High throughput flow cytometry analysis of highly multiplex samples using sample indices with specific binding element-fluorescence conjugates

Abstract

Methods are provided for generating a plurality of samples of particles (e.g., cells, microspheres, etc.) that are fluorescently barcoded in a distinguishable manner, e.g., for use in a multiplex flow cytometry workflow. Aspects of the methods include providing a sample of a plurality of particles (e.g., cells, microspheres, etc.); and labeling different ones of the plurality of samples of particles (e.g., cells, microspheres, etc.) with a unique fluorescent barcode, where a given fluorescent barcode includes one or more fluorescently labeled specific binding elements that specifically bind to a particle marker. Compositions useful in practicing the methods of the invention are also provided.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority under 35 USC § 119(e) to the filing date of U.S. Provisional Patent Application Serial No. 63 / 332,085, filed on April 18, 2022, the disclosure of which is incorporated herein by reference in its entirety. Background Art

[0002] Characterization of analytes in biological fluids has become an important part of biological research, medical diagnostics, and assessment of a patient's overall health and well-being. Detection of analytes in biological fluids such as human blood or blood-derived products can provide results that can play a role in determining treatment regimens for patients with a variety of disease conditions.

[0003] Flow cytometry is a technique used to characterize and often sort target particles in biological matter, such as cells of a blood sample, or another type of biological or chemical sample. A flow cytometer typically includes a sample reservoir for receiving a fluid sample, such as a blood sample, and a sheath fluid reservoir containing a sheath fluid. A flow cytometer delivers particles, including cells, in a fluid sample to a flow chamber in the form of a stream of cells, while also directing a sheath fluid to the flow chamber. To characterize the components of the flow stream, the flow stream is illuminated with light. Differences in the material in the flow stream, such as morphology or the presence of fluorescent markers, can result in differences in the light observed, which allow for characterization and separation. To characterize the components in the flow stream, light must be directed onto the flow stream and collected. The light source in a flow cytometer can vary and can include one or more broad spectrum lamps, light emitting diodes, and single wavelength lasers. The light source is aligned with the flow stream, and the optical response from the illuminated particles is collected and quantified.

[0004] With high-throughput flow cytometry workflows, there is a desire to process a large number of different samples while minimizing reagent consumption and maximizing the robustness of the data obtained. Fluorescent cell barcoding (FCB) (Krutzik and Nolan, “Fluorescent cell barcoding in flow cytometry allows high-throughput drug screening and signaling profiling,” Nat. Methods (2006) 3(5):361-368) is a technology designed to meet this need. In FCB, different cell samples are encoded with unique fluorescent signatures and then combined or pooled for subsequent simultaneous antibody staining and data acquisition. In FCB, in order to encode different cell samples using unique fluorescence characteristics, fluorescent dyes are derivatized to make them reactive with suitable cell targets, such as using N-hydroxysuccinimide, so that they are reactive to amine functional groups such as lysine side chains and the N-terminus of proteins present on proteins. Cell samples are stained with reactive fluorescent dyes of different concentrations, thereby producing samples with unique dye intensity distributions. The resulting fluorescently encoded samples can be distinguished based on their fluorescence intensity in the barcoded detection channel. FCB eliminates differences between samples, which may be caused by many different sources, including chromosome volume, antibody concentration, etc. In addition, since many different samples are pooled for subsequent flow cytometric analysis, data acquisition time is minimized.

[0005] Although FCB provides many benefits in flow cytometry workflow, such as mentioned above, it is not without disadvantages. FCB usually includes only 2 or 3 colors, and relies on complex reactive chemicals associated with dyes to chemically produce covalent bonds of dyes on cell membranes or in cells. These materials are difficult to manufacture, store and transport because they are extremely unstable. Due to the inconsistency of cell sample labeling, utility is full of complexity. There are difficulties in parsing more than 20 populations based on fluorescent barcodes. Summary of the invention

[0006] The invention described herein improves the manufacturability, stability, utility and multiplicity of fluorescent cell barcoding, which is accomplished by using well-characterized specific binding elements such as antibodies, fluorophore conjugates. Embodiments of the present invention provide a way to perform ultra-high throughput flow cytometry via fluorescent barcoding (sample indexing) followed by pooled sample data acquisition. Fluorescent barcoding is achieved using specific binding elements such as antibodies coupled to fluorophores that target various ubiquitinated cell surface epitopes. The ubiquitinated properties of cell surface features can be common to all cells, species-specific, or targeted to certain target cell subsets. In the case where each sample in a particular study is assigned a unique fluorescent feature and / or the brightness of each color in the feature, the samples can be pooled and run simultaneously as a single sample in multi-parameter flow cytometry. The embodiments described herein allow 10 s or 100 s in a sample to be pooled, measured, and multiple sample data is collected in a fraction of the time required to collect data from samples that are not pooled separately. In addition, because each cell in the sample is indexed using a unique sample barcode, concerns about sample-to-sample contamination in the flow cytometer are alleviated. Based on the specific fluorescent barcode read from each cell associated with a given sample, single sample data can be identified and separated from a single data file by bioinformatics during post-collection analysis. The ability to merge 100 s in a sample into a single library before assaying and analyzing provided by the embodiments of the present invention has several additional advantages in addition to greatly improving assay and analysis throughput. Using a single tubing to assay processing and staining can eliminate differences between tubing and tubing. Compared with a mechanical automatic loader, the ability to load a single large multiple sample provides a more effortless sample data collection automation. Loading a single sample for all research data collection can also minimize human errors during data collection for several tubings or holes.

[0007] Although the above discussion focuses on cell barcoding, the present invention is not limited thereto. Instead, the present invention can also be used to barcode other types of particles (eg, microspheres) to achieve multiplexing of microsphere-based assay samples.

[0008] Methods for producing a plurality of particle (e.g., cell) samples that are fluorescently barcoded in a distinguishable manner are provided, for example, for use in a multiplexed flow cytometry workflow. Aspects of the methods include: providing a plurality of particle (e.g., cell) samples; and labeling different particle (e.g., cell) samples in the plurality with unique fluorescent barcodes, wherein a given fluorescent barcode comprises one or more fluorescently labeled specific binding elements that specifically bind to a universal surface (e.g., cell surface) marker. Compositions for practicing the methods of the invention are also provided. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] The invention is best understood from the following detailed description when read in conjunction with the accompanying figures.

[0010] Figure 1 A schematic diagram of a workflow according to an embodiment of the present invention is provided.

[0011] definition

[0012] Unless otherwise defined, technical and scientific terms used herein have the same meanings as those generally understood by those of ordinary skill in the art to which the present disclosure belongs. See, for example, Singleton et al., Dictionary of Microbiology and Molecular Biology, 2nd edition, J. Wiley & Sons (New York, NY, 1994); Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Springs Harbor Press (Cold Spring Harbor, NY, 1989). For the purposes of the present disclosure, the following terms are defined as follows.

[0013] As used herein, an antibody can be a full-length (e.g., naturally occurring or formed by normal immunoglobulin gene fragment recombination processes) immunoglobulin molecule (e.g., IgG antibody) or an immunologically active (i.e., specifically binding) portion of an immunoglobulin molecule, such as an antibody fragment. In some embodiments, the antibody is a functional antibody fragment. For example, an antibody fragment can be a part of an antibody, such as F(ab')2, Fab', Fab, Fv, sFv, etc. The antibody fragment can bind to the same antigen recognized by the full-length antibody. The antibody fragment can include a separate fragment consisting of the variable region of the antibody, such as an "Fv" fragment consisting of the variable region of the heavy chain and the light chain, and a recombinant single-chain polypeptide molecule ("scFv protein") in which the light chain variable region and the heavy chain variable region are connected by a peptide linker. Exemplary antibodies may include, but are not limited to, antibodies against cancer cells, antibodies against viruses, antibodies that bind to cell surface receptors (e.g., CD8, CD34, and CD45), and therapeutic antibodies.

[0014] As used herein, the term "association" or "associated with..." can mean that two or more substances can be identified as being co-located at a certain point in time. Association can mean that two or more substances are or have been in similar containers. Association can be an informatics association. For example, digital information about two or more substances can be stored and can be used to determine that one or more of the substances are co-located at a certain point in time. Association can also be physical association. In some embodiments, two or more associated substances are "bound", "attached" or "fixed" to each other or to a common solid or semi-solid surface. Association can refer to covalent or non-covalent means for attaching a marker to a solid or semi-solid support such as a microsphere. Association can be a covalent bond between a target and a marker. Association can include hybridization between two molecules (such as a target molecule and a marker).

[0015] As used herein, the term "complementarity" can refer to the ability of accurate pairing between two nucleotides. For example, if the nucleotide at a given position of a nucleic acid can hydrogen bond with the nucleotide of another nucleic acid, it is considered that the two nucleic acids are complementary to each other at that position. The complementarity between two single-stranded nucleic acid molecules can be "partial", with only some bonding in the nucleotides, or it can be complete when there is complete complementarity between single-stranded molecules. If the first nucleotide sequence is complementary to the second nucleotide sequence, the first nucleotide sequence can be referred to as the "complement" of the second sequence. If the first nucleotide sequence is complementary to a sequence that is reversed with the second sequence (i.e., the nucleotide order is reversed), the first nucleotide sequence can be referred to as the "reverse complement" of the second sequence. As used herein, the terms "complement", "complementarity" and "reverse complement" can be used interchangeably. It can be understood from the present disclosure that if a molecule can hybridize with another molecule, it can be the complement of the molecule being hybridized.

[0016] As used herein, the term "sample" may refer to a composition comprising a target. Suitable samples for analysis by the disclosed methods, devices and systems include cells, tissues, organs or organisms. A cell sample is a composition composed of a plurality of cells, such as a composition comprising a plurality of distinct cells, such as an aqueous composition of a single cell, wherein the number of cells may vary.

[0017] As used herein, the term "sampling device" or "device" may refer to a device that can obtain a portion of a sample and / or place the portion on a substrate. The sampling device may refer to, for example, a fluorescence activated cell sorter (FACS) instrument, a cell sorter, a biopsy needle, a biopsy device, a tissue sectioning device, a microfluidic device, a blade grid, and / or a slicer.

[0018] As used herein, the term "solid support" may refer to a discrete solid or semi-solid surface to which nucleic acids can be attached. A solid support may encompass any type of solid, porous or hollow sphere, ball, bearing, cylinder or other similar structure, which is composed of plastic, ceramic, metal or polymeric material (e.g., hydrogel) on which nucleic acids can be immobilized (e.g., covalently or non-covalently). A solid support may include discrete particles, which may be spherical (e.g., microspheres) or have a non-spherical or irregular shape, such as a cube, a cuboid, a pyramid, a cylinder, a cone, an ellipse or a disc. The shape of the microsphere may be non-spherical. Multiple solid supports spaced apart in an array may not include a substrate. A solid support may be used interchangeably with the term "microsphere". A microsphere sample is a composition consisting of a plurality of different microspheres, which can be individually distinguishable based on size and / or fluorescence characteristics (e.g., as provided by emission maximum and / or brightness), wherein different microspheres can specifically bind to different analytes (e.g., proteins), and wherein the number of microspheres can vary. DETAILED DESCRIPTION

[0019] Methods are provided for generating a plurality of particle (e.g., cell) samples that are fluorescently barcoded in a distinguishable manner, e.g., for use in a multiplexed flow cytometry workflow. Aspects of the methods include: providing a plurality of particle (e.g., cell) samples; and labeling different particle (e.g., cell) samples in the plurality with unique fluorescent barcodes, wherein a given fluorescent barcode comprises one or more fluorescently labeled specific binding elements that specifically bind to a universal particle surface (e.g., cell surface) marker. Compositions for practicing the methods of the invention are also provided.

[0020] Before describing the present invention in detail below, it should be understood that the present invention is not limited to the specific embodiments described, and of course, these embodiments can be varied. It should also be understood that the terms used herein are only for the purpose of describing specific embodiments, and are not intended to be limiting, because the scope of the present invention will be limited only by the appended claims.

[0021] Where a numerical range is provided, it is understood that each intervening value between the upper and lower limits of the range (based on one-tenth of the unit of the lower limit unless the context clearly dictates otherwise) and any other stated or intervening value within the stated range are included within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also included within the invention, subject to any explicitly excluded limits in the stated range. Where the stated range includes one or both of the limits, ranges excluding one or both of these included limits are also included within the invention.

[0022] Certain ranges appearing herein are presented by preceding the numerical value with the term "about". The term "about" is used herein to provide literal support for the exact number preceding it as well as a number that is close to or approximately the number preceding the term. In determining whether a number is close to or approximately a specifically cited number, the close or approximate unquoted number may be a number that provides a substantial equivalent of the specifically cited number in the context in which it appears.

[0023] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, representative illustrative methods and materials are now described.

[0024] All publications and patents cited in this specification are incorporated herein by reference as if each individual publication or patent was specifically and individually indicated to be incorporated by reference, and are herein incorporated by reference to disclose and describe the methods and / or materials related to the cited publication. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. In addition, the publication dates provided may be different from the actual publication dates, which may need to be independently confirmed.

[0025] It is noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. It should also be noted that the claims may be drafted to exclude any optional elements. Thus, this statement is intended to serve as antecedent basis for use of exclusive terminology, such as "only," "only," and the like, in connection with the recitation of claim elements or as a "negative" limitation.

[0026] As will be apparent to those skilled in the art upon reading this disclosure, each individual embodiment described and illustrated herein has discrete components and features that can be readily separated or combined with the features of any of the other several embodiments without departing from the scope or spirit of the invention. Any recited method may be performed in the order of events recited or in any other order that is logically possible.

[0027] Although the apparatus and method have been or will be described for the sake of grammatical fluency and functional interpretation, it should be clearly understood that unless expressly recited pursuant to 35 USC §112, the claims should not be construed as necessarily limited in any way to the "means" or "step" limitations, but should be given the full scope of meaning and equivalents provided by the claims pursuant to the judicial doctrine of equivalents, and to the extent a claim is expressly recited pursuant to 35 USC §112, it will be given full legal equivalents pursuant to 35 USC §112.

[0028] method

[0029] As outlined above, methods are provided for producing a plurality of samples of particles (e.g., cells or microspheres) that are fluorescently barcoded in a distinguishable manner. A "fluorescently barcoded sample" means a sample of particles (e.g., cells, microspheres, etc.) consisting of a plurality of particles (e.g., cells, microspheres, etc.), wherein the particles (e.g., cells, microspheres, etc.) of the sample are associated with the same fluorescent barcode. Because the particles (e.g., cells, microspheres, etc.) of the sample are associated with the same fluorescent barcode, they contain a common fluorescent barcode that can be detected during flow cytometric analysis and used to determine which sample of particles (e.g., cells, microspheres, etc.) a given particle (e.g., cell, microsphere, etc.) cell is derived from. The fluorescent barcode of a given fluorescently barcoded sample can be used to obtain a fluorescent signature (i.e., a fluorescent identifier) ​​consisting of one or more fluorescent emission signals, which are obtained based on one or more fluorophores of the fluorescent barcode associated with the particles (e.g., cells, microspheres, etc.) of the sample, such as described in more detail below. Different fluorescently barcoded samples among the plurality produced by methods of embodiments of the present invention have distinguishable fluorescent barcodes associated therewith and thus provide different fluorescent signatures, for example, when assayed by a flow cytometry protocol.

[0030] As mentioned above, the fluorescent barcodes of the present invention include one or more fluorescently labeled specific binding elements that specifically bind to particle surface markers, such as universal cell markers, markers present on the surface of microspheres, etc. When a given fluorescent barcode includes more than one fluorescently labeled specific binding element, two or more fluorescently labeled specific binding elements together constitute the fluorescent barcode. Therefore, in an embodiment of the present invention, a given fluorescent barcode can be composed of a single fluorescently labeled specific binding element or two or more fluorescently labeled specific binding elements (e.g., 2 to 20, such as 3 to 10 fluorescently labeled specific binding elements) that together constitute the fluorescent barcode. Based on the type of fluorophore and / or the signal brightness provided thereby, any given two distinguishable fluorescent barcodes can be distinguishable from each other (and produce distinguishable fluorescent features). Therefore, based on the fluorescent signal and / or its intensity of the fluorescent signal that together constitutes the fluorescent feature, any two distinguishable fluorescent features obtained from different barcodes can be distinguishable. For example, two distinguishable fluorescent barcodes can be distinguishable from each other because they are composed of a combination of different types of fluorophores, for example, one of which includes fluorophores a, b and c, and the other includes fluorophores b, c and d. Two distinguishable fluorescent barcodes can also be distinguishable from each other because they are composed of different amounts of fluorophores, for example, one of which is composed of fluorophores a, b and c present in a first amount on various specific binding elements, and the other is composed of fluorophores present in a second amount, and the second amount differs from the first amount by a value that can be detected, for example, by a difference in signal brightness. Combinations of fluorophore types and amounts can be used to provide any desired number of unique fluorescent barcodes. As outlined above, the methods of embodiments of the present invention provide a plurality of samples of particles (e.g., cells, microspheres, etc.) that are fluorescently barcoded in a distinguishable manner. Although the number of distinguishably fluorescently barcoded particle (e.g., cell, microsphere, etc.) samples produced in a given embodiment can vary, in some cases the number ranges from 5 to 5000, such as 5 to 500, including 50 to 400 particle (e.g., cell, microsphere, etc.) samples, where in some cases the number of particle (e.g., cell, microsphere, etc.) samples corresponds to the number of wells of a conventional multi-well plate, such as 6, 12, 24, 48, 96 or 384 particle (e.g., cell, microsphere, etc.) samples.

[0031] In an embodiment of the method of practice, a plurality of particle (e.g., cell, microsphere, etc.) samples to be fluorescently barcoded are provided. As discussed above, while the number of particle (e.g., cell, microsphere, etc.) samples may vary, in some cases, the number ranges from 5 to 5000, such as 5 to 500, including 50 to 400 particle (e.g., cell, microsphere, etc.) samples, wherein in some cases, the number of particle (e.g., cell, microsphere, etc.) samples corresponds to the number of wells of a conventional multi-well plate, such as 6, 12, 24, 48, 96 or 384 particle (e.g., cell, microsphere, etc.) samples. The number of particles (e.g., cell, microsphere, etc.) in a given particle (e.g., cell, microsphere, etc.) sample may vary, wherein in some cases, the number of particles (e.g., cell, microsphere, etc.) ranges from 50 to 50,000,000, such as 100 to 1,000,000 and including 500 to 100,000.

[0032] As outlined above, the particles in a given particle sample may vary, with examples of particles including cells, microspheres, and the like. The cells present in a given cell sample may be any type of cell, including prokaryotic cells and eukaryotic cells. Suitable prokaryotic cells include, but are not limited to, bacteria, such as Escherichia coli (E. coli), various Bacillus species, and extreme bacteria such as thermophiles. Suitable eukaryotic cells include, but are not limited to, fungi such as yeast and filamentous fungi, including species of Aspergillus, Trichoderma, and Neurospora; plant cells, including cells of corn, sorghum, tobacco, rapeseed, soybean, cotton, tomato, potato, alfalfa, sunflower, and the like; and animal cells, including fish, birds, and mammals. Suitable fish cells include, but are not limited to, cells from salmon, trout, tulapia, tuna, carp, flounder, halibut, swordfish, cod, and zebrafish species. Suitable avian cells include, but are not limited to, cells from chickens, ducks, quail, pheasants, and turkeys, as well as other bush foul or game birds. Suitable mammalian cells include, but are not limited to, cells from horses, cattle, buffalo, deer, sheep, rabbits, rodents (such as mice, rats, hamsters, and guinea pigs), goats, pigs, primates, marine mammals (including dolphins and whales), and cell lines (such as human cell lines of any tissue or stem cell type) and stem cells (including pluripotent and non-pluripotent, and non-human fertilized eggs).

[0033] Suitable cells also include those cell types involved in a variety of disease conditions, even when in a non-diseased state. Thus, suitable eukaryotic cell types include, but are not limited to, all types of tumor cells (e.g., melanoma, myeloid leukemia, lung cancer, breast cancer, ovarian cancer, colon cancer, kidney cancer, prostate cancer, pancreatic cancer, and testicular cancer), cardiomyocytes, dendritic cells, endothelial cells, epithelial cells, lymphocytes (T cells and B cells), mast cells, eosinophils, vascular intimal cells, macrophages, natural killer cells, erythrocytes, hepatocytes, leukocytes (including mononuclear leukocytes), stem cells (such as hematopoietic stem cells, neural stem cells, skin stem cells, lung stem cells, kidney stem cells, liver stem cells, and myocyte stem cells (for screening differentiation factors and dedifferentiation factors)), osteoclasts, chondrocytes and other connective tissue cells, keratinocytes, melanocytes, hepatocytes, kidney cells, and adipocytes. In certain embodiments, the cell is a primary disease state cell, such as a primary tumor cell. Suitable cells also include known research cells, including but not limited to Jurkat T cells, NIH3T3 cells, CHO, COS, etc. See the ATCC cell line catalog, which is expressly incorporated herein by reference.

[0034] In certain embodiments, the cells used in the present invention are taken from a subject. As used herein, "subject" refers to humans and other animals and other organisms, such as experimental animals. Therefore, the methods and compositions described herein are suitable for human and veterinary applications. In certain embodiments, the subject is a mammal, including embodiments in which the subject is a human patient suffering from (or suspected of suffering from) a disease or pathological condition.

[0035] In certain embodiments, the cells to be analyzed are enriched prior to fluorescent barcoding, e.g., as described in more detail below. For example, if the target cells are leukocytes derived from a human subject, density gradient centrifugation can be performed on whole blood from the subject to enrich peripheral blood mononuclear cells (PBMCs or leukocytes). Cells can be enriched using any convenient method known in the art, including fluorescence activated cell sorting (FACS), magnetic activated cell sorting (MACS), density gradient centrifugation, and the like. Parameters for enriching certain cells from a mixed population include, but are not limited to, physical parameters (e.g., size, shape, density, etc.), in vitro growth characteristics (e.g., in response to specific nutrients in cell culture), and molecular expression (e.g., expression of cell surface proteins or carbohydrates, reporter molecules (e.g., green fluorescent protein)).

[0036] In certain embodiments, the cell is a living cell that maintains viability during the assay. "Maintaining viability" means that a certain percentage of cells are still alive at the end of the assay, including from about 20% survival to about 100% survival. In certain other embodiments, the method of the present invention is performed in a manner that makes the cell inactive during the assay, for example, the cell can be fixed, permeabilized or otherwise maintained in a buffer or under conditions where the cell is not alive. Such parameters are generally determined by the nature of the assay performed and the reagents used.

[0037] In some cases, the cells can be treated, for example, with a stimulus. The stimulus that can be used to treat the cells can vary, ranging from culture conditions, exposure to temperature changes (e.g., heat or cold), exposure to electromagnetic radiation (e.g., light), exposure to an active agent, exposure to mechanical changes, etc. Different cell samples in the plurality can be treated with the same or different stimuli as desired. Thus, in some cases, the method includes differentially treating two or more of the plurality of cell samples, for example, wherein two or more different samples are contacted with different active agents or different concentrations of the same active agent, etc.

[0038] The particle sample used in the embodiments of the present invention can be a microsphere sample. The microsphere sample can include one or more distinguishable microspheres, wherein each of the one or more distinguishable microspheres specifically binds to different analytes, such as proteins, nucleic acids, small molecules, etc. The number of distinguishable microspheres in a given microsphere sample can be different, wherein in some cases, the number ranges from 1 to 250, such as 1 to 100, such as 1 to 50, for example 1 to 30. In some embodiments, the microsphere sample is a sample prepared by combining a biological sample (e.g., a blood-based sample (such as a plasma sample)) with one or more microspheres that specifically bind to a target analyte, wherein in some cases, the microspheres can be microspheres of multiple microsphere array assays, such as microspheres of a flow microsphere array (Cytometric Bead Array, CBA) (e.g., as commercialized by BD Biosciences), Luminex xMAP (ThermoFisher) microspheres, etc. Multiplex microsphere array assays and microsphere systems that can be used therein (which can be barcoded and processed according to embodiments of the invention) include those reviewed further in Elshal and McCoy, "Multiplex Bead Array Assays: Performance Evaluation and Comparison of Sensitivity to ELISA," Methods. 2006 Apr; 38(4): 317–323 PMID: 16481199; and Zhang et al., "Cytometry Multiplex Bead Antibody Array," Methods Mol Biol. 2021;2237:83-92; PMID 33237410.

[0039] Multiple particle (e.g., cell, microsphere, etc.) samples can be present in separate particle (e.g., cell, microsphere, etc.) composition containers. The particle (e.g., cell, microsphere, etc.) composition container can be configured to hold an aqueous particle (e.g., cell, microsphere, etc.) composition and can have any convenient volume, wherein the volume can in some cases range from 10 µl to 5 ml, such as 10 µl to 1 ml. The particle (e.g., cell, microsphere, etc.) container of interest that can be used to hold the particle (e.g., cell, microsphere, etc.) composition can vary, including tubes, vials, wells (e.g., of a multi-well plate), etc., wherein in some cases, the particle (e.g., cell, microsphere, etc.) composition is present in the wells of a standard laboratory multi-well plate (e.g., a 96-well or 384-well plate).

[0040] Any convenient scheme may be used to provide the plurality of samples. In some cases, an initial particle (e.g., cell, microsphere, etc.) sample may be divided into a plurality of particle (e.g., cell, microsphere, etc.) samples. In other cases, one or more of the plurality of samples (including all elements in the plurality of samples) may be obtained from different sources. In other embodiments, subsets of the particle (e.g., cell, microsphere, etc.) samples may be prepared from the same initial source.

[0041] After providing a plurality of particle (e.g., cell, microsphere, etc.) samples, the particle (e.g., cell, microsphere, etc.) samples in the plurality are fluorescently barcoded. In order to fluorescently barcode the particle (e.g., cell, microsphere, etc.) samples in the plurality, an embodiment of the method includes labeling each of the particle (e.g., cell, microsphere, etc.) samples in the plurality to be barcoded with a unique fluorescent barcode. A uniquely fluorescent barcoded sample is a sample in the plurality that has a fluorescent barcode that is different from any other fluorescent barcode of any other sample in the plurality. Therefore, a given fluorescent barcode of a labeled particle (e.g., cell, microsphere, etc.) sample in the plurality is distinguishable from the fluorescent barcode of any other particle (e.g., cell, microsphere, etc.) sample in the plurality.

[0042] As outlined above, a given fluorescent barcode includes one or more fluorescently labeled specific binding elements that specifically bind to a particle marker, such as a universal cell marker, a surface marker on a microsphere, etc. In some cases, a given fluorescent barcode includes a single fluorescently labeled specific binding element that specifically binds to a marker, such as a universal cell marker, a marker on the surface of a microsphere. In other cases, a given fluorescent barcode includes a plurality of distinguishably fluorescently labeled specific binding elements, each of which binds to a different marker, such as a different universal cell marker, a different marker on the surface of a microsphere, etc. In such cases, the number of different distinguishably fluorescently labeled specific binding elements that make up a given barcode may vary, ranging in some cases from 2 to 20 distinguishably fluorescently labeled specific binding elements, such as 3 to 10 distinguishably fluorescently labeled specific binding elements.

[0043] In the case where a given fluorescent barcode is comprised of two or more distinguishably fluorescently labeled specific binding elements, the distinguishably fluorescently labeled specific binding elements differ from one another by emission maxima (e.g., provided by different types of fluorophores on different specific binding elements). Thus, any two of the labeled specific binding elements may differ from one another by emission maxima (e.g., provided by different fluorophores). In such a case, a plurality of the two or more distinguishably fluorescently labeled specific binding elements together comprise the fluorescent barcode of the sample.

[0044] The specific binding elements that are fluorescently labeled in a distinguishable manner that constitute the fluorescent barcode of the present invention include specific binding elements and fluorescent labels. The specific binding element components of the fluorescently labeled specific binding elements that constitute the fluorescent barcodes used in the embodiments of the present invention may be different. The term "specific binding" refers to the direct association between two molecules due to, for example, covalent, electrostatic, hydrophobic and ionic and / or hydrogen bond interactions (including interactions such as salt bridges and water bridges). The specific binding element describes an element in a pair of molecules that has binding specificity to each other. The elements of the specific binding pair may be naturally derived or produced in whole or in part synthetically. One element in the molecular pair has a region or cavity on its surface that specifically binds to and is therefore complementary to a specific spatial and polar organization of another element in the molecular pair. Therefore, the elements of the pair have the property of specifically binding to each other. Examples of specific binding element pairs are antigen-antibody, biotin-avidin, hormone-hormone receptor, receptor-ligand, enzyme-substrate. The specific binding elements of the binding pair exhibit high affinity and binding specificity for binding to each other. Typically, the affinity between a pair of specific binding elements is characterized by K d (dissociation constant) is 10 -6 M or smaller, such as 10 -7 M or smaller, including 10 -8 M or smaller, e.g. 10 -9 M or smaller, 10 -10 M or smaller, 10 -11 M or smaller, 10 -12 M or smaller, 10 -13 M or smaller, 10 -14 M or smaller, including 10 -15M or less. "Affinity" refers to the strength of binding, and increased binding affinity is associated with a lower KD. In embodiments, affinity is determined by surface plasmon resonance (SPR), for example, as used by a Biacore system. The affinity of a molecule to another molecule is determined by measuring, for example, the binding kinetics of an interaction at 25°C. "Affinity" refers to the strength of binding, and increased binding affinity is associated with a lower KD. In embodiments, affinity is determined by surface plasmon resonance (SPR), for example, as used by a Biacore system. The affinity of a molecule to another molecule is determined by measuring, for example, the binding kinetics of an interaction at 25°C. Specific binding elements may vary, and examples of specific binding elements include, but are not limited to, polypeptides, nucleic acids, carbohydrates, lipids, peptoids, etc. In some cases, specific binding elements are proteinaceous. As used herein, the term "proteinaceous" refers to a portion consisting of amino acid residues. The portion of a proteinaceous property may be a polypeptide. In some cases, a proteinaceous specific binding element is an antibody. In certain embodiments, the proteinaceous specific binding element is an antibody fragment, for example, a binding fragment of an antibody that specifically binds to a polymer dye. As used herein, the terms "antibody" and "antibody molecule" are used interchangeably and refer to a protein consisting of one or more polypeptides that are substantially encoded by all or part of a validated immunoglobulin gene. Validated immunoglobulin genes (e.g., in humans) include κ (k), λ (l) and heavy chain loci, which together contain a large number of variable region genes, as well as constant region genes μ (u), δ (d), γ (g), σ (e) and α (a) encoding IgM, IgD, IgG, IgE and IgA isotypes, respectively. The immunoglobulin light or heavy chain variable region consists of a "framework" region (FR) interrupted by three hypervariable regions (also referred to as "complementarity determining regions" or "CDRs"). The extent of the framework regions and CDRs has been precisely defined (see "Sequences of Proteins of Immunological Interest," E. Kabat et al., US Department of Health and Human Services, (1991)). All antibody amino acid sequences discussed herein are numbered according to the Kabat system. The sequences of the framework regions of different light or heavy chains are relatively conserved within species. The framework region of an antibody, i.e., the combined framework regions of the constituent light and heavy chains, is used to locate and align the CDRs. The CDRs are primarily responsible for binding to the epitope of the antigen.The term antibody is meant to include full-length antibodies, and may refer to natural antibodies from any organism, engineered antibodies, or other purpose antibodies generated in a recombinant manner for experiments, treatments, or as further defined below. Target antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv, scFv, or other antigen-binding subsequences of antibodies, produced by modifying complete antibodies or synthesized de novo using recombinant DNA technology. Antibodies may be monoclonal or polyclonal, and may have other specific activities (e.g., antagonists, agonists, neutralizing antibodies, inhibitory antibodies, or stimulating antibodies) to cells. It should be understood that antibodies may have additional conservative amino acid substitutions that have substantially no effect on antigen binding or other antibody functions. In certain embodiments, the specific binding element is a Fab fragment, a F(ab')2 fragment, a scFv, a diabody, or a triabody. In certain embodiments, the specific binding element is an antibody. In some cases, the specific binding element is a mouse antibody or its binding fragment. In some cases, the specific binding element is a recombinant antibody or its binding fragment.

[0045] The specific binding elements constituting the fluorescent barcode can specifically bind to any convenient binding element pair, such as a particle marker, for example, a protein on the surface of a particle (such as a cell or microsphere). In the case where the particle sample is a cell sample, in some cases, the specific binding elements constituting the fluorescent barcode specifically bind to a universal cell marker. In some cases, the universal marker is a cell surface marker, wherein the target cell surface marker includes but is not limited to a ubiquitinated cell surface marker, that is, a cell surface marker on all cells of a given cell sample that is at least predicted to be processed in a given workflow according to the present invention. Examples of ubiquitinated cell surface markers to which the specific binding element can specifically bind include but are not limited to: CD44, CD45, CD47, β-2 microglobulin, etc. In the case where a given barcode is composed of two or more fluorescently labeled specific binding elements, each of the two or more fluorescently labeled specific binding elements can be specifically bound to a different universal marker as needed. Thus, if a given fluorescent barcode is comprised of four distinguishably labeled specific binding elements, the distinguishably labeled specific binding elements can bind to four different universal markers, e.g., one can bind to CD44, one can bind to CD45, one can bind to CD47, and one can bind to beta-2 microglobulin.

[0046] In the case where the particle sample is a microsphere sample, in some cases, the specific binding element constituting the fluorescent barcode specifically binds to a marker present on the surface of the microsphere. In some cases, one or more markers present on the surface of a given microsphere are markers different from the analyte-specific binding element of the microsphere, where examples of such markers include proteins or fragments thereof that are different from the specific binding element (e.g., antibody) of the microsphere and do not interfere with the desired function of the specific binding element to specifically bind to its target analyte. In some cases, the marker may be a ubiquitous cell surface marker, such as CD44, CD45, CD47, beta-2 microglobulin, etc., as described above. In the case where a given barcode is composed of two or more fluorescently labeled specific binding elements, each of the two or more fluorescently labeled specific binding elements may be specifically bound to a different microsphere marker as desired. Thus, if a given fluorescent barcode is comprised of four distinguishably labeled specific binding elements, the distinguishably labeled specific binding elements can bind to four different microsphere markers, e.g., one can bind to CD44, one can bind to CD45, one can bind to CD47, and one can bind to beta-2 microglobulin.

[0047] In addition to the specific binding element components, the specific binding elements of the fluorescent labels constituting the fluorescent barcodes also include fluorescent labels. As desired, a given fluorescent label may include one or more fluorophores. Thus, a given specific binding element may be labeled with a fluorescent label including a single type of fluorophore. Alternatively, a given specific binding element may be labeled with a fluorescent label including two or more different types of fluorophores, for example, as found in tandem dyes, for example, where the first fluorophore acts as a donor for the second fluorophore. Examples of fluorophores include, but are not limited to: acridine and derivatives such as acridine, acridine orange, acridine yellow, acridine red, and acridine isothiocyanate; 5-(2'-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS); 4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5-disulfonate (Lucifer yellow (Lucifer Yellow (VS); N-(4-amino-1-naphthyl) maleimide; o-aminobenzamide; Brilliant Yellow; Coumarin and derivatives such as coumarin, 7-amino-4-methylcoumarin (AMC, coumarin 120), 7-amino-4-trifluoromethylcoumarin (coumarin 151); cyanines and derivatives such as tetrachlorotetrabromofluorescein, Cy3, Cy5, Cy5.5 and Cy7; 4',6-diamidino-2-phenylindole indole (DAPI); 5',5''-dibromopyrogallol-sulfonphthalein (bromopyrogallol red); 7-diethylamino-3-(4'-isothiocyanatophenyl)-4-methylcoumarin; diethylaminocoumarin; diethylenetriamine pentaacetate; 4,4'-diisothiocyanatodihydro-stilbene-2,2'-disulfonic acid; 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid; 5-[dimethylamino]naphthalene-1-sulfonyl chloride (DNS, dansyl chloride); 4-(4'-dimethylamino)-3-(4'-isothiocyanatophenyl)-4-methylcoumarin; diethylaminocoumarin; diethylenetriamine pentaacetate; 4,4'-diisothiocyanatodihydro-stilbene-2,2'-disulfonic acid; 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid; 5-[dimethylamino]naphthalene-1-sulfonyl chloride (DNS, dansyl chloride); 4-Dimethylaminophenylazo)benzoic acid (DABCYL); 4-dimethylaminophenylazophenyl-4'-isothiocyanate (DABITC); Eosin and derivatives such as Eosin and Eosin isothiocyanate; Erythrosine and derivatives such as Erythrosine B and Erythrosine isothiocyanate; Ethidium bromide; Fluorescein and derivatives such as 5-carboxyfluorescein (FAM), 5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF), 2'7'-dimethoxy-4'5'-dichloro-6-carboxyfluorescein (JOE), fluorescein isothiocyanate (FITC), fluoresceinchlorotriazinyl, naphthofluorescein and QFITC ​​(XRITC); Fluorescein; IR144; IR1446; LissamineTM; Lissamine rhodamine, fluorescein yellow; Malachite Green isothiocyanate isothiocyanate); 4-methylumbelliferone; o-cresolphthalein; nitrotyrosine;pararosaniline; Nile Red; Oregon Green; phenol red; B-phycoerythrin; o-phthalaldehyde; pyrene and derivatives such as pyrene, pyrenebutyrate and succinimidyl 1-butyryl; CibacronTM Brilliant Red 4 3B-A); rhodamine and derivatives such as 6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), 4,7-dichlororhodamine limonium, rhodamine B sulfonyl chloride, rhodamine (Rhod), rhodamine B, rhodamine 123, rhodamine X isothiocyanate, sulforhodamine B, sulforhodamine 101, the sulfonyl chloride derivative of sulforhodamine 101 (Texas Red), N,N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA), tetramethylrhodamine and tetramethylrhodamine isothiocyanate (TRITC); riboflavin; rhodamine and terbium chelate derivatives; xanthenes; Alexa-Fluor dyes (e.g., Alexa Fluor 350, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, Alexa Fluor 750), Pacific Blue, Pacific Orange, Cascade Blue, Cascade Yellow; quantum dot dyes (Quantum Dot Corporation); Dylight dyes from Pierce Company (Rockford, IL), including Dylight 800, Dylight 680, Dylight 649, Dylight 633, Dylight 549, Dylight 488, Dylight 405; or a combination thereof. Other fluorophores or combinations thereof known to those skilled in the art, such as those available from Molecular Probes (Eugene, Oreg.) and Exciton (Dayton, Ohio), may also be used. ;

[0048] In some cases, the specific binding element is labeled with one or more polymer dyes (e.g., fluorescent polymer dyes). The fluorescent polymer dyes that find use in the methods and systems of the present subject matter are varied. In some cases of the method, the polymer dye includes a conjugated polymer. The characteristic of a conjugated polymer (CP) is a delocalized electronic structure, which includes a main chain of alternating unsaturated bonds (e.g., double bonds and / or triple bonds) and saturated bonds (e.g., single bonds), wherein π-electrons can move from one bond to another. Therefore, the conjugated main chain can give the polymer dye an extended linear structure with limited bond angles between the repeating units of the polymer. For example, although proteins and nucleic acids are also polymerized, they do not form extended rod structures in some cases, but fold into more advanced three-dimensional shapes. In addition, CP can form a "rigid rod" polymer main chain and experience limited twisting (e.g., torsion) angles between monomer repeating units along the polymer main chain. In some cases, the polymer dye includes a CP with a rigid rod structure. The structural features of the polymer dye can have an effect on the fluorescent properties of the molecule.

[0049] Any convenient polymer dye can be used in the apparatus and methods of the present subject matter. In some cases, the polymer dye is a multi-chromophore having a structure capable of collecting light to amplify the fluorescence output of a fluorophore. In some cases, the polymer dye is capable of collecting light and efficiently converting it into emitted light of a longer wavelength. In some cases, the polymer dye has a light-collecting multi-chromophore system that can effectively transfer energy to a nearby luminescent substance (e.g., a "signaling chromophore"). Mechanisms for energy transfer include, for example, resonance energy transfer (e.g., Foster (or fluorescence) resonance energy transfer, FRET), quantum charge exchange (Dexter energy transfer), etc. In some cases, these energy transfer mechanisms are relatively short-range; that is, the close proximity of the light-collecting multi-chromophore system to the signaling chromophore provides efficient energy transfer. Under conditions of efficient energy transfer, emission from the signaling chromophore occurs amplified when the number of individual chromophores in the light-harvesting multichromophore system is large; that is, when the incident light ("excitation light") is at a wavelength absorbed by the light-harvesting multichromophore system, emission from the signaling chromophore is more intense than when the signaling chromophore is directly excited by the pump light.

[0050] The multichromophore may be a conjugated polymer. Conjugated polymers (CPs) are characterized by a delocalized electronic structure and can be used as highly responsive optical reporters for chemical and biological targets. Because the effective conjugation length is substantially shorter than the length of the polymer chain, the backbone contains a large number of conjugated segments in close proximity. Therefore, conjugated polymers are efficient for light harvesting and are capable of optical amplification via Förster energy transfer.

[0051] Polymeric dyes of interest include, but are not limited to, those described in U.S. Pat. Nos. 7,270,956, 7,629,448, 8,158,444, 8,227,187, 8,455,613, 8,575,303, 8,802,450, 8,969,509, 9,139,869, 9,371,559, 9,547,008, 10,094,838, 10,302,648, 10,458,989, 10,641,775, and 10,962,546, the disclosures of which are incorporated herein by reference in their entireties; and Gaylord et al., J. Am. Chem. Soc., 2001, 123 (26), pp. 6417-6418; Feng et al., Chem. Soc. Rev., 2010, 39, 2411-2419; and Traina et al., Journal of the American Chemical Society, 2011, 133 (32), pp. 12600-12607, the disclosures of which are incorporated herein by reference in their entirety. Specific polymer dyes that can be used include, but are not limited to, BD Horizon Brilliant™ dyes, such as BD Horizon Brilliant™ violet dyes (e.g., BV421, BV510, BV605, BV650, BV711, BV786); BD Horizon Brilliant™ ultraviolet dyes (e.g., BUV395, BUV496, BUV737, BUV805); and BD Horizon Brilliant™ blue dyes (e.g., BB515) (Biosciences, San Jose, CA). Any fluorescent dye known to those skilled in the art (including, but not limited to, those described above) or fluorescent dyes not yet discovered can be used in the methods of the present subject matter.

[0052] In some cases, each of the multiple distinguishably fluorescently labeled specific binding elements that make up a given barcode is excitable by a common light source, such as a common laser. In such cases, each of the multiple distinguishably fluorescently labeled specific binding elements that make up a given barcode can have a common excitation maximum, but differ from one another in terms of emission maxima.

[0053] As reviewed above, any given two distinguishable fluorescent barcodes may be distinguishable from each other (and produce distinguishable fluorescent signatures) based on the type of fluorophores that make up the barcodes and / or the signal brightness provided thereby. Thus, any two distinguishable fluorescent signatures obtained from different barcodes may be distinguishable based on the fluorescent signals and / or their intensities that together make up the fluorescent signature. For example, two distinguishable fluorescent barcodes may be distinguishable from each other because they are composed of a combination of different types of fluorophores, for example, one of which includes fluorophores a, b, and c, while the other includes fluorophores b, c, and d. Two distinguishable fluorescent barcodes may also be distinguishable from each other because they are composed of different amounts of fluorophores, for example, one of which is composed of fluorophores a, b, and c present on various specific binding elements in a first amount, while the other is composed of fluorophores present in a second amount, the second amount differing from the first amount by a value that can be detected, for example, by a difference in signal brightness. Different brightnesses can be easily provided by associating different amounts of fluorophores with specific binding elements. Combinations of fluorophore types and amounts can be used to provide any desired number of unique fluorescent barcodes.As outlined above, methods of embodiments of the present invention provide a plurality of samples of particles (eg, cells, microspheres, etc.) that are fluorescently barcoded in a distinguishable manner.

[0054] Fluorescent barcodes can be associated with a given sample of particles (e.g., cells, microspheres, etc.) so that the sample of particles (e.g., cells, microspheres, etc.) can be labeled with the fluorescent barcode using any convenient protocol. For example, a sample of particles (e.g., cells, microspheres, etc.) can be contacted with a barcode labeling composition including specific binding elements of different fluorescent labels, which together constitute the barcode of the composition. In other cases, a sample of particles (e.g., cells, microspheres, etc.) can be contacted sequentially with different fluorescently labeled specific binding elements that constitute the barcode of the sample. For example, in order to label multiple samples of particles (e.g., cells, microspheres, etc.), all samples that include a specific binding element of a given specific label in their expected fluorescent barcode can be first contacted with the specific binding element of the label. Then, all samples including the specific binding element of the second given specific label in its expected fluorescent barcode can be contacted with the specific binding element of the label, wherein one or more samples contacted with the specific binding element of the second label can be samples that are also contacted with the specific binding element of the first label, depending on the fluorescent barcode used for those samples. For example, in those samples with fluorescent barcodes including both the first binding element and the second binding element, those samples will contact both the specific binding element of the first label and the specific binding element of the second label. Contact can be achieved under any suitable conditions that provide the specific binding element of the fluorescent label and its corresponding universal marker specific binding. The specific binding element of the label can be contacted with the particles (e.g., cells, microspheres, etc.) of the cell sample, for example, by introducing the specific binding element of the label into the container of the particle (e.g., cells, microspheres, etc.) sample, such as by manual or automated fluid distribution. In some cases, different combinations of different fluorescently labeled binding elements can be distributed to different particles (e.g., cells, microspheres, etc.) samples using an automated liquid distribution system to provide samples that are fluorescently barcoded in a distinguishable manner.

[0055] After generating a plurality of samples of particles (e.g., cells, microspheres, etc.) that are fluorescently barcoded in a distinguishable manner, the resulting plurality can be pooled for subsequent processing as desired. Thus, disparate fluorescently barcoded samples can be combined into a single composition. A single composition can be prepared from different fluorescently barcoded particle (e.g., cell, microsphere, etc.) samples using any convenient scheme, such as by transferring the contents of each container (e.g., a well of a well plate) to a single container (e.g., a tube or vial) of a suitable volume. The resulting pooled composition can then be further processed as desired.

[0056] In certain embodiments of the invention, for example, where the particle sample is a cell sample, the method may include detecting one or more phenotypic characteristics of the cell, which are separate from the fluorescent barcode. Detectable phenotypic characteristics include, but are not limited to, the presence of an analyte (e.g., a cell surface or internal marker), physical characteristics (e.g., size, shape, granularity, etc.), cell number (or frequency), etc. In fact, any detectable target feature can be determined as a detectable target phenotypic feature. In certain embodiments, the methods of the invention are used to qualitatively or quantitatively detect the presence of an analyte (e.g., a marker) associated with (e.g., in, on, or attached to) a cell being assayed.

[0057] In some of these embodiments, the method includes contacting the merged or pooled cell sample with a detectable analyte-specific binding agent. "Analyte-specific binding agent" and its grammatical equivalents mean any molecule (e.g., nucleic acid, small organic molecule and protein, nucleic acid binding dye (e.g., ethidium bromide)) that can surpass any other molecule to associate with a specific analyte (or a specific isotype of analyte) in a cell. The target analyte includes any molecule associated with the cell analyzed in the method of this theme or present in the cell. Therefore, the target analyte includes but is not limited to proteins, carbohydrates, organelles, nucleic acids, infectious particles (e.g., viruses, bacteria, parasites), metabolites, etc. In certain embodiments, the analyte-specific binding agent is a protein. In some of these embodiments, the analyte-specific binding agent is an antibody or its binding fragment, for example, as described above. Therefore, the methods and compositions of the present invention can be used to detect any specific element isotype that is detectable in a sample and can be distinguished from other isotypes of activatable elements present in a sample in terms of antigenicity.

[0058] In certain embodiments, multiple detectable analyte-specific binding agents are used in the methods according to the present invention. "Multiple analyte-specific binding agents" means the use of at least 2 or more analyte-specific binding agents, including 3 or more, 4 or more, 5 or more, etc. In certain embodiments, each of the different analyte-specific binding agents is labeled (again, directly or indirectly) with a distinct detectable label (e.g., a fluorophore with an emission wavelength that can be detected in different channels on a flow cytometer with or without compensation). Multiple analyte-specific binding agents can bind to the same analyte in or on a cell (e.g., two antibodies that bind to different epitopes on the same protein), bind to different analytes in or on a cell, or bind in any combination (e.g., two agents bind to the same analyte and a third agent binds to a different analyte). The upper limit of the number of analyte-specific binding agents will depend largely on the parameters of the assay and the detection capabilities of the detection system employed.

[0059] After merging or pooling and any desired subsequent processing, such as by contacting with a labeling reagent for a phenotypic marker (e.g., as described above), the method may include subjecting the assay composition to an assay by flow cytometry. "Assaying by flow cytometry" means subjecting a composition (e.g., an assay composition as described above) to a flow cytometric assay. A flow cytometric assay may include characterizing a sample (e.g., a sample comprising an assay composition) using a flow cytometer system. A flow cytometric assay may include introducing the assay composition into a flow cytometer. A flow cytometer typically includes a sample reservoir for receiving a fluid sample (such as a sample comprising an assay composition) and a sheath fluid reservoir containing a sheath fluid. The flow cytometer transports particles (including cells, such as cells from an assay composition) in the fluid sample as a stream of cells to a flow chamber, while also directing a sheath fluid to the flow chamber. In order to characterize the components of the flow stream, the flow stream is illuminated with light. Differences in the material in the flow stream, such as the presence of morphology or fluorescent markers, can result in differences in the light observed, and these differences allow for characterization and separation. For example, particles in a fluid suspension (such as microspheres or single cells that bind molecules, analytes) are passed through a detection area where the particles are exposed to excitation light, typically from one or more lasers, and the light scattering and fluorescence properties of the particles are measured. Particles or their components are typically labeled with fluorescent dyes for ease of detection. By labeling different particles or components with spectrally different fluorescent dyes, multiple different particles or components can be detected simultaneously. In some implementations, the analyzer includes multiple detectors, one for each of the scattering parameters to be measured, and one or more for each of the different dyes to be detected. For example, some embodiments include spectral configurations in which each dye uses more than one sensor or detector. The data obtained includes signals and fluorescence emissions measured for each light scattering detector. In certain embodiments, flow cytometry can detect signals indicating the presence of a labeled second antibody in a sample. In the case where a signal is detected, the sample may include antibodies to antigenic determinants of a coronavirus antigen.

[0060] As outlined above, the sample (e.g., in the flow stream of a flow cytometer) can be irradiated with light from a light source. In some embodiments, the light source is a broadband light source, emitting light with a wide wavelength range, such as, for example, a span of 50 nm or greater, such as 100 nm or greater, such as 150 nm or greater, such as 200 nm or greater, such as 250 nm or greater, such as 300 nm or greater, such as 350 nm or greater, such as 400 nm or greater and including a span of 500 nm or greater. For example, a suitable broadband light source emits light with a wavelength of 200 nm to 1500 nm. Another example of a suitable broadband light source includes a light source that emits light with a wavelength of 400 nm to 1000 nm. In the case where the method includes irradiating with a broadband light source, the target broadband light source scheme may include, but is not limited to, a halogen lamp, a deuterium arc lamp, a xenon arc lamp, a stable fiber-coupled broadband light source, a broadband LED with a continuous spectrum, a superluminescent diode, a semiconductor light emitting diode, a wide spectrum LED white light source, a multi-LED integrated white light source, and other broadband light sources or any combination thereof.

[0061] In other embodiments, the method includes irradiating with a narrowband light source emitting a specific wavelength or narrow wavelength range, such as, for example, irradiating with a light source emitting a narrow wavelength range such as 50 nm or less, such as 40 nm or less, such as 30 nm or less, such as 25 nm or less, such as 20 nm or less, such as 15 nm or less, such as 10 nm or less, such as 5 nm or less, such as 2 nm or less and including a light source emitting light of a specific wavelength (i.e., monochromatic light). Where the method includes irradiating with a narrowband light source, the target narrowband light source scheme may include, but is not limited to, a narrow wavelength LED, a laser diode or broadband light source coupled to one or more optical bandpass filters, a diffraction grating, a monochromator, or any combination thereof.

[0062] In certain embodiments, the method includes irradiating the sample with one or more lasers. As discussed above, the type and number of lasers will vary depending on the sample and the desired light collected, and may be a gas laser such as a helium-neon laser, an argon laser, a krypton laser, a xenon laser, a nitrogen laser, a CO2 laser, a CO laser, an argon fluorine (ArF) excimer laser, a krypton fluorine (KrF) excimer laser, a xenon chlorine (XeCl) excimer laser, or a xenon fluorine (XeF) excimer laser, or a combination thereof. In other cases, the method includes irradiating the flow stream with a dye laser such as a stilbene, coumarin, or rhodamine laser. In other cases, the method includes irradiating the flow stream with a metal vapor laser, such as a helium-cadmium (HeCd) laser, a helium-mercury (HeHg) laser, a helium-selenium (HeSe) laser, a helium-silver (HeAg) laser, a strontium laser, a neon-copper (NeCu) laser, a copper laser, or a gold laser, and combinations thereof. In yet other instances, the method includes irradiating the flow stream with a solid-state laser, such as a ruby ​​laser, a Nd:YAG laser, a NdCrYAG laser, an Er:YAG laser, a Nd:YLF laser, a Nd:YVO4 laser, a Nd:YCa4O(BO3)3 laser, a Nd:YCOB laser, a titanium sapphire laser, a thulium YAG laser, an ytterbium YAG laser, a Yb2O3 laser or a cerium-doped laser and combinations thereof.

[0063] The sample can be irradiated with one or more of the above-mentioned light sources, such as 2 or more light sources, such as 3 or more light sources, such as 4 or more light sources, such as 5 or more light sources, and including 10 or more light sources. The light source can include any combination of light source types. For example, in some embodiments, the method includes irradiating the sample in the flow stream with a laser array, such as an array with one or more gas lasers, one or more dye lasers, and one or more solid-state lasers. Where necessary, at least one laser is used for excitation of the fluorescent barcode, and other lasers are used for other fluorophores associated with the cell.

[0064] In some cases, the flow stream is illuminated with multiple beams of frequency-shifted light, and cells in the flow stream are imaged using fluorescence imaging with radiofrequency tag excitation (FIRE) to generate frequency-encoded images, such as those described in Diebold et al., Nature Photonics, Vol. 7(10); 806-810 (2013) and U.S. Pat. Nos. 9,423,353, 9,784,661, and 10,006,852, and U.S. Patent Publication Nos. 2017 / 0133857 and 2017 / 0350803, the disclosures of which are incorporated herein by reference.

[0065] Aspects of the inventive method include collecting fluorescence using a fluorescence detector. In some cases, the fluorescence detector can be configured to detect the fluorescence emission of fluorescent molecules, for example, a labeled specific binding element (such as a labeled antibody specifically bound to a target marker) associated with the particles in the flow cell. In certain embodiments, the method includes detecting the fluorescence of the sample with one or more fluorescence detectors, such as 2 or more, such as 3 or more, such as 4 or more, such as 5 or more, such as 6 or more, such as 7 or more, such as 8 or more, such as 9 or more, such as 10 or more, such as 15 or more and including 25 or more fluorescence detectors. In an embodiment, each of the fluorescence detectors is configured to generate a fluorescence data signal. The fluorescence of the sample can be detected independently by each fluorescence detector in one or more of the wavelength range of 200 nm-1200 nm. In some cases, the method is included in the fluorescence of detecting sample in the wavelength range of 200 nm to 1200 nm, such as 300 nm to 1100 nm, such as 400 nm to 1000 nm, such as 500 nm to 900 nm and including 600 nm to 800 nm. In other cases, the method includes utilizing each fluorescence detector to detect fluorescence under one or more specific wavelengths. For example, fluorescence can be detected under one or more of 450 nm, 518 nm, 519 nm, 561 nm, 578 nm, 605 nm, 607 nm, 625 nm, 650 nm, 660 nm, 667 nm, 670 nm, 668 nm, 695 nm, 710 nm, 723 nm, 780 nm, 785 nm, 647 nm, 617 nm and any combination thereof, depending on the quantity of different fluorescence detectors in the light detection system of this theme. In certain embodiments, the method includes detecting the wavelength of light corresponding to the fluorescence peak wavelength of some fluorophores present in the sample. In an embodiment, fluorescence flow cytometry data is received from one or more fluorescence detectors (e.g., one or more detection channels), such as 2 or more, such as 3 or more, such as 4 or more, such as 5 or more, such as 6 or more and including 8 or more fluorescence detectors (e.g., 8 or more detection channels).

[0066] Light from the sample may be measured at one or more wavelengths, such as at 5 or more different wavelengths, such as at 10 or more different wavelengths, such as at 25 or more different wavelengths, such as at 50 or more different wavelengths, such as at 100 or more different wavelengths, such as at 200 or more different wavelengths, such as at 300 or more different wavelengths, and including measuring the collected light at 400 or more different wavelengths.

[0067] In certain embodiments, the method includes spectrally resolving the light of each fluorophore of a fluorophore-biomolecule agent pair in a sample. In some embodiments, the overlap between each different fluorophore is determined, and the contribution of each fluorophore to the overlapping fluorescence is calculated. In some embodiments, spectrally resolving the light of each fluorophore includes calculating a spectral unmixing matrix for the fluorescence spectrum of each of the multiple fluorophores with overlapping fluorescence in the sample detected by the light detection system. In some cases, spectrally resolving the light of each fluorophore and calculating a spectral unmixing matrix for each fluorophore can be used to estimate the abundance of each fluorophore, such as, for example, for resolving the abundance of target cells in a sample.

[0068] In certain embodiments, the method includes spectrally parsing the light detected by the plurality of photodetectors, such as described in International Patent Application No. PCT / US2019 / 068395, filed on December 23, 2019; U.S. Provisional Patent Application No. 62 / 971,840, filed on February 7, 2020, and U.S. Provisional Patent Application No. 63 / 010,890, filed on April 16, 2020, the disclosures of which are incorporated herein by reference in their entireties. For example, spectrally parsing the light detected by the plurality of photodetectors of the second set of photodetectors may include solving a spectral unmixing matrix using one or more of: 1) a weighted least squares algorithm; 2) a Sherman-Morrison iterative inverse updater; 3) an LU matrix decomposition, such as where the matrix is ​​decomposed into the product of a lower triangular (L) matrix and an upper triangular (U) matrix; 4) a modified Cholesky decomposition; 5) by QR factorization; and 6) computing a weighted least squares algorithm by singular value decomposition.

[0069] In certain embodiments, the method further includes characterizing the spillover spread of light detected by the plurality of photodetectors, such as described in US Patent Application No. 17 / 237,504, the disclosure of which is incorporated herein by reference.

[0070] In some cases, the abundance of fluorophores associated (e.g., chemically associated (i.e., covalently, ionically) or physically associated) with a target particle is calculated based on the spectrally resolved light of each fluorophore associated with the particle. For example, in one instance, the relative abundance of each fluorophore associated with a target particle is calculated based on the spectrally resolved light of each fluorophore. In another instance, the absolute abundance of each fluorophore associated with a target particle is calculated based on the spectrally resolved light of each fluorophore. In certain embodiments, particles can be identified or classified based on the relative abundance of each fluorophore determined to be associated with the particle. In these embodiments, particles can be identified or classified by any convenient scheme, such as by comparing the relative or absolute abundance of each fluorophore associated with the particle to a control sample of particles with known identities; or by performing a spectroscopic or other measurement analysis on a population of particles (e.g., cells) having the calculated relative or absolute abundance of associated fluorophores.

[0071] In certain embodiments, the method includes sorting one or more particles (e.g., cells) of a sample that is identified based on the estimated abundance of a fluorophore associated with the particle. The term "sorting" is used herein in its conventional sense to refer to separating components of a sample (e.g., droplets containing cells, droplets containing non-cellular particles such as biomacromolecules), and in some cases, delivering the separated components to one or more sample collection containers. For example, the method may include sorting 2 or more components of a sample, such as 3 or more components, such as 4 or more components, such as 5 or more components, such as 10 or more components, such as 15 or more components, and including sorting 25 or more components of a sample. When sorting particles identified based on the abundance of fluorophores associated with particles, the method includes data acquisition, analysis, and recording (such as using a computer), wherein multiple data channels record data from each detector for obtaining overlapping spectra of multiple fluorophore-biomolecule reagent pairs associated with particles. In these embodiments, the analysis includes spectrally resolving light from multiple fluorophores of fluorophore-biomolecule agent pairs with overlapping spectra associated with the particles (e.g., by computing a spectral unmixing matrix) and identifying the particles based on the estimated abundance of each fluorophore associated with the particles. This analysis can be transmitted to a sorting system configured to generate a set of digital parameters based on the classification of the particles. In some embodiments, methods for sorting components of a sample include sorting particles (e.g., cells in a biological sample), such as described in U.S. Pat. Nos. 3,960,449, 4,347,935, 4,667,830, 5,245,318, 5,464,581, 5,483,469, 5,602,039, 5,643,796, 5,700,692, 6,372,506, and 6,809,804, the disclosures of which are incorporated herein by reference. In some embodiments, the method includes sorting the components of the sample using a particle sorting module, such as those described in U.S. Patent Nos. 9,551,643 and 10,324,019, U.S. Patent Publication No. 2017 / 0299493, and International Patent Publication No. WO / 2017 / 040151, the disclosures of which are incorporated herein by reference. In certain embodiments, cells of the sample are sorted using a sorting decision module having a plurality of sorting decision units, such as those described in U.S. Patent Application No. 16 / 725,756 filed on December 23, 2019, the disclosures of which are incorporated herein by reference.

[0072] Flow cytometry assay procedures are well known in the art. See, e.g., Ormerod (ed.), Flow Cytometry: A Practical Approach, Oxford Univ. Press (1997); Jaroszeski et al. (eds.), Flow Cytometry Protocols, Methods in Molecular Biology, Vol. 91, Humana Press (1997); Practical Flow Cytometry, 3rd ed., Wiley-Liss (1995); Virgo et al. (2012) Ann Clin Biochem. Jan;49(pt 1):17-28; Linden et al., Semin Throm Hemost. Oct 2004;30(5):502-11; Alison et al., J Pathol. Pathol, Dec 2010; 222(4):335-344; and Herbig et al., (2007) Crit Rev Ther Drug Carrier Syst. 24(3):203-255; the disclosures of which are incorporated herein by reference. In certain aspects, measuring the composition by flow cytometry involves using a flow cytometer capable of simultaneously exciting and detecting multiple fluorophores, such as the FACSCanto by BD Biosciences. TM Flow cytometer, used essentially according to the manufacturer's instructions. The methods of the present disclosure may involve image cytometry, such as described in Holden et al. (2005) Nature Methods 2:773 and Valet et al. 2004 Cytometry 59:167-171, the disclosures of which are incorporated herein by reference.

[0073] As discussed above, the methods include cytometric analysis, which may include sorting. Target cells identified in the sample may be sorted and subsequently analyzed by any convenient analytical technique. Subsequent targeted analysis techniques include, but are not limited to, sequencing; assays performed by CellSearch, as described in the Food and Drug Administration (2004) final rule, Federal Register 69: 26036-26038; assays performed by CTC chips, as described in Nagrath et al. (2007) Nature 450: 1235-1239; assays performed by MagSweeper, as described in Talasaz et al. (2009). Proc Natl Acad Sci U SA 106: 3970-3975; and assays performed by nanostructured substrates, as described in Wang S et al. (2011) Angew Chem Int Ed Engl 50: 3084-3088; the disclosures of which are incorporated herein by reference. If desired, the sorting scheme may include distinguishing live from dead cells, wherein any convenient staining scheme for identifying such cells may be incorporated into the method.

[0074] Analysis of data collected from the barcoded samples of the present invention may include deconvolution. "Deconvolution" means a process, whether performed manually or in an automated system, by which the fluorescent barcode of each detected cell is used to determine which original sample it originated from. Because the type and amount of each fluorescent barcode of each starting sample is known, the fluorescent barcode characteristics of each detected cell (i.e., its barcode characteristics) can be used to positively identify its source sample. Deconvolution of multiple data can be performed using any convenient method, including the use of computer-based analysis software known in the art (e.g., FlowJo software package available from Becton Dickinson Biosciences). Deconvolution can be performed manually (e.g., manually reviewing the data and classifying the cells), automatically (e.g., by using data analysis software specifically configured to deconvolute barcode data), or a combination thereof. In certain embodiments, a computer program can be used to create a separate data file for each of the deconvoluted barcoded samples corresponding to the original starting sample to facilitate data manipulation and / or interpretation.

[0075] Analysis of data collected from the barcoded multiple samples of the present invention involves analysis of detectable target features of the cells (e.g., as described in more detail above). Analysis of the detectable features can be performed at any convenient step in the data analysis phase, including before, during, or after deconvolution. In fact, because the collected data can be analyzed and re-analyzed at will, there is no intention to limit the order of deconvolution and analysis of the detectable features.

[0076] Reagent test kit

[0077] Aspects of the present invention further include kits and compositions for finding use in various embodiments of practicing the methods of the present invention. The kit of the present invention may include a variety of specific binding elements that are fluorescently labeled in a distinguishable manner that specifically bind to universal cell surface markers, such as described above. Depending on the needs, different specific binding elements that are fluorescently labeled in a distinguishable manner may be separate or present as a pre-combined labeling composition. The kit may further include one or more additional components for finding use in embodiments of the practice method. For example, the kit may include microspheres for multiple microsphere array determinations, such as described above, wherein such microspheres may include specific binding elements for target analytes and one or more surface markers, such as described above. The kit may also include components that use various workflows, such as multiwell plates, liquid containers (e.g., tubing, etc.). In addition, the kit may include one or more reagents used in the flow cytometry workflow, such as labeling reagents, buffers, dyes, etc. The components of the kit may be present in separate containers, or multiple components may be present in a single container.

[0078] In addition to the above-mentioned components, the test kit of this theme may further include (in certain embodiments) instructions for practicing the method for this theme. These instructions can be present in the test kit of this theme in various forms, one or more of which can be present in the test kit. A form in which these instructions can exist is the form of printed information on a suitable medium or substrate, for example, one or more sheets of paper, the packaging of the test kit, the packaging insert, etc. having printed information thereon. Another form of these instructions is a computer-readable medium, such as a floppy disk, a compact disk (CD), a portable flash drive, etc., which has recorded information thereon. Another form in which these instructions can exist is a website address, which can be used to access the information at the site removed via the Internet.

[0079] The following is offered by way of illustration and not limitation. experiment

[0080] Figure 1An embodiment of the workflow according to the present invention is shown in FIG. As shown in the figure, cell samples in different wells of a multi-well plate are barcoded with different combinations of fluorescently labeled antibodies that specifically bind to different universal cell surface markers. Different fluorophores are all violet-exciting.

[0081] Notwithstanding the appended claims, the present disclosure is also defined by the following terms:

[0082] 1. A method for producing a plurality of distinguishably fluorescently barcoded cell samples, the method comprising:

[0083] providing a plurality of cell samples; and

[0084] labeling different cell samples in the plurality with unique fluorescent barcodes, wherein a given fluorescent barcode comprises one or more fluorescently labeled specific binding elements that specifically bind to a universal cell marker;

[0085] To generate multiple cell samples that are fluorescently barcoded in a distinguishable manner.

[0086] 2. The method according to clause 1, wherein the plurality of cell samples comprises 5 to 500 cell samples.

[0087] 3. A method according to clause 2, wherein the multiple cell samples comprise 50 to 400 cell samples.

[0088] 4. The method according to clause 3, wherein the plurality of cell samples comprises 96 cell samples.

[0089] 5. The method according to clause 3, wherein the plurality of cell samples comprises 384 cell samples.

[0090] 6. A method according to any of the preceding clauses, wherein the cell sample is provided in a well of a multi-well plate.

[0091] 7. A method according to any of the preceding clauses, wherein each unique fluorescent barcode comprises a plurality of specific binding elements that are fluorescently labeled in a distinguishable manner.

[0092] 8. The method according to clause 7, wherein the plurality of distinguishably fluorescently labeled specific binding elements comprises 2 to 20 distinguishably fluorescently labeled specific binding elements.

[0093] 9. The method according to clause 8, wherein the plurality of distinguishably fluorescently labeled specific binding elements comprises 3 to 10 distinguishably fluorescently labeled specific binding elements.

[0094] 10. The method according to any one of clauses 7 to 9, wherein each of the plurality of distinguishably fluorescently labeled specific binding members differ from each other in one or more of emission maximum and brightness.

[0095] 11. The method according to any one of clauses 7 to 10, wherein each of the plurality of distinguishably fluorescently labeled specific binding members is excitable by a common light source.

[0096] 12. The method of clause 11, wherein the common light source is a laser.

[0097] 13. A method according to any of the preceding clauses, wherein the universal cell marker is a non-phenotypic marker.

[0098] 14. The method according to clause 13, wherein the universal cell marker is selected from the group consisting of: CD44, CD45, CD47 and beta-2 microglobulin.

[0099] 15. A method according to any of the preceding clauses, wherein the specific binding element is an antibody or a binding fragment thereof.

[0100] 16. A method according to any of the preceding clauses, wherein each cell sample comprises 50 to 50,000,000 cells.

[0101] 17. A method according to any of the preceding clauses, wherein the method further comprises pooling the plurality of distinguishably fluorescently labeled barcoded samples to produce a pooled sample.

[0102] 18. The method according to clause 16, wherein the method further comprises assaying the pooled samples by flow cytometry.

[0103] 19. The method according to clause 18, wherein the method further comprises attributing cells having the same fluorescent barcode as originating from the same cell sample.

[0104] 20. A method according to any of the preceding clauses, wherein the method further comprises differentially processing two or more of the multiple cell samples.

[0105] 21. A plurality of distinguishably fluorescently barcoded cell samples, each labeled with a unique fluorescent barcode, wherein a given fluorescent barcode comprises one or more fluorescently labeled specific binding elements that specifically bind to a universal cell marker.

[0106] 22. The plurality of distinguishably fluorescently barcoded cell samples of clause 21, wherein the plurality comprises 5 to 500 distinguishably fluorescently barcoded sample cell samples.

[0107] 23. The plurality of distinguishably fluorescently barcoded cell samples according to clause 22, wherein the plurality comprises 50 to 400 distinguishably fluorescently barcoded cell samples.

[0108] 24. The plurality of distinguishably fluorescently barcoded cell samples of clause 23, wherein the plurality comprises 96 distinguishably fluorescently barcoded cell samples.

[0109] 25. The plurality of distinguishably fluorescently barcoded cell samples of clause 23, wherein the plurality comprises 384 distinguishably fluorescently barcoded cell samples.

[0110] 26. A plurality of distinguishably fluorescently barcoded cell samples according to any one of clauses 21 to 25, wherein the cell samples are provided in wells of a multi-well plate.

[0111] 27. A plurality of distinguishably fluorescently barcoded cell samples according to any one of clauses 21 to 26, wherein each unique fluorescent barcode comprises a plurality of distinguishably fluorescently labeled specific binding elements.

[0112] 28. The plurality of distinguishably fluorescently barcoded cell samples of clause 27, wherein the plurality of distinguishably fluorescently labeled specific binding elements comprises 2 to 20 distinguishably fluorescently labeled specific binding elements.

[0113] 29. The plurality of distinguishably fluorescently barcoded cell samples of clause 28, wherein the plurality of distinguishably fluorescently labeled specific binding elements comprises 5 to 10 distinguishably fluorescently labeled specific binding elements.

[0114] 30. A plurality of distinguishably fluorescently barcoded cell samples according to any one of clauses 27 to 29, wherein each of the plurality of distinguishably fluorescently labeled specific binding elements differ from each other in one or more of emission maximum and brightness.

[0115] 31. A plurality of distinguishably fluorescently barcoded cell samples according to any one of clauses 27 to 30, wherein each of the plurality of distinguishably fluorescently labeled specific binding elements is excitable by a common light source.

[0116] 32. The plurality of distinguishably fluorescently barcoded cell samples of clause 31, wherein the common light source is a laser.

[0117] 33. A plurality of distinguishably fluorescently barcoded cell samples according to any one of clauses 21 to 32, wherein the universal cell marker is a non-phenotypic marker.

[0118] 34. The plurality of distinguishably fluorescently barcoded cell samples of clause 33, wherein the universal cell marker is selected from the group consisting of: CD44, CD45, CD47, and beta-2 microglobulin.

[0119] 35. A plurality of distinguishably fluorescently barcoded cell samples according to any one of clauses 21 to 34, wherein the specific binding element is an antibody or a binding fragment thereof.

[0120] 36. A plurality of distinguishably fluorescently barcoded cell samples according to any one of clauses 21 to 35, wherein each cell sample comprises 50 to 50,000,000 cells.

[0121] 37. A pooled sample comprising a plurality of distinguishably fluorescently barcoded cell samples according to any one of clauses 21 to 36.

[0122] 38. A flow cytometer comprising the pooled sample according to clause 37.

[0123] 39. A kit comprising:

[0124] A plurality of distinguishably fluorescently labeled specific binding elements that specifically bind to universal cell surface markers.

[0125] 40. The kit according to clause 39, wherein the plurality comprises from 2 to 20 specific binding elements that are distinguishably fluorescently labeled.

[0126] 41. The kit according to clause 40, wherein the plurality comprises 3 to 10 specific binding elements that are fluorescently labeled in a distinguishable manner.

[0127] 42. A kit according to any one of clauses 39 to 41, wherein each of the plurality of distinguishably fluorescently labelled specific binding members differ from one another in one or more of emission maximum and brightness.

[0128] 43. The kit according to any one of clauses 39 to 42, wherein each of the plurality of distinguishably fluorescently labeled specific binding members is excitable by a common light source.

[0129] 44. A kit according to clause 43, wherein the common light source is a laser.

[0130] 45. A kit according to any one of clauses 39 to 44, wherein the universal cellular marker is a non-phenotypic marker.

[0131] 46. ​​The kit according to clause 45, wherein the universal cell marker is selected from the group consisting of: CD44, CD45, CD47 and beta-2 microglobulin.

[0132] 47. A kit according to any one of clauses 39 to 46, wherein the plurality of distinguishably fluorescently labelled specific binding elements comprises distinguishably fluorescently labelled specific binding elements that specifically bind to different universal cell surface markers.

[0133] 48. A kit according to any one of clauses 39 to 47, wherein the specific binding member is an antibody or a binding fragment thereof.

[0134] Notwithstanding the appended claims, the present disclosure is also defined by the following terms:

[0135] 1. A method for producing a plurality of distinguishably fluorescently barcoded particle samples, the method comprising:

[0136] providing a plurality of particle samples; and

[0137] labeling different particle samples in the plurality with unique fluorescent barcodes, wherein a given fluorescent barcode comprises one or more fluorescently labeled specific binding elements that specifically bind to a particle marker;

[0138] To generate multiple samples of particles that are fluorescently barcoded in a distinguishable manner.

[0139] 2. The method according to clause 1, wherein the plurality of particle samples comprises 5 to 500 particle samples.

[0140] 3. The method according to clause 2, wherein the plurality of particle samples comprises 50 to 400 particle samples.

[0141] 4. The method of clause 3, wherein the plurality of particle samples comprises 96 particle samples.

[0142] 5. The method of clause 3, wherein the plurality of particle samples comprises 384 particle samples.

[0143] 6. A method according to any of the preceding clauses, wherein the particle sample is provided in the wells of a multi-well plate.

[0144] 7. A method according to any of the preceding clauses, wherein each unique fluorescent barcode comprises a plurality of specific binding elements that are fluorescently labeled in a distinguishable manner.

[0145] 8. The method according to clause 7, wherein the plurality of distinguishably fluorescently labeled specific binding elements comprises 2 to 20 distinguishably fluorescently labeled specific binding elements.

[0146] 9. The method according to clause 8, wherein the plurality of distinguishably fluorescently labeled specific binding elements comprises 3 to 10 distinguishably fluorescently labeled specific binding elements.

[0147] 10. The method according to any one of clauses 7 to 9, wherein each of the plurality of distinguishably fluorescently labeled specific binding members differ from each other in one or more of emission maximum and brightness.

[0148] 11. The method according to any one of clauses 7 to 10, wherein each of the plurality of distinguishably fluorescently labeled specific binding members is excitable by a common light source.

[0149] 12. The method of clause 11, wherein the common light source is a laser.

[0150] 13. A method according to any of the preceding clauses, wherein the marker is a non-phenotypic marker.

[0151] 14. The method according to clause 13, wherein the marker is selected from the group consisting of: CD44, CD45, CD47 and beta-2 microglobulin.

[0152] 15. A method according to any of the preceding clauses, wherein the specific binding element is an antibody or a binding fragment thereof.

[0153] 16. A method according to any of the preceding clauses, wherein each particle sample comprises 50 to 50,000,000 particles.

[0154] 17. A method according to any of the preceding clauses, wherein the method further comprises pooling the plurality of distinguishably fluorescently labeled barcoded samples to produce a pooled sample.

[0155] 18. The method according to clause 16, wherein the method further comprises assaying the pooled samples by flow cytometry.

[0156] 19. The method of clause 18, wherein the method further comprises attributing particles having the same fluorescent barcode as originating from the same particle sample.

[0157] 20. A method according to any of the preceding clauses, wherein the method further comprises differentially processing two or more of the plurality of particle samples.

[0158] 21. A plurality of distinguishably fluorescently barcoded particle samples, each labeled with a unique fluorescent barcode, wherein a given fluorescent barcode comprises one or more fluorescently labeled specific binding elements that specifically bind to a particle marker.

[0159] 22. The plurality of distinguishably fluorescently barcoded particle samples of clause 21, wherein the plurality comprises 5 to 500 distinguishably fluorescently barcoded particle samples.

[0160] 23. The plurality of distinguishably fluorescently barcoded particle samples of clause 22, wherein the plurality comprises 50 to 400 distinguishably fluorescently barcoded particle samples.

[0161] 24. The plurality of distinguishably fluorescently barcoded particle samples of clause 23, wherein the plurality comprises up to 96 distinguishably fluorescently barcoded particle samples.

[0162] 25. The plurality of distinguishably fluorescently barcoded particle samples of clause 23, wherein the plurality comprises 384 distinguishably fluorescently barcoded particle samples.

[0163] 26. A plurality of distinguishably fluorescently barcoded particle samples according to any one of clauses 21 to 25, wherein the particle samples are provided in wells of a multi-well plate.

[0164] 27. A plurality of distinguishably fluorescently barcoded particle samples according to any one of clauses 21 to 26, wherein each unique fluorescent barcode comprises a plurality of distinguishably fluorescently labeled specific binding elements.

[0165] 28. The plurality of distinguishably fluorescently barcoded particle samples of clause 27, wherein the plurality of distinguishably fluorescently labeled specific binding elements comprises 2 to 20 distinguishably fluorescently labeled specific binding elements.

[0166] 29. The plurality of distinguishably fluorescently barcoded particle samples of clause 28, wherein the plurality of distinguishably fluorescently labeled specific binding elements comprises 5 to 10 distinguishably fluorescently labeled specific binding elements.

[0167] 30. A plurality of distinguishably fluorescently barcoded particle samples according to any one of clauses 27 to 29, wherein each of the plurality of distinguishably fluorescently labeled specific binding elements differs from one another in one or more of emission maximum and brightness.

[0168] 31. A plurality of distinguishably fluorescently barcoded particle samples according to any one of clauses 27 to 30, wherein each of the plurality of distinguishably fluorescently labeled specific binding elements is excitable by a common light source.

[0169] 32. A plurality of distinguishably fluorescently barcoded particle samples according to clause 31, wherein the common light source is a laser.

[0170] 33. A plurality of distinguishably fluorescently barcoded particle samples according to any one of clauses 21 to 32, wherein the particle marker is a non-phenotypic marker.

[0171] 34. The plurality of distinguishably fluorescently barcoded particle samples of clause 33, wherein the particle markers are selected from the group consisting of: CD44, CD45, CD47, and beta-2 microglobulin.

[0172] 35. A plurality of distinguishably fluorescently barcoded particle samples according to any one of clauses 21 to 34, wherein the specific binding element is an antibody or a binding fragment thereof.

[0173] 36. A plurality of distinguishably fluorescently barcoded particle samples according to any one of clauses 21 to 35, wherein each cell sample comprises 50 to 50,000,000 cells.

[0174] 37. A pooled sample comprising a plurality of distinguishably fluorescently barcoded particle samples according to any one of clauses 21 to 36.

[0175] 38. A flow cytometer comprising the pooled sample according to clause 37.

[0176] 39. A kit comprising:

[0177] A plurality of distinguishably fluorescently labeled specific binding elements that specifically bind to the particle marker.

[0178] 40. The kit according to clause 39, wherein the plurality comprises from 2 to 20 specific binding elements that are distinguishably fluorescently labeled.

[0179] 41. The kit according to clause 40, wherein the plurality comprises 3 to 10 specific binding elements that are fluorescently labeled in a distinguishable manner.

[0180] 42. A kit according to any one of clauses 39 to 41, wherein each of the plurality of distinguishably fluorescently labelled specific binding members differ from one another in one or more of emission maximum and brightness.

[0181] 43. The kit according to any one of clauses 39 to 42, wherein each of the plurality of distinguishably fluorescently labeled specific binding members is excitable by a common light source.

[0182] 44. A kit according to clause 43, wherein the common light source is a laser.

[0183] 45. A kit according to any one of clauses 39 to 44, wherein the particle marker is a non-phenotypic marker.

[0184] 46. ​​A kit according to clause 45, wherein the particle marker is selected from the group consisting of: CD44, CD45, CD47 and beta-2 microglobulin.

[0185] 47. A kit according to any one of clauses 39 to 46, wherein the plurality of distinguishably fluorescently labelled specific binding elements comprises distinguishably fluorescently labelled specific binding elements that specifically bind to different particle markers.

[0186] 48. A kit according to any one of clauses 39 to 47, wherein the specific binding member is an antibody or a binding fragment thereof.

[0187] 49. A kit according to any one of clauses 39 to 47, wherein the kit further comprises microspheres for a multiplex microsphere array assay.

[0188] Notwithstanding the appended claims, the present disclosure is also defined by the following terms:

[0189] 1. A method for producing a plurality of microsphere samples that are fluorescently barcoded in a distinguishable manner, the method comprising:

[0190] providing a plurality of microsphere samples; and

[0191] labeling different microsphere samples in the plurality with unique fluorescent barcodes, wherein a given fluorescent barcode comprises one or more fluorescently labeled specific binding elements that specifically bind to a microsphere marker;

[0192] This allows the generation of multiple samples of microspheres that are fluorescently barcoded in a distinguishable manner.

[0193] 2. The method according to clause 1, wherein the plurality of microsphere samples comprises 5 to 500 microsphere samples.

[0194] 3. The method according to clause 2, wherein the plurality of microsphere samples comprises 50 to 400 microsphere samples.

[0195] 4. The method of clause 3, wherein the plurality of microsphere samples comprises 96 microsphere samples.

[0196] 5. The method of clause 3, wherein the plurality of microsphere samples comprises 384 microsphere samples.

[0197] 6. A method according to any of the preceding clauses, wherein the microsphere sample is provided in the wells of a multi-well plate.

[0198] 7. A method according to any of the preceding clauses, wherein each unique fluorescent barcode comprises a plurality of specific binding elements that are fluorescently labeled in a distinguishable manner.

[0199] 8. The method according to clause 7, wherein the plurality of distinguishably fluorescently labeled specific binding elements comprises 2 to 20 distinguishably fluorescently labeled specific binding elements.

[0200] 9. The method according to clause 8, wherein the plurality of distinguishably fluorescently labeled specific binding elements comprises 3 to 10 distinguishably fluorescently labeled specific binding elements.

[0201] 10. The method according to any one of clauses 7 to 9, wherein each of the plurality of distinguishably fluorescently labeled specific binding members differ from each other in one or more of emission maximum and brightness.

[0202] 11. The method according to any one of clauses 7 to 10, wherein each of the plurality of distinguishably fluorescently labeled specific binding members is excitable by a common light source.

[0203] 12. The method of clause 11, wherein the common light source is a laser.

[0204] 13. A method according to any of the preceding clauses, wherein the marker is a non-phenotypic marker.

[0205] 14. The method according to clause 13, wherein the marker is selected from the group consisting of: CD44, CD45, CD47 and beta-2 microglobulin.

[0206] 15. A method according to any of the preceding clauses, wherein the specific binding element is an antibody or a binding fragment thereof.

[0207] 16. The method of any of the preceding clauses, wherein each microsphere sample comprises 50 to 50,000,000 microspheres.

[0208] 17. A method according to any of the preceding clauses, wherein the method further comprises pooling the plurality of distinguishably fluorescently labeled barcoded samples to produce a pooled sample.

[0209] 18. The method according to clause 16, wherein the method further comprises assaying the pooled samples by flow cytometry.

[0210] 19. The method of clause 18, wherein the method further comprises attributing microspheres having the same fluorescent barcode as originating from the same microsphere sample.

[0211] 20. The method of any of the preceding clauses, wherein the method further comprises differentially processing two or more of the plurality of microsphere samples.

[0212] 21. A plurality of distinguishably fluorescently barcoded microsphere samples, each labeled with a unique fluorescent barcode, wherein a given fluorescent barcode comprises one or more fluorescently labeled specific binding elements that specifically bind to a microsphere marker.

[0213] 22. The plurality of distinguishably fluorescently barcoded microsphere samples of clause 21, wherein the plurality comprises 5 to 500 distinguishably fluorescently barcoded microsphere samples.

[0214] 23. The plurality of distinguishably fluorescently barcoded microsphere samples of clause 22, wherein the plurality comprises 50 to 400 distinguishably fluorescently barcoded microsphere samples.

[0215] 24. The plurality of distinguishably fluorescently barcoded microsphere samples of clause 23, wherein the plurality comprises 96 distinguishably fluorescently barcoded microsphere samples.

[0216] 25. The plurality of distinguishably fluorescently barcoded microsphere samples of clause 23, wherein the plurality comprises 384 distinguishably fluorescently barcoded microsphere samples.

[0217] 26. A plurality of distinguishably fluorescently barcoded microsphere samples according to any one of clauses 21 to 25, wherein the microsphere samples are provided in wells of a multi-well plate.

[0218] 27. A plurality of distinguishably fluorescently barcoded microsphere samples according to any one of clauses 21 to 26, wherein each unique fluorescent barcode comprises a plurality of distinguishably fluorescently labeled specific binding elements.

[0219] 28. The plurality of distinguishably fluorescently barcoded microsphere samples of clause 27, wherein the plurality of distinguishably fluorescently labeled specific binding elements comprises 2 to 20 distinguishably fluorescently labeled specific binding elements.

[0220] 29. The plurality of distinguishably fluorescently barcoded microsphere samples of clause 28, wherein the plurality of distinguishably fluorescently labeled specific binding elements comprises 5 to 10 distinguishably fluorescently labeled specific binding elements.

[0221] 30. A plurality of distinguishably fluorescently barcoded microsphere samples according to any one of clauses 27 to 29, wherein each of the plurality of distinguishably fluorescently labeled specific binding elements differ from each other in one or more of emission maximum and brightness.

[0222] 31. A plurality of distinguishably fluorescently barcoded microsphere samples according to any one of clauses 27 to 30, wherein each of the plurality of distinguishably fluorescently labeled specific binding elements is excitable by a common light source.

[0223] 32. The plurality of distinguishably fluorescently barcoded microsphere samples of clause 31, wherein the common light source is a laser.

[0224] 33. A plurality of distinguishably fluorescently barcoded microsphere samples according to any one of clauses 21 to 32, wherein the particle marker is a non-phenotypic marker.

[0225] 34. The plurality of distinguishably fluorescently barcoded microsphere samples of clause 33, wherein the particle marker is selected from the group consisting of: CD44, CD45, CD47, and beta-2 microglobulin.

[0226] 35. A plurality of distinguishably fluorescently barcoded microsphere samples according to any one of clauses 21 to 34, wherein the specific binding element is an antibody or a binding fragment thereof.

[0227] 36. A plurality of distinguishably fluorescently barcoded microsphere samples according to any one of clauses 21 to 35, wherein each microsphere sample comprises 50 to 50,000,000 microspheres.

[0228] 37. A pooled sample comprising a plurality of distinguishably fluorescently barcoded microsphere samples according to any one of clauses 21 to 36.

[0229] 38. A flow cytometer comprising the pooled sample according to clause 37.

[0230] 39. A kit comprising:

[0231] A plurality of distinguishably fluorescently labeled specific binding elements that specifically bind to the particle marker.

[0232] 40. The kit according to clause 39, wherein the plurality comprises from 2 to 20 specific binding elements that are distinguishably fluorescently labeled.

[0233] 41. The kit according to clause 40, wherein the plurality comprises 3 to 10 specific binding elements that are fluorescently labeled in a distinguishable manner.

[0234] 42. A kit according to any one of clauses 39 to 41, wherein each of the plurality of distinguishably fluorescently labelled specific binding members differ from one another in one or more of emission maximum and brightness.

[0235] 43. The kit according to any one of clauses 39 to 42, wherein each of the plurality of distinguishably fluorescently labeled specific binding members is excitable by a common light source.

[0236] 44. A kit according to clause 43, wherein the common light source is a laser.

[0237] 45. A kit according to any one of clauses 39 to 44, wherein the particle marker is a non-phenotypic marker.

[0238] 46. ​​A kit according to clause 45, wherein the particle marker is selected from the group consisting of: CD44, CD45, CD47 and beta-2 microglobulin.

[0239] 47. A kit according to any one of clauses 39 to 46, wherein the plurality of distinguishably fluorescently labelled specific binding elements comprises distinguishably fluorescently labelled specific binding elements that specifically bind to different particle markers.

[0240] 48. A kit according to any one of clauses 39 to 47, wherein the specific binding member is an antibody or a binding fragment thereof.

[0241] 49. A kit according to any one of clauses 39 to 47, wherein the kit further comprises microspheres for a multiplex microsphere array assay.

[0242] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be apparent to one skilled in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

[0243] Therefore, the above only illustrates the principle of the present invention.It should be understood that those skilled in the art will be able to design various arrangements, which, although not explicitly described or shown in this article, embody the principle of the present invention and are included in its spirit and scope.In addition, all examples and conditional language described in this article are mainly intended to help readers understand the principles of the present invention and the concepts contributed by the inventor to promote this area, and should be interpreted as not being limited to such specific examples and conditions.In addition, all statements describing the principles, aspects and embodiments of the present invention and their specific examples are intended to cover both their structural equivalents and functional equivalents.In addition, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e., any element that performs the same function developed, regardless of the structure.In addition, regardless of whether such disclosure is explicitly described in the claims, any content disclosed herein is not intended to be dedicated to the public.

[0244] Therefore, the scope of the present invention is not intended to be limited to the exemplary embodiments shown and described herein. Instead, the scope and spirit of the present invention are embodied by the appended claims. In the claims, 35 USC §112(f) or 35 USC §112(6) is expressly defined as a limitation in a claim only when the exact phrase "means for..." or the exact phrase "step for..." is cited at the beginning of such limitation in the claim; if such exact phrase is not used in the limitation of the claim, 35 USC §112(f) or 35 USC §112(6) is not cited.

Claims

1. A method of producing a plurality of distinguishably fluorescently barcoded particle samples, the method comprising: Provide multiple particle samples; as well as labeling different particle samples in the plurality with unique fluorescent barcodes, wherein a given fluorescent barcode comprises one or more fluorescently labeled specific binding elements that specifically bind to a particle marker; To generate multiple samples of particles that are fluorescently barcoded in a distinguishable manner.

2. The method according to any one of the preceding claims, wherein the plurality of particle samples comprises a plurality of cell samples or a plurality of microsphere samples.

3. A method according to any one of the preceding claims, wherein the particle sample is provided in the wells of a multi-well plate.

4. The method according to any one of the preceding claims, wherein each unique fluorescent barcode comprises a plurality of specific binding elements that are fluorescently labeled in a distinguishable manner.

5. The method of claim 4, wherein each of the plurality of distinguishably fluorescently labeled specific binding members differ from one another in one or more of emission maximum and brightness.

6. The method according to any one of claims 4 to 5, wherein each of the plurality of distinguishably fluorescently labeled specific binding elements is excitable by a common light source, preferably a laser.

7. The method according to any one of the preceding claims, wherein the particulate marker is a non-phenotypic marker.

8. The method according to any one of the preceding claims, wherein the specific binding member is an antibody or a binding fragment thereof.

9. The method of any of the preceding claims, wherein the method further comprises pooling the plurality of distinguishably fluorescently labeled barcoded samples to produce a pooled sample.

10. The method of claim 9, wherein the method further comprises assaying the pooled samples by flow cytometry.

11. The method of claim 10, wherein the method further comprises attributing cells having the same fluorescent barcode as originating from the same particle sample.

12. The method of any one of the preceding claims, wherein the method further comprises differentially processing two or more of the plurality of particle samples.

13. A plurality of distinguishably fluorescently barcoded particle samples, each labeled with a unique fluorescent barcode, wherein a given fluorescent barcode comprises one or more fluorescently labeled specific binding elements that specifically bind to a particle marker. 14 . The plurality of distinguishably fluorescently barcoded particle samples of claim 13 , wherein the plurality comprises fluorescently barcoded cell samples or fluorescently barcoded microsphere samples.

15. A kit comprising: A plurality of distinguishably fluorescently labeled specific binding elements that specifically bind to the particle marker.

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