL-10, TNF̲̲α, and P24.
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