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-12105084-B2
Publication Date
2024-10-01
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 assay detects a protein or polypeptide across biological samples using antibody-based immunocomplexes and proximity-dependent nucleic acid reporters. First and second antibody-based binding moieties associate protein-specific identity barcodes with a sample-specific identity barcode in each nucleic acid reporter. Reporters from separate assay reactions are pooled and detected by PCR or next-generation sequencing, supporting multiplex analysis across analytes and pooled biological samples.
The method uses sequential capture, release, and recapture of immunocomplexes on paramagnetic beads to reduce nonspecific background and improve sensitivity. Selective partial capture reduces signals from abundant targets and manages detection bandwidth during readout. Because the background section is not included in the provided content, the specific problem being addressed is not explicitly described in patent.
Claims Coverage
The provided claims identify one independent claim directed to detecting a protein or polypeptide across at least two biological samples, comprising four inventive features.
Antibody-based immunocomplex formation with protein-specific target labels
The claim forms an immunocomplex comprising the protein or polypeptide and first and second antibody or antibody-fragment binding moieties, wherein each binding moiety carries a nucleic acid target label comprising an identity barcode specific to the protein or polypeptide.
Sequential capture and release of immunocomplexes on paramagnetic beads
The claim captures the immunocomplex on first paramagnetic beads through nucleic acid hybridization, washes to remove unbound molecules, releases the immunocomplex into solution, and recaptures the immunocomplex on second paramagnetic beads through biotin binding to streptavidin or avidin.
Sample-specific barcoding and proximity-based reporter generation
The claim joins a double-stranded nucleic acid sample label comprising a sample-specific identity barcode via proximity-based ligation with the first and second protein-specific target labels, generating a reporter containing the sample-specific identity barcode positioned between the two protein-specific identity barcodes.
Pooled reporter detection across biological samples
The claim pools nucleic acid reporters from separate assay reactions and detects the protein or polypeptide in the respective biological samples by detecting the pooled reporters using next-generation sequencing or PCR amplification.
The independent claim combines antibody-based target recognition, sequential bead capture and release, dual-barcode reporter generation, and pooled reporter detection across multiple biological samples.
Stated Advantages
Releasing bead-captured immunocomplexes into solution before signal generation and sequential capture and recapture reduce nonspecific background and improve sensitivity.
In the reported five-analyte demonstration, assays achieve attomolar-level sensitivity, with three assays having limits of detection below 50 aM and average coefficients of variation below 30%.
The assay enables multiplexing across analytes and pooled samples, including up to thousands of analytes.
Paired target IDs distinguish true signals from mismatched-binding noise.
Selective partial capture is proposed to reduce signals from abundant targets and manage detection bandwidth.
Reported examples demonstrate attomolar EGFR detection and a five-analyte NULISAseq assay with average CVs below 30% and LODs of 16.7–299.7 aM across the tested analytes.
Documented Applications
Detection of a protein or polypeptide in at least two biological samples, including serum, plasma, and cerebrospinal fluid.
A five-analyte NULISAseq demonstration measuring IL-4, IL-6, IL-10, TNFα, and P24.
Multiplex protein detection assays, including detection of EGFR.
Detection and quantification of pooled nucleic acid reporters using PCR, qPCR, digital PCR, or next-generation sequencing.
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