General detection and isolation of specific cells by binding of labeled molecules
Inventors
Pedersen, Henrik • Jakobsen, Søren • HADRUP, Sine Reker • BENTZEN, Amalie Kai • JOHANSEN, Kristoffer Haurum
Assignees
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Publication Number
US-11402373-B2
Publication Date
2022-08-02
Expiration Date
Abstract
The present invention relates to detection molecules comprising at least one binding molecule, at least one linker and at least one label, and detection methods making use of same. The invention provides a high-throughput method for detection, isolation and/or identification of specific entities or cells.
Core Innovation
The invention provides a cell detection method that combines a sample comprising cells with a composition comprising two or more different sets of detection molecules to form a mixture. Each detection molecule comprises at least two binding molecules that are peptide-MHC complexes and at least one nucleic acid label comprising a 5′ first primer region, a barcode region unique to and specific for the binding molecules of a set, a 3′ second primer region, and a random nucleotide region between the primer regions, with the random nucleotide region unique for each detection molecule and the primer regions identical for all detection molecules in the composition.
The detection molecules further include a multimerization domain associated with the peptide-MHC binding molecules and the nucleic acid label, optionally via one or more connector molecules. The multimerization domain is selected from peptide, protein, streptactin, a polysaccharide, dextran, avidin, and streptavidin.
The method incubates the mixture so that the detection molecules bind to the cells to form labeled cells, then isolates the labeled cells and detects one or more labels bound to the labeled cells. After detection, the identity of the detected labeled cells is determined using the barcode region as an identification tag for the at least two binding molecules of a set and the unique random nucleotide region to distinguish individual detection molecules.
Claims Coverage
The provided material identifies one independent claim, supported by dependent refinements. The inventive features center on peptide-MHC multimeric detection molecules carrying nucleic-acid labels with unique barcodes and shared primer regions, followed by incubation, isolation of labeled cells, label detection, and determination of cell identity.
Multiplexed peptide-MHC detection molecules with nucleic-acid barcode labels
A method combining a sample comprising cells with a composition comprising two or more different sets of detection molecules to form a mixture, wherein each detection molecule comprises at least two peptide-MHC complex binding molecules and at least one nucleic acid label. The nucleic acid label includes a 5′ first primer region, a barcode region unique to and specific for a set of binding molecules, a 3′ second primer region, and a random nucleotide region between the primer regions, with the random region unique for each detection molecule and the primer regions identical across detection molecules.
Multimerization domain associated with peptide-MHC and nucleic-acid label
Each detection molecule includes a multimerization domain associated with the peptide-MHC binding molecules and the nucleic acid label, optionally via one or more connector molecules. The multimerization domain is selected from peptide, protein, streptactin, a polysaccharide, dextran, avidin, and streptavidin.
Labeling, isolation, label detection, and identity determination
Incubating the mixture so that detection molecules bind to cells to form labeled cells, isolating the labeled cells, detecting one or more labels bound to the labeled cells, and determining an identity of the detected labeled cells.
Across the identified claim material, cell labeling is achieved using peptide-MHC complex multimer detection molecules carrying nucleic-acid labels with set-specific barcodes and detection-molecule-specific random nucleotide regions, followed by isolation and label-based detection to determine cell identity.
Stated Advantages
High-throughput multiplexing using DNA-barcoded peptide-MHC multimers, enabling multiplex barcode readout after washing and cell capture.
Fingerprinting antigen-responsive T cells and enabling association with TCR sequencing.
Documented Applications
Detection of antigen-responsive T cells and association with TCR sequencing using DNA-barcoded peptide-MHC multimers.
Multiplex barcode detection of labeled cells after washing and cell capture, as demonstrated by multiplex barcode readout and qPCR readout in the examples narrative.
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