Detecting antigen responsive cells in a sample
Inventors
Reker-Hadrup, Sine • Bakker, Arnold Hendrik • Shu, Cheng Yi Jenny • Schumacher, Antonius Nicolaas Maria
Assignees
Netherlands Cancer Institute • Stichting Sanquin Bloedvoorziening
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Publication Number
US-9151757-B2
Publication Date
2015-10-06
Expiration Date
Abstract
The present invention relates to methods for detecting antigen responsive cells in a sample using multidimensional labeled antigen presenting compounds, such as antigen-major histocompatibility complexes (NHC). Further, the present invention relates to the use of the present multidimensional labeled antigen presenting compounds, such as antigen-major histocompatibility complexes (MHC), for detecting antigen responsive cells in a sample, preferably a single sample, such as a blood sample. The present method allows high-throughput analysis of specific antigen responsive cells, such as T- and B-cells, thereby providing, for example, high-throughput methods for monitoring of diseases or conditions and the development of immunotherapeutics, vaccines, or the identification epitopes or immunogenic amino acid sequences.
Core Innovation
The invention provides a method for detecting antigen responsive cells in a sample by using antigen-containing antigen presenting compounds carrying multiple labels. The compounds include two or more predetermined antigens, and each antigen is represented by at least two different labels. After contacting the antigen-containing antigen presenting compounds with the sample, binding to antigen responsive cells is detected by emitted signals from the labels associated with the bound cells.
Presence of an antigen is detected by detecting a signal emitted by the at least two different labels bound to an antigen responsive cell through the antigen presenting compounds loaded with the at least one of the antigens. The disclosed framework supports parallel detection of many antigen specificities and is described as reducing background signals, including dual-color encoded pMHC staining.
The disclosed concept is further demonstrated for antigen recognition involving antigen-presenting structures such as major histocompatibility complex (MHC) and for detecting ligand binding to a cell or a cell-bound receptor. In the ligand-binding embodiment, the ligand is labelled with at least two different labels via a ligand-binding compound carrying the labels.
Claims Coverage
The document contains two independent methods. Across these independent claims, the inventive coverage centers on multi-label encoding of each antigen (or ligand) such that the presence of a target is detected by signals emitted from at least two different labels bound to the same antigen responsive cell (or binding event).
Multidimensional labeled antigen presenting compounds for antigen-responsive cell detection
A method for detecting antigen responsive cells in a sample by providing antigen-containing antigen presenting compounds carrying at least one label, where two or more predetermined antigens are provided and each antigen is represented by at least two different labels; contacting the antigen-containing antigen presenting compounds with the sample; detecting binding to antigen responsive cells; and detecting the presence of at least one of the antigens by detecting a signal emitted by the at least two different labels bound to an antigen responsive cell through said antigen presenting compounds loaded with the at least one of said antigens.
Multi-label ligand binding detection to a cell or cell-bound receptor
A method for detecting binding of a ligand to a cell or a cell-bound receptor comprising labelling said ligand with at least one ligand-binding compound having at least one label, wherein the ligand is labelled with at least two different labels; and detecting said cell or cell-bound receptor by detecting a signal emitted by the at least two different labels based on binding of the cell or cell-bound receptor to said ligand.
Overall, the claim coverage is grounded in the use of at least two different labels per antigen (or ligand) and the detection of binding events by emitted signals from those at least two labels bound to the same antigen responsive cell (or produced by the binding of a cell or cell-bound receptor to the labelled ligand).
Stated Advantages
Enables multiplex analysis in a single sample with higher throughput and sensitivity.
Reduces background signals, including in dual-color encoded pMHC staining.
Enables parallel detection of many antigen specificities.
Documented Applications
Multiplex identification of virus- and cancer-associated epitopes.
Epitope discovery for HLA-A3 associated melanoma antigens with functional IFN-γ confirmation.
Detecting binding of antigen-presenting compounds to antigen-responsive T/B cells in a sample such as blood or blood-derived samples (e.g., PBMC).
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