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-11668705-B2
Publication Date
2023-06-06
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 describes a B-cell detection method where a sample comprising cells is combined with a composition comprising two or more different sets of detection molecules to form a mixture. Each detection molecule includes at least two identical binding molecules capable of binding to a B-cell receptor, and at least one nucleic acid label comprising a 5′ first primer region, a barcode region, a 3′ second primer region, and a random nucleotide region between the primer regions.
The barcode region is unique to and specific for the binding molecules of a set, while the random nucleotide region is unique for each detection molecule. The detection molecules in a set have identical binding molecules and identical barcodes, and the primer regions are identical for all detection molecules in the composition. The at least two identical binding molecules and the at least one nucleic acid label are associated via a multimerization domain, optionally via one or more connector molecules, where the multimerization domain is selected from peptide, protein, streptactin, a polysaccharide, dextran, avidin, and streptavidin.
After incubation, the detection molecules bind to B-cells from the sample to form labeled B-cells. The labeled B-cells are then isolated, one or more labels bound to the labeled B-cells are detected, and an identity of the detected labeled B-cells is determined based on the detected labels. The approach supports multiplexing by using two or more different sets of detection molecules in a single mixture while maintaining set-specific barcodes and molecule-specific random regions.
Claims Coverage
The independent claim covers a B-cell detection method with three inventive features: multiplex detection molecules with set-specific barcodes and molecule-specific random regions, multimerization-domain association of binding molecules and nucleic-acid labels, and incubation, isolation, label detection, and identity determination.
Multiplex detection molecule mixture with set-specific barcodes and molecule-specific random regions
A sample comprising cells is combined with a composition comprising two or more different sets of detection molecules to form a mixture, where each detection molecule comprises at least two identical binding molecules capable of binding to a B-cell receptor and at least one nucleic acid label that includes 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 that is unique for each detection molecule.
Multimerization-domain association between binding molecules and nucleic-acid labels
The at least two identical binding molecules and the at least one nucleic acid label are associated via a multimerization domain, optionally via one or more connector molecules, where the multimerization domain is selected from peptide, protein, streptactin, a polysaccharide, dextran, avidin, and streptavidin.
Incubation to form labeled B-cells followed by label detection and B-cell identity determination
The mixture is incubated so that the detection molecules bind to B-cells from the sample to form labeled B-cells, the labeled B-cells are isolated, one or more labels bound to the labeled B-cells are detected, and an identity of the detected labeled B-cells is determined based on the detected labels.
The claim coverage is directed to multiplex B-cell detection using detection molecules with set-specific barcode regions and molecule-specific random nucleotide regions, linked by a multimerization domain, followed by incubation, isolation, label detection, and identity determination.
Stated Advantages
Multiplex detection enabled by unique barcode identification for binding molecules of different sets.
Supports antigen specificity/epitope mapping and fine specificity/consensus recognition via unique barcode and random nucleotide regions [procedural detail omitted for safety].
Barcode-based nucleic-acid label readout enables detection after isolation of labeled B-cells using label detection modalities including QPCR and deep sequencing [procedural detail omitted for safety].
Documented Applications
Multiplex detection of antigen-specific T cells using DNA-tagged MHC dextramers/multimers and barcode readout via qPCR and deep sequencing [procedural detail omitted for safety].
DNA-tagged MHC dextramers/multimers showing barcode stability in blood and enrichment after cell capture/sorting, as part of a proof-of-feasibility demonstration [procedural detail omitted for safety].
Antigen-responsive T cells detection using multimeric MHC and nucleic-acid barcode readout [procedural detail omitted for safety].
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