Lyophilized antibody panel

Abstract

To solve the problem in which conventional techniques for creating and storing element-labeled assays can be susceptible to damage and other error-inducing effects, which can lead to inaccurate results.SOLUTION: A lyophilized antibody panel for interrogation using elemental analysis is disclosed. An antibody may be a fragment thereof, such as a nanobody or Fab fragment. Further, affinity reagents in general (including not only antibodies but also other affinity reagents such as lectins, aptamers and non-antibody protein-ligand pairs such as streptavidin-biotin) may be used in place of antibodies in embodiments discussed herein.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 824,917, filed March 27, 2019, and U.S. Provisional Patent Application No. 62 / 951,546, filed December 20, 2019, the contents of both of which are incorporated herein by reference for all purposes.

[0002]

[0002] This disclosure relates generally to biological assays, and more particularly to biological assays investigated by elemental analysis. [Background technology]

[0003]

[0003] Biological assays can be used to determine information related to a sample. For example, certain biological assays can be performed to determine the presence of various cell types in a blood sample. The results of biological assays can be used throughout research and medicine.

[0004] In certain current assays, various antibodies are tagged with different fluorescent substances. Each antibody may be specific for a particular protein (e.g., an antigen) that may or may not be present in the sample. When these fluorescently tagged antibodies are mixed with the sample, the antibodies specific for proteins in the sample can bind to those proteins. The sample can then be washed, and the resulting sample can be optically interrogated to identify the presence of the fluorescent substances.

[0005]

[0005] However, such fluorescence assays are limited in the number of distinguishable channels and the time required to perform the assay. For example, the multiplexing of traditional fluorescence microscopy is limited by the spectral overlap of fluorophore emissions. Recent developments in imaging mass spectrometry of samples stained with element-labeled antibodies allow for the simultaneous imaging of even more targets, as each target can be associated with a unique isotope via an antibody intermediate. However, traditional techniques for creating and storing element-labeled assays can be susceptible to damage and other error-inducing effects, which can lead to inaccurate results.

[0006]

[0006] This specification makes reference to the following accompanying figures, in which the use of like reference numerals in different figures is intended to indicate like or similar components. [Brief explanation of the drawings]

[0007] [Figure 1] FIG. 1 is a schematic diagram of an element tagging moiety according to certain aspects of the present disclosure. [Figure 2A] FIG. 1 is a schematic diagram of a barcoded reagent having distinct sample barcodes, according to certain aspects of the present disclosure. [Figure 2B] FIG. 2B is a schematic diagram of the barcoded reagent of FIG. 2A after attachment of a sample barcode, according to certain embodiments of the present disclosure. [Figure 3] FIG. 3 is a schematic diagram of a barcoded reagent 308 having a sample barcode 312 integrated therein, according to certain embodiments of the present disclosure. [Figure 4] FIG. 1 is a schematic diagram of a system for assaying one or more samples using an elemental analyzer and a lyophilized antibody panel, according to certain embodiments of the present disclosure. [Figure 5] FIG. 1 is a schematic diagram illustrating the analysis and processing of unknown particles according to certain aspects of the present disclosure. [Figure 6] FIG. 1 is a schematic diagram illustrating three exemplary element tags with charts showing their respective elemental data, according to certain embodiments of the present disclosure. [Figure 7]FIG. 1 is a schematic diagram of a sample cell tagged with an element tagging moiety, a sample barcode, and a barcoding reagent, according to certain aspects of the present disclosure. [Figure 8] FIG. 1 is a schematic diagram illustrating sample cells tagged with multi-isotope sample barcodes, according to certain aspects of the present disclosure. [Figure 9] FIG. 1 is a schematic diagram showing sample cells tagged with dispersed sample barcode moieties, according to certain aspects of the present disclosure. [Figure 10] FIG. 1 is a schematic diagram of a barcoding reagent and a reporter antibody according to certain aspects of the present disclosure. [Figure 11] FIG. 1 is a schematic diagram showing the preparation of a lyophilized antibody panel according to certain aspects of the present disclosure. [Figure 12] FIG. 1 is a schematic diagram of a process for preparing barcoded reagents according to certain aspects of the present disclosure. [Figure 13] 1 is a flowchart illustrating a process for staining and analyzing a blood sample according to certain aspects of the present disclosure. [Figure 14] FIG. 1 is a schematic diagram illustrating an exemplary gating strategy for automatically analyzing elemental data, according to certain aspects of the present disclosure. [Figure 15] FIG. 1 is a schematic diagram illustrating a technique for labeling samples with barcoded reagents and analyzing a set of samples, according to certain aspects of the present disclosure. [Figure 16] FIG. 1 is a schematic diagram illustrating a technique for labeling samples and analyzing a set of samples, according to certain aspects of the present disclosure. [Figure 17] FIG. 1 is a schematic diagram illustrating the preparation of a pre-configured set of sample-barcode labeled barcoding reagents according to certain embodiments of the present disclosure. DETAILED DESCRIPTION OF THE INVENTION

[0008]

[0025] Certain aspects and features of the present disclosure relate to lyophilized antibody panels for investigation using elemental analysis. The antibodies may be fragments thereof, such as nanobodies or Fab fragments. Furthermore, in the embodiments described herein, general affinity reagents (including not only antibodies but also other affinity reagents, such as lectins, aptamers, and non-antibody protein-ligand pairs, such as streptavidin-biotin) may be used in place of antibodies.

[0009]

[0026] An antibody panel can include multiple different antibodies, each elementally tagged or labeled with one or more isotopes, so that each different antibody is isotopically distinguishable from the others. Each elemental tag can contain one or more unique elements, isotopes, or unique combinations of isotopes. A set of elementally tagged antibodies can be lyophilized in admixture. Thus, the lyophilized elementally tagged antibody panel can be easily and efficiently resuspended and mixed with a sample before interrogation using an elemental analysis device such as a mass spectrometer. This lyophilized elementally tagged antibody panel can provide the advantages of elemental tagging assays while being easy to use and remaining stable for extended periods of time. The elemental tags of any of the subject methods or kits can be mass tags, for example, when the detection method is mass spectrometry and / or when the elemental tags include enriched isotopes. The same mass tag or overlapping mass tags refers to mass tags that have the same isotopically (or isotopic mass) labeled atom and cannot be distinguished by mass spectrometry.

[0010]

[0027] Certain aspects and features of the present disclosure also relate to techniques for uniquely barcoding different samples, allowing them to be combined before interrogation by an elemental analyzer. Each different sample barcoding reagent can contain a unique combination of isotopes that can be used to distinguish that sample barcoding reagent from other sample barcoding reagents. Multiple samples can be individually mixed with different sample barcoding reagents, allowing the sample barcoding reagents to bind to targets in the samples (e.g., assay barcoded beads and / or cells), thereby labeling each sample with its own unique barcode. Even after the samples are mixed together, data about each sample can be extracted based on the presence of unique combinations of isotopes in data collected from the elemental analyzer. In some cases, samples can be mixed together before performing an assay, such as an assay using a lyophilized element-tagged antibody panel.

[0011]

[0028] Elemental analysis is the process by which a sample is analyzed for its elemental and sometimes isotopic composition. Elemental analysis can be accomplished by several methods, including optical atomic spectroscopy (e.g., flame atomic absorption, graphite furnace atomic absorption, and inductively coupled plasma atomic emission), which probe the outer electronic structure of atoms; mass spectrometry (e.g., inductively coupled mass spectrometry), which probes the mass of atoms; x-ray fluorescence, particle-induced x-ray emission, x-ray photoelectron spectroscopy, and Auger electron spectroscopy, which probes the inner electronic structure of atoms. Other elemental analysis techniques may also be used.

[0012]

[0029] A mass spectrometer for use in the present invention can be selected based on the needs of the operator or a particular application. Exemplary types of mass spectrometers include quadrupole, time-of-flight, magnetic sector, high-resolution, single- or multi-collector mass spectrometers. Typically, time-of-flight or magnetic sector mass spectrometers are used to record fast transient events with predicted transit times from particles (e.g., beads, cells, or laser ablation plumes). In certain embodiments, time-of-flight mass spectrometers can have a high-pass filter with a mass cutoff of 80 amu or greater. In certain embodiments, an atomization and ionization source, such as an inductively coupled plasma (ICP), can be upstream of the time-of-flight or magnetic sector mass detector.

[0013]

[0030] Certain embodiments of the present disclosure are particularly suitable for use with a type of elemental analysis called mass spectrometry, in which the mass of an isotope or element is determined. Mass spectrometry can include atomic mass spectrometry, such as mass cytometry. Mass spectrometry can be particularly suitable for identifying elemental isotopes, thereby distinguishing one isotope from another distinct isotope, or distinguishing one combination of isotopes from another distinct combination of isotopes. Thus, mass spectrometry can be a desired form of elemental analysis for any embodiment of the present disclosure disclosed herein, where appropriate.

[0014]

[0031] An elemental analyzer is an instrument for quantifying the atomic composition of a sample. The elemental analyzer can use one of the elemental analysis techniques described herein. An elemental analyzer can have a set number of channels for isotope detection or differentiation. For example, a mass spectrometer can have multiple mass channels available for detecting different isotopes. The number of channels available for a particular elemental analyzer can be limited. Therefore, any subset of channels can be used for a specific purpose (e.g., labeling antibodies in a lyophilized antibody panel or barcoding samples), leaving the remaining channels available for other purposes. For example, empty channels can be used to perform additional assays or other barcoding.

[0015]

[0032] In some cases, elemental analysis can be performed on an individual particle basis, known as particle elemental analysis. Particle elemental analysis involves determining the elemental composition of individual particles (e.g., per cell) using, for example, a mass spectrometer-based flow cytometer. Certain aspects of the present disclosure utilize cell-by-cell particle elemental analysis, which may be known as cytometric elemental analysis. In some cases, elemental analysis can be performed on a bulk basis, known as bulk elemental analysis or solution elemental analysis. Bulk elemental analysis involves determining the elemental composition of the entire volume of a sample.

[0016]

[0033] Elemental analysis can be used to investigate samples, such as biological samples. If the sample is labeled with a known elemental tag, detection of the elemental tag during elemental analysis can indicate a characteristic of the sample associated with the elemental tag.

[0017]

[0034] As referred to herein, mass cytometry is any method for detecting elemental tags (mass tags) in biological samples, such as simultaneously detecting multiple distinguishable mass tags with single cell resolution.Mass cytometry can include the analysis of mass-tagged beads separately from cells or in addition to cells.All of the subject kits and methods can include or be adapted to mass cytometry.Mass cytometry includes suspension mass cytometry and imaging mass cytometry (IMC).

[0018]

[0035] Suspension mass cytometry involves the analysis of suspended elementally tagged cells and / or beads by mass spectrometry (e.g., by atomic mass spectrometry) and is described in U.S. patent publications, including U.S. Patent Nos. 20050218319, 20150183895, and 20150122991, all of which are incorporated herein by reference.

[0019]

[0036] Imaging mass cytometry includes any imaging mass spectrometry (e.g., imaging atomic mass spectrometry) of elementally tagged biological samples such as tissue sections or cell smears. IMC can atomize and ionize mass tags from cell samples using one or more of laser radiation, ion beam radiation, electron beam radiation, and / or inductively coupled plasma (ICP). Mass cytometry can simultaneously detect distinct mass tags from single cells using time-of-flight (TOF) or magnetic sector mass spectrometry (MS). Examples of mass cytometry include suspension mass cytometry, in which cells flow through, as well as ICP-MS and imaging mass cytometry, in which cell samples (e.g., tissue sections) are sampled, for example, by laser ablation (LA-ICP-MS) or a primary ion beam (e.g., in the case of SIMS). Laser-based IMC is described in U.S. Patent Publications such as U.S. Patent Nos. 20160131635, 20170148619, 20180306695, and 20180306695, all of which are incorporated herein by reference. In certain embodiments, when the sample is a cell smear for analysis by IMC, the cells can be processed as described herein, for example, by staining with a lyophilized panel, sample barcoding, and / or assay barcoding. Similarly, the assay beads described herein can be analyzed by IMC, either separately or mixed with cells.

[0020]

[0037] Mass tags can be sampled, atomized, and ionized prior to elemental analysis. For example, mass tags in biological samples can be sampled, atomized, and / or ionized by radiation, such as a laser beam, ion beam, or electron beam. Alternatively or additionally, mass tags can be atomized and ionized by plasma, such as inductively coupled plasma (ICP). In suspension mass cytometry, whole cells containing mass tags can be flowed into an ICP-MS, such as an ICP-TOF-MS. In imaging mass cytometry, a form of radiation can remove (and optionally ionize and atomize) a portion (e.g., a pixel, region of interest) of a solid biological sample, such as a tissue sample, containing mass tags. Examples of IMC include LA-ICP-MS and SIMS-MS of mass-tagged samples. In certain embodiments, ion optics can deplete ions other than isotopes of the mass tags. For example, ion optics can remove relatively light ions (e.g., C, N, O) and organic molecular ions. In ICP applications, the ion optics may remove gases such as Ar and / or Xe, for example, via a high-pass quadrupole filter. In certain embodiments, IMC may provide images of mass tags (e.g., targets associated with mass tags) with cellular or subcellular resolution.

[0021]

[0038] Similar to fluorescence immunohistochemistry, mass cytometry (including imaging mass cytometry) workflows may include cell (e.g., tissue) fixation and / or permeabilization prior to staining with antibodies and / or other specific binding partners. In contrast to fluorescence methods, in mass cytometry, mass tags (e.g., containing heavy metals not endogenous to cells) associate with target analytes via specific binding partners, such as antibodies. Similar to fluorescence microscopy, imaging mass cytometry may include an antigen retrieval step in which the sample is exposed to conditions, such as heat, to expose the target analyte for binding by biomolecules. Unbound biomolecules are typically washed away before detection of the mass tags by mass spectrometry. Of note, other detection methods, such as elemental analysis (e.g., emission spectroscopy or X-ray dispersion spectroscopy), are also within the scope of this application.

[0022]

[0039] Of note, antigen retrieval conditions may be particularly important for IMC compared to other imaging methods, because elemental tags (e.g., mass tags) can cause greater steric hindrance in tissues than other labels, such as fluorophores. However, highly stringent retrieval conditions (such as prolonged exposure of tissue sections to high heat) can denature the sample and damage the epitope to be detected. In certain embodiments, a metal heating block, such as an aluminum heating block, may be provided in the kit or used to heat the tissue section for antigen retrieval. The inventors have found that such a block provides uniform heat distribution and allows for suitably rapid temperature transitions for controlled antigen retrieval. Therefore, the methods or kits of the present application can be used for any imaging mass cytometry application or other imaging application, such as light microscopy.

[0023]

[0040] Additional reagents for mass cytometry include metal-containing biosensors (e.g., deposited or bound under conditions such as hypoxia, protein synthesis, cell cycle, and / or cell death) and / or metal-containing histochemical compounds that bind to structures (e.g., DNA, cell membranes, layers) based on their chemical properties. Additionally, mass tags (e.g., of the present application or other mass tags) may be combined to provide a unique barcode for labeling specific samples or experimental conditions prior to pooling with other samples or experimental conditions.

[0024]

[0041] In IHC, tissue samples can be sections with thicknesses ranging from 1 to 10 μm, e.g., 2 to 6 μm. In some cases, ultrathin sections with thicknesses of 500 nm, 400 nm, 200 nm, 100 nm, or less than 50 nm can be used, such as samples cut from resin-embedded tissue blocks. Techniques for preparing such sections, including dehydration, fixation, embedding, permeabilization, and sectioning, are well known from the field of IHC, e.g., using a microtome. Thus, tissues can be chemically fixed, and then sections can be prepared at the desired plane. Frozen sectioning or laser capture microdissection can also be used to prepare tissue samples. Samples can be permeabilized, for example, to allow the addition of reagents for labeling intracellular targets. Even after antigen retrieval (e.g., by heating), access to analytes by biomolecules can be sterically hindered. Therefore, small biomolecules and specific mass tags may best enable biomolecules to access their target analytes.

[0025]

[0042] In imaging mass cytometry, cell segmentation may enable automated cell classification and other aspects of this application, although different tissues and cell types have different surface markers, making a single universal membrane stain difficult.

[0026]

[0043] A kit for cell segmentation may include a membrane stain comprising multiple antibodies against different cell surface targets, wherein the antibodies are conjugated to the same mass tag. In certain aspects, the membrane stain comprises an antibody against a junction protein and an antibody against a non-junction protein.

[0027]

[0044] Membrane stains do not include antibodies that stain targets in compartments other than the plasma membrane. Membrane stains may bind to membranes of more cell types than any individual antibody in the membrane stain. Multiple antibodies may be mixed.

[0028]

[0045] The kit may further include nuclear and / or cytosolic stains, for example, to better identify cells individually and guide cell segmentation along the membrane. The kit may further include a panel of antibodies to cell surface targets, the antibodies of the panel conjugated to different mass tags, and be useful for identifying the cell populations described herein (e.g., by automating the sorting of the populations).

[0029]

[0046] In certain embodiments, membrane stains can be applied before, beside, or after other antibody panels. Cell segmentation can be performed on images obtained by imaging mass cytometry. Segmentation techniques are known in the art and are described, for example, by Wang et al. in "Cell Segmentation for Image Cytometry: Advances, Insufficiencies, and Challenges," Cytometry. Part A (2019): 708-711. In general, cell segmentation benefits from clear labeling of the cell membrane and the cell interior (e.g., the cell nucleus, visualized by a nuclear stain).

[0030]

[0047] Protein localization information is centralized in the Human Protein Atlas, which can be searched to identify protein targets that specifically localize to the plasma membrane (e.g., cell junctions) across various tissues and cell types. Thus, a complementary set of protein targets that identify cell membranes in and across multiple tissues can be identified, and proteins for those targets can be tagged with the same element tag to provide the membrane stains of the present application. The membrane stain may include one, two, three, four, or more antibodies that specifically bind to syntaxin 4, solute carrier family 16 member 1, erythrocyte membrane protein band 4.1-like 3, adaptor-associated protein complex 2 mu 1 subunit, G protein subunit β2, moesin, EZR, CTNNB1, ATPase Na+ / K+ transport subunit β3, phosphatidylethanolamine-binding protein 1, catenin β1, solute carrier family 1 member 5, ezrin, S100 calcium-binding protein A4, and ankyrin 3, which have been reported as membrane proteins that are conserved and well-expressed across various cell lines. Alternatively or additionally, the membrane stain may include one, two, three, four, or more antibodies that specifically bind to CDH17, CTNNA1, DNAJC18, GJB6, TJP3, and C4of19, which have been reported as cell junction proteins that are conserved and well-expressed across various cell lines and / or tissues.

[0031]

[0048] To detect RNA, cells in a biological sample described herein may be prepared for analysis of RNA and protein content using the methods and devices described herein. In certain embodiments, cells are fixed and permeabilized before the hybridization step. The cells may be provided as fixed and / or permeabilized. Cells may be fixed with a cross-linking fixative, such as formaldehyde or glutaraldehyde. Alternatively, or additionally, cells may be fixed using a precipitating fixative, such as ethanol, methanol, or acetone. Cells may be permeabilized with a detergent, such as polyethylene glycol (e.g., Triton X-100), polyoxyethylene (20) sorbitan monolaurate (Tween-20), saponin (a group of amphiphilic glycosides), or chemicals, such as methanol or acetone. In certain cases, fixation and permeabilization may be performed using the same reagent or set of reagents. Fixation and permeabilization techniques are described by Jamur et al. in "Permeabilization of Cell Membranes" (Methods Mol. Biol., 2010).

[0032]

[0049] Biological samples can include any sample of biological nature requiring analysis. For example, samples can include biomolecules, tissues, body fluids, and cells of animal, plant, fungal, or bacterial origin. They can also include molecules of viral origin. Typical samples include, but are not limited to, sputum, blood, blood cells (e.g., white blood cells), tissue or fine needle biopsy samples, urine, peritoneal fluid, and pleural effusion, or cells therefrom. Biological samples can also include tissue sections, such as frozen sections taken for histological purposes. Another typical source of biological samples is virus and cell cultures of animals, plants, bacteria, and fungi, where gene expression states can be manipulated to explore relationships between genes. In some cases, other samples, such as artificial samples, can be investigated. Certain aspects of the present disclosure are particularly useful when investigating samples of human origin, especially when investigating samples of human peripheral blood.

[0033]

[0050] To facilitate the determination of useful information during elemental analysis, samples can be tagged or labeled with elemental tags. An elemental tag is a detectable isotope (e.g., an element or an isotope of an element) that can be detected by elemental analysis. In some cases, an elemental tag can contain only the detectable isotope itself, but this is not necessarily the case. In some cases, an elemental tag can contain a substrate to which one or more isotopes are attached or which contains one or more isotopes. In this manner, to create an elementally tagged moiety, a detectable isotope can be attached to the substrate separately (e.g., before, simultaneously with, or after) from the substrate being attached to the moiety. For example, an elemental tag can include a polymer chain containing multiple pendant groups (e.g., chelating groups containing a metal) containing detectable isotopes, and the polymer chain can be attached to a moiety corresponding to the elemental tag of the moiety. In another example, an elemental tag can include a bead or nanoparticle that can contain one or more detectable isotopes within or on its surface, and the bead or nanoparticle can be attached to a moiety corresponding to the elemental tag of the moiety. As used herein, an isotope, or composition of isotopes, located on the surface of a bead or nanoparticle includes an isotope, or composition of isotopes, captured by a polymer on the surface or bound to the surface (e.g., covalently or otherwise). In some cases, the bead or nanoparticle can include a solid metal core, optionally encapsulated by a silicon oxide shell, or a polymer core that captures and / or chelates the metal, and a polymer surface (e.g., using poly-L-lysine, PEG (polyethylene glycol), PEG MEA (methyl ether acrylate), PVMS (polyvinylmethylsiloxane), polydopamine, polystyrene, and / or other suitable polymers). In certain embodiments, the polymer (e.g., and / or its monomer precursor) can be hydrophobic and can include, for example, acrylic, amide and imide, carbonate, diene, ester, ether, fluorocarbon olefin, and / or aromatic groups, e.g., phenolic groups such as catechol.The polymer may also provide reactive groups, such as amine and / or thiol reactive groups. For example, the reactive group may be a reactive functional group (e.g., a thiol group, an amine group, a thiol-reactive group, an amine-reactive group, or a click chemistry functional group) such as a quinone that forms upon polymerization. The polymer may form a thin film, such as a monolayer film.

[0034]

[0051] The beads of the present application can be element-encoded particles suitable for attachment of biomolecules, enabling massively multiplexed biological analysis. For example, polymer beads can be made according to one or more aspects of U.S. Patent Publication No. 20100144056, which is incorporated herein by reference and summarized below.

[0035]

[0052] One approach involves synthesizing polymer particles containing embedded metal ions or atoms. The polymer matrix of the particles can help encapsulate the metal ions while providing colloidal stability in aqueous media. The polymer matrix minimizes direct contact between the metal ions and the aqueous phase, and functional groups on the particle surface are available for attaching antibodies or other biomolecules to the particles. These functional groups can also be used to attach linker arms or spacer groups to which biomolecules can be attached. The desired polymer particles have diameters ranging from about a few nanometers to about 20 μm, with the largest desired polymer particles having diameters of about 50 nm to about 500 nm.

[0036]

[0053] Polymer particles may be synthesized that contain metal ions or atoms embedded therein. The polymer matrix of the particles may serve to encapsulate the metal ions while providing colloidal stability in aqueous media. In certain embodiments, chelated lanthanide (or other metal) ions may be used.

[0037]

[0054] The polymer matrix of the particles minimizes direct contact between the metal ions and the aqueous phase, and functional groups on the particle surface can be used to attach antibodies or other biomolecules to the particles. These functional groups can also be used to attach linker arms or spacer groups to which biomolecules can be attached. Alternatively, as described herein, polymer films of different structures can be synthesized on the surface of the beads to allow for the attachment of biomolecules. In certain embodiments, the beads are assay barcoded beads described herein. The interior of the beads can contain assay barcodes, such as distinguishable combinations of metal isotopes. The interior of the beads can be any of a variety of suitable structures, such as a solid metal core, a metal-chelating polymer interior, a nanocomposite interior, or a hybrid interior. A solid metal core can be formed by subjecting a mixture (e.g., a solution) of one or more metal elements and / or isotopes to high heat and / or pressure. A nanocomposite structure can include a combination (e.g., a matrix) of nanoparticles / nanostructures (e.g., each with different physical properties and contributing one or more assay barcode elements / isotopes and / or providing a scaffold for other nanoparticles containing assay barcode elements / isotopes). The interior of the beads can include a polymer that captures the assay barcode metal and / or chelates the assay barcode metal (e.g., via pendant groups such as DOTA, DTPA, or derivatives thereof). Suitable polymer backbones can be branched (e.g., hyperbranched) or form a matrix. In some embodiments, the polymer can be formed in an emulsion. The polymer may comprise styrene, acrylate, and any derivatives thereof, or other polymer subunits known in the art. In certain embodiments, the interior of the assay bead may present an inert surface (e.g., a solid metal surface) that must be functionalized (e.g., by over-surface polymerization) before attachment to an assay biomolecule (e.g., an oligonucleotide or antibody). Element tags are provided for mass cytometry for conjugation or preconjugation to affinity reagents, such as biomolecules, e.g., antibodies (e.g., antibodies or derivatives thereof, e.g., antibody Fab fragments) that specifically bind to target analytes. Element tags for conjugation may comprise a polymer preloaded with a metal, or a polymer with a separate metal solution for loading onto the polymer (e.g., prior to conjugation to a biomolecule). The polymer may comprise a backbone and multiple metal-binding pendant groups, such as pendant groups containing a chelator (e.g., DTPA, DOTA, or their derivatives).Elemental tags conjugated to biomolecules, such as antibodies, may be preloaded with a metal. In certain embodiments, the metal is an enriched isotope, such as an isotope of a lanthanide. Lanthanides are chemically similar, and lanthanide elemental tags (including those conjugated to antibodies) have been found to be stable in solution to a degree suitable for mass cytometry. However, non-lanthanide metals may not have the same stability. In certain embodiments, the metal element or its isotope can be outside the lanthanide family, such as a non-lanthanide transition metal or a post-transition metal. Post-transition metals suitable for mass cytometry include elements with atomic numbers 48-50 (e.g., cadmium, indium, and / or tin) and 80-84. The stability of post-transition metal-loaded polymers can be improved by lyophilization. The subject methods and kits include lyophilized non-lanthanide elemental tags described herein, provided separately from other elemental tags or admixed with other element-tagged biomolecules. Medium-weight elements (e.g., cadmium, cadmium-1, cadmium-2, cadmium-3, cadmium-4, cadmium-5, cadmium-6, cadmium-7, cadmium-8, cadmium-9, cadmium-10, cadmium-11, cadmium-12, cadmium-13, cadmium-14, cadmium-15, cadmium-16, cadmium-17, cadmium-18, cadmium-19, cadmium-20, cadmium-21, cadmium-22, cadmium-23, cadmium-24, cadmium-25, cadmium-26, cadmium-27, cadmium-28, cadmium-29 ...

[0038]

[0055] The surface of the assay beads may include polymers, linkers to separate the assay biomolecules from the surface, and / or linkers to add colloidal stability (e.g., PEG linkers), functional groups for attaching (or having attached to) the assay biomolecules and / or sample barcodes.

[0039]

[0056] Elemental tags can be made using many different isotopes or unique combinations of isotopes. A unique elemental tag can have a distinct (e.g., distinguishable by elemental analysis) element, isotope, or combination of isotopes. For example, in a set of elemental tags, an elemental tag can be unique if it is distinguishable by mass from other elemental tags in the set (e.g., distinguishable by its composition of one or more isotopes). For clarity, while elemental tags or unique elemental tags are referenced throughout, it will be understood that certain aspects of the present disclosure utilize multiple copies of unique elemental tags. An elemental tag can be one or more isotopes of a single element (e.g., 142 Nd and 143 Nd), or one or more isotopes across multiple elements (e.g., 142 Nd and 141 Pr). In certain embodiments, the elemental tag may comprise a metal atom having an atomic mass greater than 80 amu. Such metals may be selected from noble metals, lanthanides, transition metals, and / or post-transition metals. The elemental tag may comprise a polymer. For example, the polymer may chelate the metal (e.g., lanthanide) to a metal-binding pendant group (such as DOTA or DTPA) on the polymer backbone, may incorporate the metal (e.g., tellurium) into the carbon backbone of the polymer itself, or may entrap the metal in its environment. The elemental tag may comprise a metal nanoparticle, such as a metal nanocrystal (e.g., functionalized on its surface to bind an affinity reagent). The metal of the elemental tag may be isotopically pure. The elemental tag may bind to or be functionalized to bind to a biomolecule (e.g., an affinity reagent such as an antibody).

[0040]

[0057] Each elemental tag can contain one or more distinguishable isotopes that can be detected by elemental analysis to determine the presence of the elemental tag. Because different elemental tags can have unique isotopes or combinations of isotopes, elemental analysis can be used to identify elemental tags based on their unique isotopic characteristics. The distinguishable isotopes can be selected to facilitate detection using elemental analysis. For example, the distinguishable isotopes can be selected to be isotopes outside the expected range of intrinsic isotopes for the sample or isotopes introduced for a particular elemental analysis technique. For example, biological samples generally contain essentially no metals greater than 80 amu, and therefore, elemental tags for use with such samples may contain metals greater than 80 amu. In another example, because certain types of inductively coupled plasma mass spectrometry (ICP-MS) utilize argon, elemental tags for analysis by ICP-MS may utilize elements with masses greater than argon, such as metals greater than 80 amu. Filtering techniques can be used to exclude elements outside of those selected as identifiable elements in the element tag being used, thereby automatically excluding elements endogenous to the sample and any elements introduced during elemental analysis. For example, in ICP-MS, a quadrupole filter can be applied to exclude ions with masses less than 80 amu, thereby preventing the detector from being overwhelmed by ions of no interest (e.g., ions unlikely to be detectable isotopes).

[0041]

[0058] When a combination of isotopes for a given elemental tag is mentioned, that combination may be present on each instance of the elemental tag or on various instances of the elemental tag that are mixed together.

[0042]

[0059] An element-tagging moiety is a chemical moiety that includes an element tag. While any suitable moiety can be used, certain aspects of the present disclosure utilize a moiety with a targeting function. The targeting function is the ability to bind to a target (e.g., a target protein or target molecule). As used herein, a reference to a moiety that binds to a target should be interpreted as a moiety that can bind to a target, regardless of whether actual binding has already occurred. The targeting function can occur through specific binding (e.g., as in the case of an antibody specific for a particular antigen), covalent binding, hybridization (e.g., of nucleic acids), or other suitable binding mechanisms. The metal-containing moiety can be an element-tagged moiety tagged with a metal. Examples of suitable metal-containing moieties include metal-containing small molecules or drugs (e.g., cisplatin), histochemical stains (e.g., ruthenium red, trichrome stain, or osmium tetroxide), metal-tagged oligonucleotides, and metal-tagged antibodies. In some cases, the element-tagging moiety can be a biomolecule, but this is not necessarily the case. The biomolecule can be any biomolecule, such as an affinity reagent (eg, an antibody, an aptamer), a nucleic acid (eg, that hybridizes to a target), a lectin, a sugar, or other such molecule.

[0043]

[0060] As used herein, the term affinity reagent can refer to a biomolecule (e.g., an antibody, an aptamer, a lectin, or a sequence-specific binding peptide) that is known to form a highly specific non-covalent bond with its respective target molecule (e.g., a peptide, an antigen, or a small molecule). Affinity reagents labeled with unique element tags are affinity reagents labeled with element tags that are unique and distinguishable from multiple other element tags in the same sample. When an affinity reagent is conjugated to an element tag, it can be considered an element-tagged moiety. Although some embodiments and examples herein recite antibodies, in any such embodiment, an affinity reagent (e.g., an antibody affinity reagent or a non-antibody affinity reagent) can be used instead of an antibody.

[0044]

[0061] The elemental tagging moiety can include or be bound to an elemental tag. In some cases, the elemental tagging moiety can include an elemental tag, for example, in the case of cisplatin, which contains platinum as the elemental tag. In some cases, the elemental tagging moiety can be bound to an elemental tag, such as an antibody bound to an elemental tag comprising a polymer scaffold having metal chelating groups to which distinguishable isotopes (e.g., metals) are bound. By using a polymer scaffold, multiple isotopes can be bound to a single elemental tagging moiety. Thus, a polymer scaffold can be used to provide an even stronger elemental signal by including more copies of distinguishable isotopes. In some cases, a polymer scaffold can be used to facilitate the provision of distinguishable combinations of isotopes by having different isotopes contained at different locations along the polymer scaffold. Such a polymer scaffold can include several metal chelating ligands attached to multiple subunits of the polymer. The number of metal chelating groups that can be bound to at least one metal atom in the polymer can be about 1 to 10,000, e.g., 5 to 100, 10 to 250, 250 to 5,000, 500 to 2,500, or 500 to 1,000. At least one metal atom can be bound to at least one of the metal chelating groups. The polymer can have a degree of polymerization of about 1 to 10,000, e.g., 5 to 100, 10 to 250, 250 to 5,000, 500 to 2,500, or 500 to 1,000. Thus, a polymer-based element tag can contain about 1 to 10,000, e.g., 5 to 100, 10 to 250, 250 to 5,000, 500 to 2,500, or 500 to 1,000 labeled atoms.

[0045]

[0062] In some cases, the element tag can be a polymer selected from the group consisting of linear polymers, copolymers, branched polymers, graft copolymers, block polymers, star polymers, and hyperbranched polymers. The polymer backbone can be derived from substituted polyacrylamides, polymethacrylates, or polymethacrylamides, and can be substituted derivatives of acrylamides, methacrylamides, acrylate esters, methacrylate esters, homopolymers, or copolymers of acrylic acid or methacrylic acid. The polymer can be synthesized using reversible addition-fragmentation polymerization (RAFT), atom transfer radical polymerization (ATRP), and anionic polymerization. The step of providing the polymer can include synthesizing the polymer from a compound selected from the group consisting of N-alkylacrylamides, N,N-dialkylacrylamides, N-arylacrylamides, N-alkylmethacrylamides, N,N-dialkylmethacrylamides, N-arylmethacrylamides, methacrylate esters, acrylate esters, and functional equivalents thereof. The polymer can be water-soluble. This moiety is not limited by its chemical content. However, analysis is simplified if the backbone has a relatively reproducible size (e.g., length, number of tag atoms, reproducible dendrimer characteristics, etc.). Stability, solubility, and non-toxicity are also important considerations. Therefore, the preparation and characterization of functionalized water-soluble polymers involves synthetic strategies that place multiple functional groups along the backbone, as well as different reactive groups (linking groups) that can be used to attach the polymer to molecules (e.g., affinity reagents) via linkers and, optionally, spacers. The size of the polymer can be controlled by controlling the polymerization reaction. Typically, the size of the polymer is selected to minimize the gyration of the polymer, e.g., between 2 and 11 nanometers. The length of the moiety attached to the elemental tag can be approximately 10 nanometers; therefore, a polymer tag that is excessively large relative to the size of the moiety to which it is attached may sterically interfere with the targeting function of that moiety.

[0046]

[0063] In some cases, the metal chelating group capable of binding to at least one metal atom can contain at least four acetate groups. For example, the metal chelating group can be a diethylenetriaminepentaacetic acid (DTPA) group; a 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) group; or a DTPA or DOTA derivative group. Alternative groups include ethylenediaminetetraacetic acid (EDTA) and ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA). The metal chelating group can be attached to the polymer scaffold via an ester or amide. Examples of suitable metal chelating polymers include X8 and DM3 polymers available from Fluidigm Canada, Inc.

[0047]

[0064] The elemental tagging moiety can be labeled with an elemental tag that can be identified by elemental analysis.The elemental tagging moiety can be selected to bind or link to any suitable structure (e.g., target) that is desired to be labeled with an elemental tag, and then be detected by elemental analysis.For example, suitable elemental tagging moieties can include oligonucleotide probes for hybridization, cell state probes (e.g., cisplatin as a survival marker, or organotellurium as a hypoxia or synthesis marker), or other suitable molecules.

[0048]

[0065] In certain aspects of the present disclosure, element-tagged antibodies are used as element tagging moieties. The antibodies may be specific to a particular cell type. A cell type (e.g., a cell population) may refer to a population of cells or a specific subset of a cell population. A cell type-specific antibody may have an antigen-binding site (e.g., a paratope) that can bind to a molecule or antigen (e.g., an epitope of an antigen) present in or on that particular cell type. In some cases, the antibody may be specific to a target on or within peripheral blood, such as a surface target or intracellular target of peripheral blood mononuclear cells (PBMCs).

[0049]

[0066] Certain aspects of the present disclosure describe components as being mixed or blended. As used herein, the terms mixture or blend include components that are combined together (e.g., physically placed together in the same container or enclosure). A container or enclosure includes any suitable volume of space that can be separated from another volume of space. A container or enclosure can be sealed, e.g., hermetically sealed. Examples of suitable containers or enclosures include tubes (e.g., test tubes), boxes, pouches, wells of a multi-well plate, or other such containers.

[0050]

[0067] In some cases, various aspects of the present disclosure may utilize a calibration material to facilitate calibration of an elemental analyzer. The calibration material may be any suitable metal or metal-containing material that can be used to provide calibration for an elemental analyzer. In some cases, the calibration material may be metal-containing beads having a mixture of different isotopes from different elements across a range of elemental masses. The calibration material may contain known amounts of one or more known isotopes.

[0051]

[0068] Certain aspects of the present disclosure utilize element-tagged antibodies to enable highly multiplexed single-cell analysis of biological samples using elemental analysis, such as mass cytometry. Such techniques can enable the resolution of multiple markers within a single panel without the need for compensation. In some cases, more than 30 markers can be resolved within a single panel.

[0052]

[0069] The large number of detection channels (e.g., greater than 20 and less than 60) enabled by mass cytometry offers the opportunity to utilize combinatorial assays. For example, the number of targets detected is typically equal to the number of channels, but this can be increased by linking antibody panels to each other through a shared subset of biomolecules (e.g., antibodies) and using the same mass tag for different antibodies across the panel. For example, the number of targets detected in a given sample (e.g., data set) of cells can be at least 1.2, 1.5, or 2 times the number of detection channels used.

[0053]

[0070] If mass tag channels are reserved for separate panels for individual aliquots, cells may be stained with a shared panel so that cell populations identified within the separate panels can be related to each other. Because a panel uses several mass tags for different antibodies (to detect different targets), a sample barcode can be applied to the cells in the aliquot as a "panel barcode" that identifies which mass tag is associated with which antibody. Alternatively, a "master" shared panel can be used (and may be necessary) to stain a section (aliquot) of cells in a sample and to relate populations identified by other panels to each other. The master shared panel can have different or only partially overlapping subsets of antibodies shared with each of multiple separate panels, each of which stains a different aliquot of cells. There can be two or more, three or more, four or more, five or more, or even ten or more separate panels that share antibodies with the shared panel. A separate panel can have more mass tags in common with another separate panel than it has common antibodies.

[0054]

[0071] The workflow involves dividing a single sample (e.g., human PBMCs) into separate aliquots and staining each aliquot with a different cell-type-specific panel. The same subset of mass channels is used for different cell-type-specific markers depending on the panel (e.g., a T-cell panel may not have any or some of the B-cell markers in a B-cell panel, and vice versa). Amazon-type software solutions can identify cells stained by different panels and combine them into the same dataset.

[0055]

[0072] Barcoding cells by panel can allow software to automatically identify which panel each cell was stained with. Aspects of the present application include such software, including software that also performs other automated analyses described herein.

[0056]

[0073] A shared subset of markers that identifies every basic cell type can be a panel in its own right, or a subset of the total panel, which can be used to identify the relative abundance of each of the major cell types, and then used in combination with cell type-specific panels to identify the relative abundance of specific cell types (populations) across the panel.

[0057]

[0074] Software solutions may reject cellular events that are not the cell type focused within a panel, resulting in each cellular event staining for the target of interest. For example, any B cell events within a T cell panel may be removed. This can be done after calculating the relative abundance of the major cell types, as described in the bullet points above. One or more separate panels may contain investigational markers, some or all of which may be shared between panels. There may also be one or more unused channels for users to detect their own targets of interest.

[0058]

[0075] In certain embodiments, the elemental analysis method includes separating cells of a sample into multiple compartments; staining the cells with a shared panel of mass-tagged antibodies; staining the cells of separate compartments with separate panels of mass-tagged biomolecules, where each separate panel contains biomolecules tagged with a mass tag that is present in the other separate panels but not in the other separate panels; and / or using elemental analysis to interrogate the sample to detect the presence of separate mass tags on individual cells. The shared panel may be stored across two or more compartments, for example, three or more, four or more, or five or more compartments. The shared panel may detect positively expressed surface targets that distinguish between parent populations, which together comprise the majority of immune cells. One or more (e.g., two or more, three or more, or four or more) individual separate panels each detect positive markers that distinguish between subpopulations within one of the parent populations, but do not distinguish between subpopulations for the majority of immune cells.

[0059]

[0076] A shared panel may not stain all compartments (eg, it may stain only one), and the shared panel contains a different subset of the same antibodies as the antibodies of two or more separate panels.

[0060]

[0077] Each distinct panel may be a subpopulation within a parent population identified by a shared panel, where the parent population is selected from a population comprising or consisting of T cells, CD4 T cells, regulatory T cells, CD8 T cells, NK cells, B cells, dendritic cells, and / or monocytes / macrophages. Alternatively or additionally, a distinct panel may identify a cellular function, for example, if the distinct panel detects targets involved in intracellular signaling, cytokine production, and / or the cell cycle.

[0061]

[0078] In certain embodiments, oligonucleotides are used in addition to or in place of antibodies in a separate panel, e.g., where the oligonucleotide specifically hybridizes, directly or indirectly, to a target RNA. For example, an oligonucleotide may hybridize to an RNA encoding a cytokine as described herein.

[0062]

[0079] Here, staining with separate panels involves staining separate compartments with oligonucleotide-tagged antibodies and signal amplification by hybridizing mass-tagged oligonucleotides directly or indirectly to the oligonucleotides tagging the antibodies.

[0063]

[0080] As described for the panels herein, one or more of the shared panel and the separate panels may be freeze-dried.

[0064]

[0081] The cells may be stained with a shared panel before being separated into compartments, or the cells may be compartmentalized before staining with the shared panel (e.g., the shared panel is mixed with each individual panel). The method may further include labeling the compartmented cells (and thus the targets associated with the panel's mass tags) with a panel barcode that identifies the individual panel, e.g., the panel barcode is provided mixed with the individual panel before being added to the compartmented cells. The compartments are combined after labeling with the panel barcode and before interrogation. The panel barcode may be applied in a manner similar to the sample barcode shown in FIG. 13, or may be administered in addition to the sample barcode.

[0065]

[0082] The above method can be performed on one or more additional samples (themselves divided into aliquots for separate staining), labeling the samples with distinct sample barcodes, and combining the barcoded samples prior to interrogation. The barcoded samples can be combined prior to separation into compartments.

[0066]

[0083] The method may further comprise classifying each investigated cell into a cell population based on the shared panel and the separate panel. For example, the shared panel may identify a parent T cell population, while the separate T cell panel used in one of the compartments may identify a subpopulation within that parent population. Other populations and panels suitable for these methods are described in the present application.

[0067]

[0084] Classification can be by automated software trained on shared and separate panels (e.g., on similar samples, such as samples containing PBMCs). Classification can be by gating, by a trained clustering algorithm operating in a high-dimensional space (the dimensionality is related to the number of surface markers used for classification), or by a neural network.

[0068]

[0085] Separate panels may be used to identify subpopulations of the parent population identified by the shared panel.The method may further comprise: integrating the investigated cell populations identified based on different separate panels into the same data set based on the shared panel.Integrating may include: discarding the data of cells identified as belonging to the parent population for which the shared panel does not identify subpopulations; identifying the proportion of cells in the original sample that are in the subpopulations identified by the separate panels of separate compartments; and / or representing the expression of targets detected by different separate panels for the same parent population detected by the shared panel.

[0069]

[0086] The method may further comprise stimulating cells of different samples or sample compartments under different conditions prior to staining.

[0070]

[0087] The kit may include a shared panel and separate panels packaged for any of the above methods. The shared panel and separate panels may be packaged into kits as described for the above methods.

[0071]

[0088] In certain embodiments, a kit for elemental analysis (e.g., mass spectrometry) may include a shared panel containing multiple conjugated antibodies. The shared panel may include multiple antibodies conjugated to distinct mass tags, each distinct mass tag being distinguishable based on its isotopic composition. The multiple antibodies may be mixed. The multiple separate panels may include mass-tagged biomolecules (e.g., antibodies and / or oligonucleotides), each of which contains a biomolecule tagged with a mass tag that is present in one or more other panels but not in the other panels.

[0072]

[0089] Certain aspects of the present disclosure enable lyophilization of antibody panels to achieve panels with high stability. The stability of a lyophilized antibody panel can be described in terms of a comparison of the ion counts of elemental tag isotopes of a sample assayed using a lyophilized antibody panel with the ion counts of elemental tag isotopes of the same sample assayed using a non-lyophilized antibody panel. The increased stability of a lyophilized antibody panel over a non-lyophilized antibody panel can be seen as a higher ion count of elemental tags from the lyophilized antibody panel than from a non-lyophilized antibody panel. Furthermore, stability can be described as a function of damage to the antibody (e.g., damage affecting the binding activity of the antibody) and / or metal retention of distinguishable isotopic metal atoms on the elemental tag and / or element-tagged moiety. For example, a stable panel exhibits little or no damage affecting the binding activity of the antibody and / or little or no loss of metal atoms from the elemental tag and / or element-tagged moiety. The lyophilized panels described herein exhibit better stability than non-lyophilized panels of the same configuration.

[0073]

[0090] Element tags for mass cytometry are provided for conjugation or pre-conjugation to antibody biomolecules. The element tags for conjugation may include a polymer preloaded with a metal, or a polymer with a separate metal solution for loading onto the polymer (e.g., prior to conjugation to a biomolecule). The polymer may include a backbone and multiple metal-binding pendant groups, such as pendant groups comprising a chelator (e.g., DTPA, DOTA, or a derivative thereof). The element tag conjugated to a biomolecule such as an antibody may be preloaded with a metal. In certain embodiments, the metal is an enriched isotope, such as an isotope of a lanthanide. Lanthanides are chemically similar, and lanthanide element tags (including those conjugated to antibodies) have been found to be stable in solution to a degree suitable for mass cytometry. However, non-lanthanide metals may not have the same stability. In certain embodiments, the metal element or its isotope may be outside the lanthanide family, such as a non-lanthanide transition metal or a post-transition metal. Post-transition metals suitable for mass cytometry include elements with atomic numbers 48-50 (e.g., cadmium, indium, and / or tin) and 80-84. The stability of post-transition metal-loaded polymers can be improved by lyophilization. The subject methods and kits include lyophilized such non-lanthanide element tags described herein, provided separately from other element tags or admixed with other element-tagged biomolecules.

[0074]

[0091] Medium-weight elements (e.g., cadmium, cadmium-1, cadmium-2, cadmium-3, cadmium-4, cadmium-5, cadmium-6, cadmium-7, cadmium-8, cadmium-9, cadmium-10, cadmium-11, cadmium-12, cadmium-13, cadmium-14, cadmium-15, cadmium-16, cadmium-17, cadmium-18, cadmium-19, cadmium-20, cadmium-21, cadmium-22, cadmium-23, cadmium-24, cadmium-25, cadmium-26, cadmium-27, cadmium-28, cadmium-29 ...

[0075]

[0092] Generally, the generation and acquisition of large multiplexed panels can involve antibody titer optimization, thorough panel validation, sample preparation, and analysis. Preparing staining cocktails from numerous individual tubes (e.g., 30 different tubes) can be laborious and error-prone. Furthermore, pre-prepared staining cocktails, especially those based on metal-tagged antibodies, can be susceptible to damage over time and may be less stable. For example, while non-lyophilized metal-tagged antibody panels containing multiple antibodies may remain stable for approximately one week or less, certain embodiments of the present disclosure relate to similar lyophilized antibody panels that may remain stable for approximately one year or more. Thus, lyophilized antibody panels facilitate the production and distribution of larger antibody panels, as well as enable long-term storage of the antibody panel, improving uniformity in subsequent assays utilizing the same panel.

[0076]

[0093] Sample preparation according to the subject methods can be performed at least in part by an automated sample preparation system, which may process the sample prior to staining, contact the sample with one or more antibody panels, assay beads and / or barcodes, perform centrifugation and washing steps, and / or deliver the sample to a mass cytometry system.

[0077]

[0094] Thus, certain aspects of the present disclosure relate to a lyophilized, multiplexed (e.g., 30-plex) immunophenotyping panel housed in a single enclosure (e.g., a single tube) that can be used with efficient workflows and automated software solutions for human whole blood analysis using mass cytometry. In some cases, the panel can focus on the T cell lineage while also capturing other relevant immune populations. Blood can be added directly to lyophilized antibody tubes, followed by red blood cell (RBC) lysis, washing, and fixation steps, and finally, data acquisition of the stained sample using an elemental analyzer (e.g., an ICP-MS system).

[0078]

[0095] Software tools can accept elemental analyzer data and automatically generate reports on various cytometry information, such as the number of live cells, percentages of specific cell populations, staining intensity, histograms, 2D dot plots, and t-SNE (t-STEM) graphs. The software can automatically calculate the frequency of populations equivalent to manual gating. Automated software can eliminate user bias that can occur during manual analysis (e.g., manual gating) and significantly reduce the time required to analyze each data file. Panels and software tools can streamline whole blood immunophenotyping while enabling researchers to accurately and reproducibly monitor changes in immune cell subsets in patient samples. In some cases, software tools can automatically load appropriate elemental tags, enabling rapid decoding of elemental analyzer data into useful results.

[0079]

[0096] Antibodies and other element tagging moieties can be used to identify the presence of cellular targets in a sample. The antibody or element tagging moiety has a targeting function for the cellular target, allowing it to remain with the sample after the sample is washed. Therefore, any detection of a distinct isotope or combination of isotopes associated with a specific element tag on the antibody or element tagging moiety indicates the presence of the cellular target. The cellular target can be a surface target or an intracellular target. Examples of intracellular targets can include cell cycle and proliferation targets, intracellular signaling targets, and intracellular cytokine targets. Because cell permeabilization can destroy cell surface markers, staining of the sample with an antibody or element tagging moiety associated with a surface target can be performed before permeabilization. However, because cell permeabilization may be necessary for proper access to intracellular targets, staining of the sample with an antibody or element tagging moiety associated with an intracellular target can be performed after permeabilization.

[0080]

[0097] In some cases, an intercalator can be used. In some cases, the intercalator can be an element tagging moiety. The intercalator can contain or bind to the element of the element tag. For example, iridium can be used as an intercalator and simultaneously function as its own element tag. Thus, detection of iridium during the investigation indicates the presence of the intercalator. Other suitable intercalators include rhodium and cisplatin. The intercalator can be included in the lyophilized panel or provided separately from the lyophilized panel. If the intercalator is mixed with the sample cells before permeabilization, the intercalator can be a cell viability stain indicating whether the cells are viable. In such cases, the presence of the intercalator detected during the investigation indicates dead cells. However, if the intercalator is mixed with the sample cells after permeabilization, the intercalator should enter every permeabilized cell, and therefore the intercalator can function as a cell identification or presence stain that can indicate the presence of cells. In such cases, the presence of the intercalator detected during the investigation indicates that the cells are being analyzed by the elemental analyzer. Optionally, an intercalator such as rhodium may be included with or mixed with the lyophilized antibody panel.

[0081]

[0098] The antibodies selected for use in the lyophilized panel can be selected to perform a particular assay. In some cases, lyophilized panels or lyophilized panel subsets can be generated for various assays, thereby facilitating the desired assay by simply selecting the appropriate panel or panel subset combination. Various panels or panel subsets are described herein for use with certain aspects of the present disclosure. Each panel or panel subset described with reference to a list of possible antibodies can include two or more antibodies from that list, or any number of antibodies from that list, up to all antibodies from that list. As used herein, a panel subset can include any combination of antibodies from the panels described herein. In some cases, a panel subset can be combined with a different panel or panel subset to generate one or more of the panels described herein.

[0082]

[0099] In some cases, the lyophilized panel can include two or more antibodies from the following list: Cd45, CD45RA, CD45RO, Cd123, CD4, CD8a, CD11C, CD57, CXCR3, CD185, CD38, CD56, CD3, CD20, CD66b, HLA-DR, IgD, CD27, CD28, CD127, CD19, CD16, CD161, CD194, CD25, CD294, CD197, CD14, CCR6, and TCR delta gamma. In some cases, the lyophilized panel can include at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 antibodies from this list. As disclosed herein, certain aspects of the present disclosure are useful for incorporating a variety of different element-tagged antibodies into a single lyophilized panel. In some cases, this panel may be particularly useful for immunophenotyping human peripheral blood. In some cases, this panel may be a shared panel in a kit or method that includes separate panels for labeling cellular compartments (e.g., aliquots) from the same sample, as further described herein. Such separate panels may include one or more of the following panels:

[0083]

[0100] In some cases, the lyophilized panel may include a leukemia panel and / or lymphoma panel that includes antibodies that are particularly suited to determining cytometric information about leukemia and / or lymphoma cells.

[0084]

[0101] Various exemplary lyophilized panels are described herein.For the purpose of explanation, the panel may be described in terms of antibody and target, with the target being indicated in parentheses.In some cases, various antibodies can be substituted for any given antibody, as long as they are specific to the target of the substituted antibody.In certain embodiments, multiple lyophilized panels can be provided (e.g., in a kit) so that they can be combined according to sample and / or application.

[0085]

[0102] In some cases, a lyophilized panel for human acute myeloid leukemia (AML) phenotyping can include antibodies (and targets) from a list including HIB19 (CD19), 104D2 (CD117), ICRF44 (CD11b), 10.1 (CD64), CD7-6B7 (CD7), 6H6 (CD123), HI30 (CD45), WM53 (CD33), clone (target), W6D3 (CD15), 581 (CD34), UCHT1 (CD3), IM7 (CD44), HIT2 (CD38), L243 (HLA-DR), and 12G5 (CXCR4). In some cases, this panel can be particularly useful for phenotyping AML. AML is the most common type of acute leukemia in adults and is a malignant tumor that arises in the bone marrow due to the destruction of normal hematopoiesis. AML arises from chromosomal rearrangements and the acquisition of multiple genetic mutations within precursors of the myeloid, erythroid, megakaryocytic, and monocytic cell lineages. The immunophenotype of AML is highly heterogeneous. Markers frequently expressed by AML include CD15, CD33, CD34, and CD64.

[0086]

[0103] In some cases, a lyophilized panel for human B cell phenotyping can include antibodies (and targets) from a list including HIB19 (CD19), 1A6-2 (IgD), 2H7 (CD20), polyclonal (IgA), BL13 (CD21), L128 (CD27), HIB22 (CD22), CB3-1 (CD79b), HIT2 (CD38), ML5 (CD24), MHM-88 (IgM), and L243 (HLA-DR). In some cases, this panel can facilitate the identification and phenotyping of human B cells, including naive, memory, transitional, and plasma B cell populations.

[0087]

[0104] In some cases, a lyophilized panel for identifying cell cycle information can include antibodies (and targets) from a list including N / A (S phase (IdU)), J112-906 (pRb [Ser807 / 811]), GNS-1 (cyclin B1), B56 (Ki67), and HTA28 (pHistone H3 [Ser28]). In some cases, this panel can facilitate assessment of cell cycle state, i.e., proliferation, G0 (senescence), G1, S phase, G2, and M phase (mitosis). This panel can be particularly useful in combination with other panels to identify cell cycle state and other cytometric information.

[0088]

[0105] In some cases, a lyophilized panel for human T helper cell phenotyping can include antibodies (and targets) from a list including G034E3 (CCR6), NP-6G4 (CCR5), RPA-T8 (CD8), C398.4A (ICOS), HI100 (CD45RA), UCHT1 (CD38), G025H7 (CXCR3), 205410 (CCR4), GP-3G10 (CD161), UCHL1 (CD45RO), 2A3 (CD25), RF8B2 (CXCR5), SK3 (CD4), EH12.2H7 (PD-1), and A019D5 (CD127). In some cases, the panel can include antibodies (and targets) from a list including T helper 1 (T H 1), T H 2. T H 17, T H 22, follicular T helper (T FH ) and T controllability (T REG It can be used to identify and phenotype human CD4+ helper T cell subsets, including CD4+ T cells (including CD4+ T cells). Differentiation of CD4+ T cells into functionally distinct helper T subsets may be important for normal immune regulation. These subsets are specified by exogenous and endogenous cues, and the resulting cell populations acquire stable phenotypes defined by the expression of signature cytokines, "master regulator" transcription factors, and characteristic cell surface phenotypes.

[0089]

[0106] In some cases, a lyophilized panel for basic human peripheral blood phenotyping can include antibodies (and targets) from a list including 2H7 (CD20), HI30 (CD45), M5E2 (CD14), 3G8 (CD16), SK1 (CD8), UCHT1 (CD3), and SK3 (CD4). This panel can be useful for assaying fresh or frozen human whole blood or PBMCs. This panel can be used to identify CD4+ T, CD8+ T, B cells, monocytes, NK cells, and granulocytes.

[0090]

[0107] In some cases, an additional lyophilized panel for basic human peripheral blood phenotyping can include antibodies (and targets) from a list including RPA-T4 (CD4), RPA-T8 (CD8a), 2H7 (CD20), 3G8 (CD16), HI30 (CD45), M5E2 (CD14), and UCHT1 (CD3). This panel can be useful for assaying fresh or frozen human whole blood or PBMCs. This panel can be used to identify CD4+ T, CD8+ T, B cells, monocytes, NK cells, and granulocytes.

[0091]

[0108] In some cases, a lyophilized panel for human peripheral blood phenotyping can include antibodies (and targets) from a list including UCHT1 (CD3), RPA-T4 (CD4), RPA-T8 (CD8a), Bu15 (CD11c), M5E2 (CD14), 3G8 (CD16), HIB19 (CD19), 2H7 (CD20), O323 (CD27), HIT2 (CD38), HI30 (CD45), HI100 (CD45RA), VI-PL2 (CD61), CD66a-B1.1 (CD66), 6H6 (CD123), HIR2 (CD235a / b), and L243 (HLA-DR). This panel can be useful for assaying fresh or frozen human whole blood or PBMCs. This panel can be useful for identifying major peripheral blood cell subsets including granulocytes, basophils, plasmacytoid dendritic cells, natural killer cells, effector T killer cells, naive T killer cells, activated T killer cells, memory T killer cells, effector T helper cells, naive T helper cells, activated T helper cells, memory T helper cells, memory B cells, naive B cells, plasma B cells, myeloid dendritic cells, non-classical monocytes, classical monocytes, and platelets.

[0092]

[0109] In some cases, a lyophilized panel for human T cell phenotyping can include antibodies (and targets) from a list including HI111 (CD11a), RPA-T4 (CD4), RPA-T8 (CD8a), 3G8 (CD16), 2A3 (CD25), HI30 (CD45), G043H7 (CCR7), FN50 (CD69), UCHL1 (CD45RO), BJ18 (CD44), O323 (CD27), HI100 (CD45RA), UCHT1 (CD3), HCD57 (CD57), L243 (HLA-DR), and A019D5 (CD127). This panel can be useful for assaying fresh or frozen human whole blood or PBMCs. This panel is useful for identifying major T cell subsets, including naive, central memory, effector and effector memory CD4+ and CD8+ cells, and can classify the activation and homing status of these subtypes.

[0093]

[0110] In some cases, a lyophilized panel for expanded human T cell phenotyping can include antibodies (and targets) from a list including TS1 / 8 (CD2), UCHT2 (CD5), CD7-6B7 (CD7), SN4 C3-3A2 (CD9), CD28.2 (CD28), 9F10 (CD49d), HP-3G10 (CD161), 205410 (CCR4), NP-6G4 (CCR5), and G025H7 (CXCR3). This panel can be useful for assaying fresh or frozen human whole blood or PBMCs. This panel may be particularly useful when combined with a human T cell phenotyping classification panel, where the combined panel can be used to identify all major T cell subsets, including naive, central memory, effector and effector memory CD4+ and CD8+ cells, naive and memory Tregs, TH1 and TH2 cells, and classify the activation and homing status of these subtypes.

[0094]

[0111] In some cases, a lyophilized panel for phenotyping human embryonic stem (ES) cells or induced pluripotent stem (iPS) cells can include antibodies (and targets) from a list including TRA-1-60 (TRA-1-60), O3O-678 (Sox2), 40 / Oct-3 (Oct-3 / 4), N31-355 (Nanog), IM7 (CD44), and 9E10 (c-Myc).

[0095]

[0112] In some cases, a lyophilized panel for phenotyping human hematopoietic stem and progenitor cells can include antibodies (and targets) from a list including HI10a (CD10), WM15 (CD13), 581 (CD34), G0H3 (CD49f), 104D2 (CD117), DL-101 (CD138), and 12G5 (CXCR4). This panel can be useful for identifying and phenotyping hematopoietic progenitor cell populations, including hematopoietic stem cells (HSCs), within human bone marrow and umbilical cord blood. This panel can be usefully combined with a human peripheral blood phenotyping panel, allowing lineage-positive cells to be excluded from the gating strategy.

[0096]

[0113] In some cases, a lyophilized panel for human intracellular cytokine I assays can include antibodies (and targets) from a list including B27 (IFNγ), MQ1-17H12 (IL-2), MP4-25D2 (IL-4), TRFK5 (IL-5), MQ2-13A5 (IL-6), N49-653 (IL-17A), SHLR17 (IL-17F), B (granzyme B), B-D48 (perforin), D21-1351 (MIP1β), and Mab11 (TNFα). This panel can be useful for assaying fresh or frozen human whole blood, PBMCs, or cell lines. This panel can facilitate measurement of 11 major cytokines as well as the cytolytic proteins granzyme B and perforin. This panel can be combined with a human peripheral blood phenotyping panel to enable comprehensive immunophenotyping of cytokine-expressing cells.

[0097]

[0114] In some cases, a lyophilized panel for human regulatory T cell phenotyping can include antibodies (and targets) from a list including 9F10 (CD49D), RPA-T4 (CD4), 205410 (CCR4), HI100 (CD45RA), UCHT1 (CD3), A1 (CD39), PCH101 (Foxp3), DX2 (CD95), UCHL1 (CD45RO), 2A3 (CD25), 14D3 (CD152), L243 (HLA-DR), and A019D5 (CD127). This panel can be useful for identifying regulatory T cells. Regulatory T cells (Tregs) are a suppressive subset of CD4+ T helper (Th) cells that are important for regulating immune responses. Tregs are defined by expression of the transcription factor Foxp3. Additional Treg markers include constitutive expression of the high-affinity IL-2 Rα chain (CD25) and cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4), along with low expression of the IL-7 Rα chain (CD127).CD4+CD25+Foxp3+ Tregs can be divided into two main types: thymus-derived Tregs (tTregs) and peripherally-derived Tregs (pTregs).

[0098]

[0115] In some cases, a lyophilized panel for human monocyte / macrophage phenotyping can include antibodies (and targets) from a list including HIB19 (CD19), ICRF44 (CD11b), CD7-6B7 (CD7), CD66a-B1.1 (CD66), 5-271 (CD36), GHI / 61 (CD163), HI30 (CD45), Bu15 (CD11c), M5E2 (CD14), 3G8 (CD16), HIT2 (CD38), 15-2 (CD206), WM53 (CD33), UCHT1 (CD3), and L243 (HLA-DR). This panel can be used to identify and phenotype monocytes and macrophages. Monocytes circulate in the blood, bone marrow, and spleen and comprise approximately 2-12% of all human white blood cells. Monocytes have been considered a systemic reservoir of myeloid precursors for tissue macrophage and dendritic cell regeneration, although DC and macrophage subpopulations exist that develop independently of monocytes. Recruited monocytes are innate effectors of the immune response to microorganisms, killing pathogens through phagocytosis, reactive oxygen species (ROS), nitric oxide (NO), myeloperoxidase, and the production of proinflammatory cytokines. Based on CD14 and CD16 expression, monocytes can be classified as "classical" (CD14+CD16-), intermediate (CD14+CD16+), and non-classical (CD14loCD16+).

[0099]

[0116] In some cases, a lyophilized panel for signal transduction assays can include antibodies (and targets) from a list including 47 (pSTAT5), 58D6 (pSTAT1), D3F9 (p38), 4 / P-Stat3 (pSTAT3), L35A5 (Iκβα), D13.14.4E (pERK1 / 2), and N7-548 (pS6). This panel can be useful for quantifying basal and induced phosphorylation of multiple important signaling pathways, namely, JAK / STAT, NFκB, and MAPK. This panel can be combined with other panels to measure cell signaling in heterogeneous samples, such as blood or splenocytes. Alternatively, it can be used as a stand-alone panel when measuring homogeneous samples, such as cell lines.

[0100]

[0117] In some cases, a lyophilized panel for basic mouse spleen / lymph node phenotyping can include antibodies (and targets) from a list including 30-F11 (CD45), M1 / 70 (CD11b (MAC1)), 145-2C11 (CD3e), 53-6.7 (CD8a), RM4-5 (CD4), and RA3-6B2 (B220). This panel can be useful for identifying CD4+ T, CD8+ T, B cells, macrophages, and monocytes within fresh or frozen isolated mouse splenocytes and thymocytes.

[0101]

[0118] In some cases, a lyophilized panel for mouse spleen / lymph node phenotyping can include antibodies (and targets) from the following list: RB6-8C5 (Ly6G / C(Gr1)), N418 (CD11c), H1.2F3 (CD69), 30-F11 (CD45), M1 / 70 (CD11b(MAC1)), 6D5 (CD19), 3C7 (CD25), 145-2C11 (CD3e), TER119 (TER-119), MEL-14 (CD62L), 53-6.7 (CD8a), H57-597 (TCRβ), PK136 (NK1.1), IM7 (CD44), RM4-5 (CD4), and RA3-6B2 (B220). This panel may be useful for identifying major mouse spleen / lymphocyte cell subsets, including effector CD4+ T, effector memory CD4+ T, central memory CD4+ T, activated CD4+ T, effector CD8+ T, effector memory CD8+ T, central memory CD8+ T, Tregs, plasmacytoid DCs, myeloid DCs, erythrocytes, macrophages, monocytes, NK cells, and granulocytes, in freshly or frozen-isolated mouse splenocytes and thymocytes.

[0102]

[0119] In some cases, a lyophilized panel for mouse intracellular cytokine I assays can include antibodies (and targets) from a list including XMG1.2 (IFNγ), JES6-5H4 (IL-2), 11B11 (IL-4), TRFK5 (IL-5), MP5-20F3 (IL-6), JES5-16E3 (IL-10), TC11-18H10.1 (IL-17A), and MP6-XT22 (TNFα). This panel can be useful for assaying key mouse cytokines in fresh or frozen sources of mouse leukocytes, including splenocytes, thymocytes, bone marrow, and lymph node cells or cell lines. This panel can be used in conjunction with a mouse spleen / lymph node phenotyping panel to enable comprehensive immunophenotyping of cytokine-expressing cells.

[0103]

[0120] In some cases, additional panels or panel subsets may include regulatory T cell surface markers (e.g., PD-1, CTLA-4, GITR, CXCR3, IL-12R, IL-4R, CRTH2, IL-17Rb, IL-23R, CCR6, IL-1Rb, OX40L, CD40L, SLAM, IL-21R, ICOS, CXCR5, TIM3, 1B11, LAG3, and / or BTLA); intracellular regulatory T cell transcription factor markers (e.g., FoxP3, RORgT, T-bet, Bcl6, and / or GATA3); intracellular cytokine markers (e.g., IL-1Rb, IL-2Rb ... The antibodies may include antibodies specific for inflammatory and chemokine markers (e.g., IFNg, IL-2, IL-4, IL-6, IL-7, IL-12, IL-15, IL-17A, IL-22, IL-23, TGFb, TNFα, perforin and / or granzyme b); druggable target markers (e.g., BTLA, GITR, 4-1BB, OX40, TIGIT, Helios and / or ICOS); and / or myeloid-derived suppressor cell markers (e.g., CD11b, CD15 and / or CD33).

[0104]

[0121] The CD4 T cell panel, in the form of clones occasionally listed in parentheses, included CCR6 (G034E3), CD45RA (HI100), CD4 (RPA-T4), LAG3 (polyclonal; R&D Systems), CCR4 (205410), CD62L (DREG-56; BioLegend), CD49b (AK-7; BD Biosciences), CXCR3 (G025H7), CD161 (HP-3G10), TIGIT (MBSA43; eBioscience), and ICOS (DX29; BD Biosciences). The panel may include at least two of the following antibodies: CD226 (11A8; BioLegend), CD8α (SK1), CD25 (2A3), CTLA-4 (14D3), CXCR5 (51505), CD3 (UCHT1; BioLegend), PD-1 (EH12.2H7). In some cases, the lyophilized panel can include at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 antibodies from this list. The panel may be provided in lyophilized form or in solution. The panel may be a separate panel as described herein.

[0105]

[0122] The NK panel may include at least two of CD27, CD4, CD8, CD57, TRAIL, KIRDL2 / L3 / S2, CD16, CD117, KIR2Ds4, LILRB1, NKp46, NKG2D, NKG2C, 2B4, NKp30, CD122, KIR3DL1, CD94, CCR7, KIRDL3, NKG2A, HLA-DR, KIR2DL4, CD56, CD45, KIR2DL5, and CD25. In some cases, the lyophilized panel may include at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 antibodies from this list. The panel may be provided in lyophilized form or in solution. The panel may be a separate panel as described herein.

[0106]

[0123] The CD8 T cell panel may include at least two of CD3, CCR7, CD11a, CD7, CD8, CD27, CD28, CD29, CD43, CD45RA, CD45RO, CD49d, CD57, CD62L, KLRG1, and HLA-DR. In some cases, the lyophilized panel may include at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 antibodies from this list. The panel may be provided in lyophilized form or in solution. The panel may be a separate panel as described herein.

[0107]

[0124] Treg panels include CCR6 G034E3, LAP TW4-2F8, CD45RA HI100, CD103 Ber-ACT8, CD31 WM59, CD8 RPA-T8, CD147 HIM6, GITR 621, CCR4 205410, CD28 CD28.2, CD49d 9F10, CD62L DREG-56, CD3 UCHT1, CXCR3 G025H7, CD73 AD2, CD161 HP-3G10, CD39 A1, ICOS C398.4A, OX40 Ber-ACT35, CCR10 6588-5, CD137 4B4-1, CD27 L128, GARP 7B11, CD25 2A3, CD25 M-A251, CTLA4 14D3, CD4 The lyophilized panel may include at least two of SK3, CD38 HIT2, HLA-DR L243, CD71 OKT-9, and KLRG1 2F1 / KLRG1 APC. In some cases, the lyophilized panel may include at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 antibodies from this list. The panel may be provided in lyophilized form or in solution. The panel may be a separate panel as described herein.

[0108]

[0125] The B cell panel may include at least two of CD10, CD117, CD11c, CD127, CD16, CD179a, CD179b, CD19, CD20, CD21, CD22, CD23, CD235, CD24, CD27, CD33, CD34, CD38, CD40, CD43, CD45, CD45RA, CD49d, CD5, CD61, CD62L, CD66b, CD7, CD72, CD79b, CXCR4, HLADR, IgD, IgMi / IgMs (IgH), kappa, lambda, Pax5, PreBCR, RAG1 and TdT. In some cases, the lyophilized panel can include at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 antibodies from this list. The panel can be provided in lyophilized form or in solution. The panel can be a discrete panel as described herein.

[0109]

[0126] The monocyte and MDSC panel may include at least two of CD14, CD16, HLA-DR, CD163, CD206, CD33, CD36, CD32, CD64, CD13, CD11b, CD11c, CD86, CD274, CCR2, CD163, CD13, CD123, and CD206. In some cases, the lyophilized panel may include at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 antibodies from this list. The panel may be provided in lyophilized form or in solution. The panel may be a separate panel as described herein.

[0110]

[0127] DC panels include EpCAM / CD326, CD19, CD117, CD11b, BDCA2 / CD303, CD16, CD127 / IL-7R, CD123 / IL3R, CD66b, CD163, CD45 , CCR7, CD14, CD11c, BDCA3 / CD141, CD335 / NKp46, BDCA-1 / CD1c, CD1a, CD172a / b / SIRPα, HLADR, CD34, CD115 / CSF 1R, CX3CR1 / CX3CR1, CD116 / GMSFR, CD275 / ICOSL, TLR4, CD274 / PDL1, CLEC9A / DNGR1, CD135 / FLT3, Dectin-1 / CLEC7 A, CD206 / MMR, CD83, Langerin / CD207, CD45RA, CD33, CD2, CD81, CD5(UCHT2), CD26, XCR1-PE, anti-APC, Siglec-6 / CD327 PE, CD100-APC, Axl, CADM1 / SynCAM, CD205 / DEC205, BDCA2 / CD303 APC, BDCA3 / CD141 PECy7, BDCA4 / CD304 APC, CD123 BUV395, CD16 BV650, CD163 FITC, CD19 PerCP / Cy5.5, CD20 PerCP / Cy5.5, CD1a Pacific Blue, CD2 APC / Cy7, CD206 / MMR APC (Klein 15-2), CD3 PerCP / Cy5.5, CD335 PerCP / Cy5.5, CD4 BV785, CD45 BV785, CD5 BV737, CD66b PerCP / Cy5.5, CD8a APC / Cy7, CD81 The panel may include at least two of PerCP / Cy5.5, CLEC9A / DNGR1 APC, CD209 / DC-SIGN APC, Dectin-1 / CLEC7A PE, HLADR BV605, Langerin / CD207 PE, TCF4 / E2-2, anti-fibroblast (TE7), CD140a, DARC, and Desmogelin-3. In some cases, the lyophilized panel may include at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 antibodies from this list. The panel may be provided in lyophilized form or in solution. The panel may be a separate panel as described herein.In certain embodiments, the panel may include element-tagged oligonucleotide probes that hybridize (directly or via a hybridization scheme) to target RNAs, such as RNAs encoding cytokines. For example, the panel may include two or more element-tagged oligonucleotide probes for RNAs encoding IFNg, IL-2, TNF, CXCL8, IL8, CCl4, IL-1B, IL-6, CCL2, ILRN, and Il1a. In some cases, the lyophilized panel may include at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 antibodies from this list. The panel may be provided in lyophilized form or in solution. The panel may be a separate panel as described herein.

[0111]

[0128] Although numerous exemplary lyophilized panels are described herein, other combinations of antibodies or other element tagging moieties can be used to facilitate performing the desired assay. In some embodiments, one or more of the above panels may be provided in a non-lyophilized form, such as a solution. Two or more of the above panels may be provided in admixture (conjugated panels).

[0112]

[0129] Each lyophilized panel disclosed herein can be associated with a specific gating strategy used to identify specific traits based on the antibodies within the panel. To facilitate rapid analysis, the gating strategy associated with a particular panel can be preloaded into software for analyzing elemental analyzer data. Once selected (e.g., manually or automatically), the software can use that gating strategy to generate results from the elemental analyzer data. For example, using a human peripheral blood phenotyping kit, the preloaded gating strategy can help distinguish different major peripheral blood cell subsets based on the presence of various antibodies in the sample (e.g., the presence of elemental tags associated with the antibodies). In some cases, combining multiple panels or panel subsets, such as those identified herein, can enable the use of new or additional gating strategies. For example, combining a human peripheral blood phenotyping panel with a human hematopoietic stem and progenitor cell phenotyping panel can use a new gating strategy to help exclude lineage-positive cells from the results.

[0113]

[0130] Within each panel, each antibody can be tagged with a unique elemental tag so that a unique isotope or combination of isotopes is associated with each antibody within the panel. In some cases, particularly for panels designed to be used in combination with other panels, the unique elemental tag of one panel will be different from the unique elemental tag of the other panel. The mapping of each antibody to each isotope or combination of isotopes can be saved as an elemental tag mapping. This elemental tag mapping can be used by the software disclosed herein to interpret data received from the elemental analyzer.

[0114]

[0131] In some cases, the lyophilized antibody panel may include one or more auxiliary reagents. Such auxiliary reagents may include any suitable lyophilizable reagent that can be used in performing assays using the antibodies of the lyophilized antibody panel. Examples of suitable auxiliary reagents include red blood cell lysis, washing buffers, fixation reagents, permeabilization reagents, etc.

[0115]

[0132] The process of preparing a lyophilized panel can include obtaining antibodies, conjugating the antibodies with their respective elemental tags, titrating the conjugated antibodies within the panel to quality control them in liquid form, diluting the liquid antibodies in a stabilizer based on the titration results, combining the diluted antibodies with excipients, combining the antibody and excipient mixtures together to form a single blend, lyophilizing the blend, and then filling and sealing the lyophilized blend in a container. In some cases, individual antibody and excipient mixtures can be lyophilized before combining them in the blend, but generally, various antibody and excipient mixtures are combined before lyophilization. Any suitable excipient, such as a sugar (e.g., trehalose, sucralose, and mannitol), can be used. The use of trehalose and / or sucrose can help inhibit protein unfolding while providing a glassy matrix. The use of mannitol can act as a bulking agent. In some cases, the excipient can include a sugar, either alone or in combination with bovine serum albumin (BSA). In some cases, the excipient can be a mixture of about 5% to 20% (e.g., 10%) sugar in PBS and 5% to 20% (e.g., 10%) BSA in PBS.

[0116]

[0133] The freeze-drying process itself can be carried out in multiple stages, including a thermal stage, a vacuum stage, a drying stage, and a holding stage. During each stage, specific settings (e.g., freeze-drying settings) for temperature, ramping time, holding time, and / or vacuum can be used to control the freeze-drying device. The freeze-drying device can be any suitable device for controlling the temperature and vacuum of the blend being freeze-dried. In some cases, each stage can include multiple separate substages, each with a separate freeze-drying setting. During the thermal stage, the temperature can be held anywhere between -60 and 0°C while the vacuum is maintained in the range of 100 to 500 Torr. During the evacuation stage, the vacuum can be lowered to a range of 100 to 500 mTorr. During the drying stage, the temperature can be operated in the range of -50 to 30°C while the vacuum is maintained in the range of 10 to 150 mTorr. During the holding stage, the temperature can be maintained at a temperature of 10 to 30°C while the vacuum is increased to a range of 100 to 500 mTorr. After the holding step, the freeze-dried blend can be filled, which can include increasing the pressure in the container, for example, to a range of 200 Torr up to 760 Torr, although in some cases the filling is carried out at a pressure below ambient pressure. In some cases, the temperature of the blend does not increase above the glass transition temperature of the blend during the entire freeze-drying step.

[0117]

[0134] During the freeze-drying process, the moisture content of the blend is reduced. In certain cases, the freeze-dried panel can have a moisture content of 5% or less by weight, although in some cases the moisture content can be 0.5% or less by weight. In some cases, the freeze-dried panel can have a moisture content of 5% or less by weight, 4.9% or less by weight, 4.8% or less by weight, 4.7% or less by weight, 4.6% or less by weight, 4.5% or less by weight, 4.4% or less by weight, 4.3% or less by weight, 4.2% or less by weight, 4.1% or less by weight, 4% or less by weight, 3.9% or less by weight, 3.8% or less by weight, 3.7% or less by weight, 3.6% or less by weight, 3.5% or less by weight, 3.4% or less by weight, 3.3% or less by weight, 3.2% or less by weight, 3.1% or less by weight, 2.9% or less by weight, 2.8% or less by weight, 2.7% or less by weight, 3.8% or less by weight, 3.7% or less by weight, 3.6% or less by weight, 3.5% or less by weight, 3.4% or less by weight, 3.3% or less by weight, 3.2% or less by weight, 3.1% or less by weight, 3% or less by weight, 2.9% or less by weight, 2.7% or less by weight, 3.8% or less by weight, 3.1 ... % or less, 2.6% by weight or less, 2.5% by weight or less, 2.4% by weight or less, 2.3% by weight or less, 2.2% by weight or less, 2.1% by weight or less, 2% by weight or less, 1.9% by weight or less, 1.8% by weight or less, 1.7% by weight or less, 1.6% by weight or less, 1.5% by weight or less, 1. 4% by weight or less, 1.3% by weight or less, 1.2% by weight or less, 1.1% by weight or less, 1% by weight or less, 0.95% by weight or less, 0.9% by weight or less, 0.85% by weight or less, 0.8% by weight or less, 0.75% by weight or less, 0.7% by weight or less, 0.65% by weight or less, 0.6% by weight % or less, 0.55% by weight or less, 0.5% by weight or less, 0.49% by weight or less, 0.48% by weight or less, 0.47% by weight or less, 0.46% by weight or less, 0.45% by weight or less, 0.44% by weight or less, 0.43% by weight or less, 0.42% by weight or less, 0.41% by weight or less Bottom, 0.4% by weight or less, 0.39% by weight or less, 0.38% by weight or less, 0.37% by weight or less, 0.36% by weight or less, 0.35% by weight or less, 0.34% by weight or less, 0.33% by weight or less, 0.32% by weight or less, 0.31% by weight or less, 0.3% by weight or less, 0. It may have a water content of 29% by weight or less, 0.28% by weight or less, 0.27% by weight or less, 0.26% by weight or less, 0.25% by weight or less, 0.24% by weight or less, 0.23% by weight or less, 0.22% by weight or less, 0.21% by weight or less, 0.2% by weight or less, 0.19% by weight or less, 0.18% by weight or less, 0.17% by weight or less, 0.16% by weight or less, 0.15% by weight or less, 0.14% by weight or less, 0.13% by weight or less, 0.12% by weight or less, 0.11% by weight or less and / or 0.1% by weight or less.In some cases, the freeze-dried panels can have a water content of 0.05% by weight or greater, such as 0.05-1% by weight.

[0118]

[0135] The lyophilized panels may be stored in any suitable container, such as a tube, a pouch, or a well of a well plate. In some cases, a single panel may be stored in a single container. In some cases, a single panel may be evenly distributed throughout multiple containers, such as multiple wells of a well plate. In some cases, the lyophilized panels may be stored in an inert atmosphere (e.g., N2) or in air (e.g., dry air). The lyophilized panels may be stored in a sealed container.

[0119]

[0136] Lyophilized panels can be used in the same way as other antibody panels. Lyophilized panels can be resuspended in solution before or after adding the sample to be analyzed. In some cases, a sample of human peripheral blood can be combined with heparin before mixing with the lyophilized panel. In some cases, the blood sample can be mixed directly into the container containing the lyophilized panel, but this is not necessary. After mixing the sample with the lyophilized panel, the mixed sample can be incubated for a period of time, washed, and then investigated using an elemental analyzer. In some cases, additional staining can be performed before investigation, such as staining for intracellular targets, which may require permeabilization and fixation of the sample.

[0120]

[0137] In certain embodiments, the kit includes an elemental standard (e.g., provided in the kit itself or with other reagents described herein, such as lyophilized panels and / or barcoded beads). The elemental standard may include microbeads containing known amounts of multiple different metal isotopes. In certain embodiments, the elemental standard may include microbeads with different amounts of one or more metal isotopes. For example, the elemental standard microbeads may include two or more, three or more, four or more, or five or more populations of microbeads, each population containing a similar amount of each of multiple metal isotopes, but with a different amount of at least one metal isotope among the populations. Similar amounts of a given isotope within a population may mean that the standard deviation of the amount of a given isotope (the number of atoms of that isotope among the beads in the population) may be less than 10%, 25%, 50%, 100%, 200%, or 300% of the average amount of the given isotope among the beads in the population. The populations of beads can have at least one isotope present in a significantly different amount compared to each other population in the kit (e.g., the significantly different isotope compared to a first other population can be different from the significantly different isotope compared to a second other population). For example, the difference in the average amount of isotope between the populations can be more than 2, 3, 4, or 5 times the standard deviation within one or both of the populations.

[0121]

[0138] In certain embodiments, the elemental standard microbeads may contain at least two, three, four, five, six, seven, eight, or nine variable isotopes that vary in amount among at least some populations of microbeads. The isotopes (e.g., variable isotopes) of the elemental standard microbeads may encompass a mass range of greater than 10 amu, greater than 20 amu, greater than 30 amu, greater than 40 amu, or greater than 50 amu. At least one isotope may remain constant across all or a portion of the population of microbeads, such that the ratio of the constant isotope to the variable isotope can be used to describe the bead population (e.g., to identify the expected amount of the variable isotope in the beads).

[0122]

[0139] Microbeads can have a size greater than 100 nm and less than 1000 μm, e.g., 500 nm to 100 μm. Thus, microbeads can have more than 1,000, 10,000, 100,000, or 1,000,000 atoms per bead. Microbeads can be made according to any of the methods described for other beads herein, such as the methods for making assay and / or sample barcoded beads (e.g., without the step of attaching a biomolecule, such as SBP, to the surface), or any suitable method.

[0123]

[0140] Elemental standards can be useful for calibrating a mass cytometer and / or normalizing signals obtained across sample runs. For example, elemental standards can be used to quantify bound antibodies, such as antibody bound per cell. Elemental standard microbeads can be mixed with cells and run through a mass cytometer (e.g., so that microbead and cell events are detected separately). Thus, the mass of at least one isotope in the microbeads can be different from any mass tags used on the cells.

[0124]

[0141] In certain embodiments, the method includes calibrating the mass cytometer (at the beginning of sample operation and / or during sample operation) based on an elemental standard described herein (e.g., a kit containing the elemental standard). Alternatively or additionally, the method may include normalizing the mass cytometry data based on an elemental standard described herein (e.g., a kit containing the elemental standard). For example, an isotope signal from a cell (i.e., from a specific mass channel) may be normalized to the intensity of an isotope of similar mass and / or amount from microbeads acquired at a similar time. For example, a signal from a metal isotope-tagged antibody bound to a specific analyte of a cell may be normalized to the intensity of the isotope signal most similar in mass and intensity, obtained within the time window of the cellular event. A standard curve spanning different masses and / or intensities may be generated to normalize the signal from a cellular event based on the elemental standard microbeads detected within the time window of that cellular event. The time window may be less than 1000 seconds, less than 100 seconds, or less than 10 seconds. Cellular events for which no elemental standard microbeads of the population are obtained within the time window may be discarded from the dataset. The cellular events may be normalized based on the most recent microbead event (e.g., based on the signal provided by the most recent microbead event for each population of microbeads). In certain embodiments, the same calibration curve may be used to calibrate every cellular event within a time window.

[0125]

[0142] In certain embodiments, the amounts (e.g., average atomic number and optional deviation) of each of multiple isotopes within an elemental standard microbead (e.g., a population of elemental standard microbeads) may be known, and the amounts (e.g., average atomic number and optional deviation) of atoms of a given isotope attached to antibodies bound to a cellular analyte may both be known and used together to calculate the antibody bound per cell (e.g., to the analyte). For example, fractionation and analysis of the labeled antibody may be performed by FPLC (e.g., size-exclusion FPLC and / or anionic ion-exchange FPLC), the bulk amount of protein may be used to back-calculate the amount of antibody, and the bulk metal may be determined by mass spectrometry. The elemental standard microbeads may be inspected (e.g., by electron microscopy) to determine size and uniformity. Instrument calibration (e.g., across and / or between sample runs) may further enable quantification of bound antibody.

[0126]

[0143] In certain embodiments, kits or methods with elemental standard microbeads can be combined with other embodiments described herein, including lyophilized mixtures of metal-tagged antibodies and / or assay barcoded beads. For example, elemental standard microbeads can be provided in the same kit or even in a container (e.g., mixed with lyophilized antibodies). Alternatively, cells stained with the lyophilized antibodies described herein can be subsequently combined with elemental standard microbeads before analysis by mass cytometry. Normalization (such as quantifying the antibody bound per cell) can improve the ability to gate cell populations, for example, by automated gating software described herein.

[0127]

[0144] The following exemplary procedure outlines the steps for staining a sample of human peripheral blood. To the aliquot of blood required for staining, add heparin sodium salt at a final concentration of 100 U / mL (10 μL of 10 U / mL to 1 mL of blood). Vortex this mixture for 2 seconds and incubate at room temperature for at least 20 minutes. Prepare an antibody cocktail mixture (e.g., a lyophilized antibody panel) in a 5 mL tube. Add a volume of heparin-blocked blood directly to a 5 mL tube so that the final volume (blood + antibody cocktail) is 300 μL. Vortex the mixture for 2 seconds to ensure the lyophilized product is resuspended. Incubate the mixture at room temperature for 30 minutes. Immediately after staining is complete, add 250 μL of Cal-Lyse lysis solution to each tube. Incubate at room temperature in the dark for 10 minutes. Add 3 mL of water to each tube. Vortex the tube for 2 seconds and incubate at room temperature for 10 minutes. The cell suspension will begin to become opaque and will become translucent after 10 minutes of incubation. If the cell suspension is not completely translucent after 10 minutes, re-vortex the sample and incubate for an additional 5 minutes at room temperature. Centrifuge the tubes and decant the supernatant into a 50 mL waste tube. Add 3 mL of cell staining buffer to each tube. Centrifuge the tubes and decant the supernatant. Repeat the washing step twice for a total of three washes. Visually inspect the cell pellet and cell supernatant after each wash. If the cell pellet or supernatant is red, repeat the wash until the supernatant is clear and the cell pellet is white. Next, fix the cells and intercalate the DNA. Prepare a new dilution of 16% formaldehyde to a 1.6% working solution using phosphate-buffered saline (PBS). Add 1 mL of 1.6% formaldehyde to each tube. Vortex the tubes for 2 seconds and incubate for 10 minutes at room temperature. Centrifuge the tubes at 800xG for 5 minutes at room temperature and decant the supernatant. Dilute the intercalator in fixation and permeabilization buffer to a final concentration of 125 nM. Add 1 mL of the intercalator + buffer mixture to each tube. Vortex the tube for 2 seconds and incubate overnight at 4 °C. Cells can be left in formaldehyde at 4 °C for up to 48 h.If necessary or available, fixed cells can be seeded onto prepared glass slides. Then, wash the cells prior to sample acquisition. Add 2 mL of cell staining buffer to each tube. Vortex the tube for 2 seconds and centrifuge at 800 x G for 5 minutes at room temperature. Once centrifugation is complete, decant the supernatant. Repeat the washing step once for a total of two washes. Add 2 mL of cell acquisition solution to each tube. Vortex the tube briefly. Count the cells to determine the final volume of the resuspension during acquisition. Centrifuge the tube at 800 x G for 5 minutes at room temperature. Once centrifugation is complete, aspirate or pipette off the supernatant. Pellet the cells and store at 4°C until ready for acquisition. Prepare a mixture of 90% by volume of cell acquisition solution and 10% by volume of four elemental calibration beads in a 15 mL tube. Once ready for acquisition, add 0.5 x 10 6 cells / mL~1.5×10 6 Resuspend the cells in the cell acquisition solution + four element calibration bead mixture at a concentration of 1000 cells / mL. Remove the cell strainer cap from a 5 mL polystyrene round-bottom tube and place it on a 5 mL polypropylene round-bottom tube. Filter the resuspended cells through the cell strainer cap. Acquire the sample using a pre-calibrated ICP-MS instrument. Acquire the sample at no more than 600 events / second. At least 400,000 events should be acquired.

[0128]

[0145] As used herein, fixation and permeabilization refers to chemical crosslinking of cellular components with agents such as glutaraldehyde, formaldehyde, formalin, ethanol, and methanol, and the formation of holes in cell membranes using detergents. Suitable detergents can be easily selected from nonionic detergents. Desirably, these detergents are used at a concentration of about 0.001% to about 0.1%. One detergent that can be used is Triton X-100 (Sigma T9284). Examples of other suitable detergents include Igepal and Nonidet P-40. Other suitable detergents can be easily selected by those skilled in the art.

[0129]

[0146] Certain embodiments of the present disclosure are useful for analyzing whole blood, such as human peripheral blood. In some cases, the whole blood can be separated into PBMCs and isolated plasma before staining with the lyophilized panel. The lyophilized panel can stain the PBMCs. In some cases, the sample or PBMCs from the sample can be tagged with a sample barcode as disclosed herein, either before or after staining with the lyophilized panel. If tagged, the samples can be pooled together before interrogation with an elemental analyzer. The stained PBMCs can be interrogated using an elemental analyzer, such as a mass spectrometer.

[0130]

[0147] Data from an elemental analyzer can include data indicating the presence of various isotopes in a sample. Specifically, this data can include the presence of detectable isotopes of elemental tags that remain bound to the sample after the sample has been washed. The elemental analyzer can interrogate the sample on a cell-by-cell or particle-by-particle basis, thereby generating data on a cell-by-cell or particle-by-particle basis. For example, data from an elemental analyzer can indicate the presence of various isotopes for each cell or particle of the sample.

[0131]

[0148] During the automated analysis process, software can decode the elemental analyzer data into useful, readable results. This software can be integrated into the elemental analyzer or provided separately (e.g., on a separate computing device). Decoding the elemental analyzer data can identify the various elemental tags detected by the elemental analyzer, which can be known as elemental tag data. Each elemental tag can be associated with a specific marker, such as an antibody, a live cell marker, or a sample barcode. These associations can be saved as an elemental tag mapping accessible to the software. The software can then apply this elemental tag mapping to the elemental tag data to generate useful, readable results. In some cases, the software can select (e.g., manually or automatically) a specific gating scheme to use in interpreting the elemental tag data. For example, for a particular lyophilized antibody panel, a specific gating scheme can be used to help interpret the elemental tag data so that the presence or absence of various antibodies from the lyophilized antibody panel helps identify specific cell types (e.g., activated T helper cells, memory T helper cells, or memory B cells). The software can then output the appropriate results, such as to a display or a storage file.

[0132]

[0149] In some cases, the software can automatically identify the cell type of the sample. The automatic identification of cell types can be based on predetermined gating of cell populations that share similar expression of a subset of surface markers. Alternatively or additionally, the identification of cell types can also be guided by a clustering algorithm.

[0133]

[0150] In some cases, the software can output cell type results, which can include relative quantification (e.g., % of total cells, % of parent cells (parent cell population), % of grand-parent cells, etc.) of various cell types, such as CD4 αβ T cells (e.g., total CD4, naive, central memory, effector, effector memory, and regulatory); CD8 αβ T cells (e.g., total CD8, naive, central memory, effector, effector memory); δγ T cells; B cells (e.g., total B cells, naive, memory, resting memory, transitional); NK cells; monocytes; and / or dendritic cells.

[0134]

[0151] In some cases, the software can output marker intensities. Marker intensities can include the intensities (e.g., median intensities) of the markers for each cell type and / or any other markers at the user's discretion. For example, a visual output can be generated showing the intensity "color" of each cell type in the phenotype tree. In another example, a comparison of each result with a user-generated dataset of reportable files containing frequency, intensity, and cell count can be shown using a visual display (e.g., if the median is 1.5 sigma higher than normal, the color can be visually "hotter," and if the median is 1.5 sigma lower than normal, the color can be visually "cooler").

[0135]

[0152] In some cases, the software can output other results, such as dot plots of any two markers and / or histograms of any markers. In some cases, the software can automatically flag any markers with a distribution with multiple modes (e.g., a bimodal distribution). In some cases, the software can output a report text file with desired reportable files (e.g., frequency, intensity, and cell count). In some cases, the software can maintain a database of reportable files (e.g., frequency, intensity, and cell count). In some cases, the software can output print-quality formatted reports with user-selected plots and tables.

[0136]

[0153] Certain aspects of the present disclosure relate to barcoding samples, and optionally assays, using elemental tags. As disclosed herein, elemental tags can be unique and distinguishable based on their isotopic composition. For example, an elemental tag (or combination of elemental tags) used in a sample or assay barcode can have a unique isotope or unique combination of isotopes, thereby distinguishing it from other elemental tags. Thus, once an assay barcode is identified during elemental analysis of a sample, it can be inferred that the particular cell or particle being investigated underwent the assay associated with the assay barcode (e.g., treatment / stimulation conditions, staining with a particular panel of elementally tagged SBPs, etc.). Notably, assay and / or sample barcodes can be used to identify (and discard data from) particle doublets containing two or more assay barcodes or two or more sample barcodes.

[0137]

[0154] Assay barcoding can be achieved by binding analytes in a sample to assay barcodes associated with specific target analytes to be detected. Assay barcodes can include a solid support (e.g., assay beads), a capture biomolecule (e.g., an assay biomolecule that specifically binds to a target analyte in the sample and attaches it to the surface of the assay bead), and a distinguishable combination of an isotope (assay barcode) associated with the target analyte. The solid support can be a planar surface / slide (e.g., comprising an assay barcode array) or an assay barcode bead. The assay biomolecule can be an oligonucleotide (e.g., that specifically hybridizes to a target RNA or target DNA), an affinity reagent (e.g., an antibody, aptamer, or lectin), or a substrate (e.g., a peptide containing an element tag that is cleaved in the presence of a target enzyme, such as a target protease). The assay biomolecule can be attached to an assay bead such that different assay biomolecules (e.g., that bind to different target analytes) are attached to beads with different assay barcodes. A reporter can provide detection of the assay biomolecule, as described herein. Such reporters may include a reporter biomolecule (e.g., an oligonucleotide or antibody) that binds (directly or indirectly) to the target analyte. The reporter may further include an element tag for detecting the presence of the target analyte associated with the assay bead (e.g., associated by binding to an assay biomolecule that is itself bound to the bead).

[0138]

[0155] In combination, because both assay biomolecules and reporters need to bind to target analytes to provide signal, it can increase specificity.In addition, the analyte that is bound by assay biomolecules is presented on the surface of beads (or at a distance from the surface), so that the reporter with bulky element tag can still bind to target.Therefore, the signal amplification method described herein can be used to detect target analytes.

[0139]

[0156] As used herein in the context of mass cytometry, signal amplification refers to the association of more than 30, more than 50, more than 100, more than 200, or more than 500 (e.g., isotopically enriched) labeled atoms with a target analyte (i.e., a single instance of the target analyte bound by a specific binding partner). In certain embodiments, the labeled atoms may be heavy metals such as lanthanides or transition metals. In certain embodiments, signal amplification may be performed for more than 2, 5, 10, or 20 target analytes. In certain embodiments, signal amplification may involve the use of branched conjugation of mass tags to biomolecules, highly sensitive polymers, large mass tag particles, mass tag nanoparticles, hybridization schemes that associate multiple element-tagged oligonucleotides (e.g., containing multiple instances of the same element tag) with the target, and / or enzymatic deposition of multiple element tags. In certain embodiments, signal amplification uses mass tag polymers as described herein.

[0140]

[0157] Mass tagged oligonucleotides can be directly or indirectly hybridized to target oligonucleotides.For example, one or more intermediate oligonucleotides can provide a scaffold to which multiple mass tagged oligonucleotides can hybridize, thereby amplifying signals.Therefore, the present application includes an embodiment of the oligonucleotide for hybridization-based signal amplification.

[0141]

[0158] The target oligonucleotide can be a DNA or RNA molecule (such as coding RNA, small interfering RNA or microRNA) endogenous to a cell. The target oligonucleotide can be single-stranded. The target oligonucleotide can have a known specific sequence (or homology to a known specific sequence). In certain embodiments, a biomolecule such as an antibody or its derivative can be conjugated to the target oligonucleotide, such as a synthetic single-stranded DNA oligonucleotide containing a known sequence. In such a case, both the antibody and the oligonucleotide can be called a biomolecule.

[0142]

[0159] After the biomolecule binds to the analyte in the sample, multiple mass-tagged oligonucleotides can be directly or indirectly hybridized to the first oligonucleotide. Hybridization can be branched or linear. In certain embodiments, polymerase can extend the first oligonucleotide along the template to provide additional sites for the attachment of element tags (such as additional hybridization sites for element-tagged oligonucleotides). Mass-tagged oligonucleotides can contain a single labeled atom or can contain a polymer containing multiple labeled atoms. Mass-tagged oligonucleotides can contain labeled atoms, such as heavy metal atoms, in the chemical structure of the oligonucleotide itself.

[0143]

[0160] Signal amplification can provide a unique advantage to bead-based assays in that the same reporter tag (labeled metal element or isotope) can be amplified and used across a variety of beads and their target analytes. In certain embodiments, mass-tagged oligonucleotides, in addition to being used in signal amplification hybridization schemes, may be mass-tagged with the highly sensitive polymers or nanoparticles described herein.

[0144]

[0161] In certain embodiments, assay beads can be customizable so that users can assay biomolecules of interest for each assay barcoded bead. This attachment can be by chemical bonding or by addressing assay biomolecules to specific assay barcoded beads in the same mixture. This addressing can be achieved by providing assay barcoded beads that display a unique oligonucleotide sequence for each assay barcode, and users can attach different addressing oligonucleotides to different biomolecules attached to the bead surface, where the addressing oligonucleotides specifically hybridize to one of the unique oligonucleotide sequences.

[0145]

[0162] If the assay biomolecule is an oligonucleotide, the element-tagged reporter oligonucleotide can hybridize to another portion of the target RNA or DNA, thereby providing a signal when the target RNA or DNA binds to the bead. If the assay biomolecule is an affinity reagent, such as an antibody, that binds to the analyte at a first epitope, the element-tagged reporter affinity reagent (e.g., a reporter antibody) can bind to another epitope on the analyte, thereby providing a signal when the target analyte binds to the bead. The analyte can be further bound by a reporter, such as an element-tagged reporter antibody or oligonucleotide. The reporter can include a highly sensitive (e.g., intense) element tag that provides a highly abundant isotope (e.g., greater than 50, 100, 200, 500, or 1000 copies of a single isotope), thereby enabling detection of small numbers of target analytes bound to the assay beads. Such highly sensitive element tags can include nanoparticles (e.g., containing metal nanocrystal surfaces functionalized to bind to biomolecules such as antibodies or oligonucleotides) or hyperbranched polymers. For example, multiple reporter biomolecules containing the same nanogold particle element tag provide a high signal, taking advantage of the fact that separate reporter biomolecules containing the same element tag can be distinguished by assay barcodes (e.g., of beads displaying the analytes for which they are specific). In certain embodiments, nanoparticle tags (e.g., gold nanoparticles) can be associated with reporter probes via biotin-avidin (e.g., biotin-streptavidin) interactions. For example, nanoparticles (e.g., gold nanoparticles) can be conjugated to streptavidin. Reporters can also contain low-sensitivity element tags that provide a low-abundance isotope (e.g., less than 100, 50, 30, 20, 10, or 5 copies of the isotope) that is different from a highly abundant isotope, thereby enabling detection / quantification of an analyte that is so abundant that the highly abundant isotope saturates the detector. In certain embodiments, the high abundance isotope and the low abundance isotope have a mass difference (e.g., greater than 5, 10, 20, 30, 40, or 50 amu) such that saturation of the detector by the high abundance isotope does not affect detection of the low abundance isotope.Reporters for different analytes (e.g., including antibodies that bind to target analytes on different assay beads) can contain the same isotope, or combination of isotopes, as the analytes are distinguished by the unique assay barcodes of the beads.

[0146]

[0163] In certain embodiments, the reporter may include a reporter system that provides signal amplification by associating multiple instances of an element tag with a single instance of a target analyte. Signal amplification may be by enzymatic deposition, hybridization (e.g., branched hybridization, strand hybridization, and / or hybridization of multiple reporter oligonucleotides to a single long intermediate oligonucleotide), extension (e.g., single extension, rolling circle extension), and / or a series of branched conjugations. In certain embodiments, multiple (e.g., all) analytes detected by assay beads may be detected by the same reporter system. In certain embodiments, the signal amplification reporter system may have a high-sensitivity element tag.

[0147]

[0164] For example, an element tag containing an enzyme substrate moiety can be deposited onto a bead (or a molecule attached to a bead) from solution by an enzyme attached to a reporter biomolecule. Such a reaction can be covalent bonding by a tyramide element tag acted upon by horseradish peroxidase bound to the reporter biomolecule.

[0148]

[0165] Embodiments include hybridization schemes in which multiple element-tagged oligonucleotides indirectly hybridize (via one or more oligonucleotide intermediates) to a single oligonucleotide target. For example, the oligonucleotide target can be a target RNA or DNA (e.g., gDNA or cDNA) sequence, or can be an oligonucleotide present on a reporter antibody.

[0149]

[0166] As described herein, mass cytometry allows for sufficient detection channels (mass channels) to detect both the sample and the assay barcode within the bead, while allowing for additional channels for reporters (e.g., to detect assay targets). Thus, the bead assays described herein can be barcoded samples and / or assays for use in mass cytometry. For example, multiple different conditions (e.g., drug candidates, e.g., enzymes, or agonists or antagonists of one or more enzymes) can be applied to a biological sample, and their effects on multiple targets can be detected by enzyme assay beads. Individual conditions can be identified by sample barcodes shared among different assay beads exposed to the same condition. Assay barcoded beads can be combined prior to analysis, e.g., before exposure to a condition. Sample barcoded beads can be combined prior to analysis.

[0150]

[0167] In certain embodiments, the enzyme can be a protease, kinase, phosphatase, or DNA-modifying protein, such as a DNA methyltransferase. The target can be a substrate acted upon by the enzyme, and the reporter (e.g., a reporter biomolecule described herein) can bind to the target only before or after it is acted upon by the enzyme. For example, a phospho-specific antibody that detects a phosphorylated form of a protein target, which can be increased in abundance when acted upon by a kinase enzyme or decreased in abundance when acted upon by a phosphatase. If the enzyme is a protease, the reporter can be associated with the end of a peptide substrate and removed from association with the bead when the substrate is cleaved (such that a decrease in the reporter element tag indicates increased protease activity).

[0151]

[0168] Sample barcodes can be used to indicate which of several enzymes (or their agonists / antagonists) were tested in a particular assay. For example, a candidate enzyme, agonist, or antagonist can be added to a biological fluid such as a cell lysate, and the sample can then be contacted with assay barcoded beads to detect the activity of the enzyme. Alternatively, the candidate can be administered to cells (e.g., directly or via genetic engineering) or to an organism such as a patient or mammalian test subject, and a sample taken from that source can be contacted with the assay beads. Sample barcoding allows for the screening of many such candidates in parallel. In either case, sample barcodes can be added as described herein for beads to identify the candidates. For example, more than 10, more than 20, more than 50, more than 100, more than 500, or more than 1000 distinct samples can be barcoded. For example, with 6 unique combinations, 12 distinct isotopes provide 924 distinct combinations (e.g., for barcoding up to 924 samples). Another 12 distinct isotopes can be used to barcode nearly 1000 assays. Thus, more than 10, 20, 50, 100, 500, or 1000 distinct assay beads can be barcoded (e.g., beads that detect the amount of different substrates acted on by the candidate). As described herein, at least one channel is left for reporter substrate detection. This can enable unprecedented screening with real-time readout by mass cytometry.

[0152]

[0169] Proteins are post-translationally modified by enzymes in living cells. Known post-translational modifications include protein phosphorylation and dephosphorylation, as well as methylation, prenylation, sulfation, and ubiquitination. The presence or absence of phosphate groups on proteins, especially enzymes, is known to play a regulatory role in many biochemical and signal transduction pathways.

[0153]

[0170] Bead-based kinase assays for mass cytometry have been described in U.S. patent publications, such as U.S. Patent No. 20070190588, which is incorporated by reference and summarized below, however, such bead-based assays do not propose both sample and assay barcoding, which offers advantages for screening and is uniquely enabled by the high plexity of mass cytometry.

[0154]

[0171] Kinase function is the transfer of a phosphate group (phosphorylation) from a high-energy donor molecule, such as ATP, to a specific target molecule (substrate). Enzymes that remove phosphate groups from targets are known as phosphatases. The largest group of kinases are protein kinases, which act on specific proteins and modify their activity. Various other kinases act on small molecules (e.g., lipids, carbohydrates, amino acids, and nucleotides) that are often named after their substrates, including adenylate kinase, creatine kinase, pyruvate kinase, hexokinase, nucleotide diphosphate kinase, and thymidine kinase.

[0155]

[0172] Protein kinases catalyze the transfer of phosphate from adenosine triphosphate (ATP) to target peptide or protein substrates at serine, threonine, or tyrosine residues. Protein kinases are distinguished by their ability to phosphorylate substrates on distinct sequences. Commercially available kinases may be active (phosphorylated by the supplier) or inactive, requiring phosphorylation by another kinase.

[0156]

[0173] Protein phosphatases hydrolyze phosphate monoesters at phosphoserine, phosphothreonine, or phosphotyrosine residues into phosphate ions and protein or peptide molecules with free hydroxyl groups. This action is the opposite of that of protein kinases. Examples include protein tyrosine phosphatases, alkaline phosphatases, serine / threonine phosphatases, and inositol monophosphatases, which hydrolyze phosphotyrosine residues.

[0157]

[0174] Another embodiment is a method for a phosphatase assay comprising incubating a plurality of elementally labeled supports having metal ion coordination complexes attached thereto in multiple solutions, each solution containing a different free phosphorylated substrate labeled with an elemental tag, which may optionally be the same elemental tag for every substrate, in a manner such that each type of phosphorylated substrate labeled with an elemental tag is attached to a single type of elementally labeled support; separating the free phosphorylated substrate from the bound substrate attached to the metal ion coordination complex attached to the multiple elementally labeled supports in the multiple separate solutions; reacting the elementally labeled supports with ADP and at least one phosphatase under conditions that allow the phosphatase to dephosphorylate the phosphorylated substrate; and performing particle elemental analysis of the bound phosphorylated substrates labeled with elemental tags, which may optionally be the same elemental tag for every substrate, attached to metal ion coordination complexes attached to the supports.

[0158]

[0175] Another aspect of the applicant's teachings is to provide a kit for detecting and measuring an element in a sample, wherein the measured element comprises an element tag attached to a phosphorylated substrate, an element of a metal ion coordination complex, and an element of a uniquely labeled support, the kit comprising: an element tag for directly tagging a phosphorylated substrate; a number of phosphorylated substrates; a uniquely labeled support; a metal ion coordination complex; and optionally, a phosphatase, a phosphatase buffer, and ADP.

[0159]

[0176] Another aspect of applicants' teachings is to provide a method for kinase assays that includes incubating ATP, at least one kinase, a free metal ion coordination complex, and a plurality of unphosphorylated substrates immobilized on an elementally labeled support under conditions that allow the kinase to phosphorylate the substrate, in a manner such that a single type of unphosphorylated substrate attaches to a single type of elementally labeled support; separating the plurality of phosphorylated substrates immobilized on the elementally labeled support having attached metal ion coordination complexes from the free metal ion coordination complexes and the plurality of immobilized unphosphorylated substrates; and measuring the plurality of phosphorylated substrates immobilized on the elementally labeled support having attached metal ion coordination complexes by elemental analysis.

[0160]

[0177] Another aspect of the applicant's teachings is to provide a kit for detecting and measuring an element in a sample, wherein the measured element comprises an element tag attached to a non-phosphorylated substrate and a metal ion coordination complex, the kit comprising: an element tag for directly tagging the non-phosphorylated substrate; a non-phosphorylated substrate; a solid support; a metal ion coordination complex; optionally, a kinase; a kinase buffer; and ATP.

[0161]

[0178] The support may be a sample or a barcoded encoded bead as described herein.

[0162]

[0179] Due to their role in maintaining animal homeostasis, enzyme assays and pharmacological modulation have become important components in identifying potential therapeutic agents. Proteases are a subclass of proteolytic enzymes that have recently been shown to play important roles in signal transduction pathways, the dysregulation of which can lead to cancer, cardiovascular disease, and neurological disorders. Of the approximately 400 known human proteases, several dozen are being tested as potential drug candidates. Small-molecule inhibitors of proteases are currently considered valuable therapeutic leads for the treatment of degenerative diseases, cancer, and as antibacterial, antiviral, and antifungal agents.

[0163]

[0180] Bead-based protease assays for mass cytometry have been described in U.S. patent publications, such as U.S. Patent No. 20170023583, which is incorporated by reference and summarized below, however, such bead-based assays have not proposed both sample and assay barcoding, which offers advantages for screening and is uniquely enabled by the high plexity of mass cytometry.

[0164]

[0181] There is a need for robust, sensitive, and quantitative enzyme assays that allow for the simultaneous measurement of multiple enzymatic reactions. Such assays would allow for the conservation of precious biological samples and reagents, achieve high throughput and shortened assay times, and reduce the overall cost of enzyme analysis.

[0165]

[0182] One aspect of the present invention is a method for detecting protease activity in a biological fluid. The method may include attaching an encoded bead to the first amino acid of a peptide substrate to form an immobilized peptide substrate, where the peptide substrate includes the first and last amino acids and is a substrate for a protease enzyme; attaching an elemental tag to the last amino acid of the peptide substrate to form a tagged peptide substrate; incubating the immobilized tagged peptide substrate with the biological fluid; and detecting the elemental tag and the encoded bead in the biological fluid by elemental analysis. The encoded bead may be both assay and sample barcoded, as described herein.

[0166]

[0183] The protease assay kit can include an assay-encoded bead attached to the first amino acid of a peptide substrate (immobilized peptide substrate), which can include the first and last amino acids and can be a substrate for a protease enzyme. An element tag can be attached to or near the last amino acid of the peptide substrate to form a tagged peptide substrate. The encoded bead can be both assay and sample barcoded, as described herein.

[0167]

[0184] A mixture of assay beads may jointly target at least 5, 10, 20, 50, 100, 200, 500, 1000 or more analytes. In certain embodiments, the sample barcodes may distinguish assay barcode beads and / or cells from at least 5, 10, 20, 50, or 100 or more different samples.

[0168]

[0185] In certain embodiments, the number and / or size of assay barcoded beads may vary depending on the analyte to which they are specific. For example, assay barcoded beads that specifically bind to a high-abundance target analyte may have fewer or more antibodies on their surface than assay barcoded beads that specifically bind to a low-abundance target analyte in the same sample. For example, in a mixture of assay barcoded beads, assay barcoded beads specific to a first target analyte may be 2, 5, 10, 20, 50, or 100 times more abundant than assay barcoded beads specific to a second target analyte. If a first analyte is more abundant than a second analyte, the first analyte will be diluted across the multiple assay barcoded beads that bind to it. An assay barcode kit may include one or more assay barcoded beads and / or reporters described herein.

[0169]

[0186] Sample barcoding can be achieved by tagging a sample with a sample barcode associated with that particular sample. Thus, once a sample barcode is identified during elemental analysis, it can be inferred that the particular cell or particle being investigated is part of that particular sample. Thus, multiple samples can be pooled together at any point between the application of the sample barcode and investigation using elemental analysis without losing the ability to associate the investigated cell or particle with each sample. By allowing samples to be pooled together, elemental analyzers can operate at much higher throughput and simplify sample handling and storage.

[0170]

[0187] The sample barcode can include an element tag attached to an element tagging moiety (e.g., an assay barcoding reagent or a sample barcoding reagent) that has targeting functionality for all, the vast majority, or most or all cells or particles in the sample. As used herein, the term predominant may include at least 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the cells or particles of a sample.

[0171]

[0188] In some cases, the barcoding reagents may be beads containing or capable of binding to elemental tags. In some cases, unique assay barcodes may be disposed within the beads, and the surface of the beads may contain, be bound to, or be functionalized to bind to unique sample barcodes. Any suitable beads containing or binding to elemental tags, such as polymer-assisted surface-functionalized beads, may be used. Such polymers may be poly-L-lysine, PEG (polyethylene glycol), PEG MEA (methyl ether acrylate), PVMS (polyvinylmethylsiloxane), polydopamine, polystyrene, and / or another polymer or derivative known to those skilled in the art. In some cases, the beads may be functionalized with a first functional group for binding to a moiety having a targeting function (e.g., an antibody) and a second functional group for binding to the sample barcode. However, in some cases, the beads may be functionalized with only a single functional group capable of both binding to a moiety having a targeting function and binding to the sample barcode. In some cases, a blocking agent (e.g., albumin) may be used to control the attachment of the moiety and sample barcode to the beads. In some cases, if the functional group reacts with the surface of cells or the cells are pre-permeabilized, reactive functional groups (e.g., thiol, amine, thiol-reactive, amine-reactive, or click chemistry functional groups, or even highly reactive functional groups such as isothiocyanates) can be used to facilitate labeling of beads blocked by a blocking agent. Such functional groups can be placed on the sample barcode to facilitate tagging of the barcoding reagent or the sample itself. Alternatively, free metals (e.g., a set of sample barcode isotopes) can be provided in solution and bound by chelating groups on the surface of the beads. In certain embodiments, beads can be functionalized to bind to (or be bound by) the same sample barcode reagent used to barcode cells in the assay. In certain embodiments, the isotope combination in the sample barcode can be the same for beads and cells from the same sample.In some cases, the assay beads may be blocked (e.g., with a blocking reagent such as BSA), in which case the sample barcode may bind to the blocking reagent.

[0172]

[0189] Sample barcoding reagents for cells may include one or more element-tagged antibodies (that bind across multiple cell types or across the majority of cells in a sample), element tags functionalized to non-specifically bind to cells (e.g., via covalent interactions), and / or metals in solution. Sample barcoding reagents for cells may further include a reagent for introducing the sample barcode into cells (e.g., DMSO, a cell permeabilization reagent, such as a detergent or alcohol, etc.). Sample barcoding reagents for assay barcoded beads may be present within the beads, on the surface of the beads, or applied to the beads. When applied to beads, the sample barcoding reagent may include functional groups described herein for binding to the surface of the beads (e.g., to functional groups displayed by the beads or to blocking reagents present on the bead surface). Sample barcoding reagents for a given sample may include a unique combination of isotopes specific to that sample. In certain embodiments, cells and assay barcoded beads from the same sample (e.g., an individual blood sample) may be labeled with the same assay barcode. The same assay barcode used to label cells and beads may contain the same combination of isotopes and / or the same means of attachment (e.g., functional groups).

[0173]

[0190] The sample barcoding reagent may be admixed with or provided together with the lyophilized antibody panel. The sample barcoding reagent may be admixed with or provided together with the assay barcoded beads. The assay barcoded beads may be admixed with or provided together with the lyophilized antibody panel. The assay barcoded beads and the sample barcoding reagent may be admixed with or provided together with the lyophilized antibody panel (e.g., where the sample barcoding reagent binds to both the assay barcoded beads and the cells in the sample). In certain of the above embodiments, the sample barcoding reagent may be in, on, or provided together with the assay barcoded beads.

[0174]

[0191] In some cases, highly reactive functional groups (e.g., thiol groups, amine groups, thiol-reactive groups, amine-reactive groups, or click chemistry functional groups) can be used to facilitate labeling of cells with sample barcodes. Such functional groups can be placed on the sample barcode to facilitate tagging of the barcoding reagent or the sample itself. Alternatively, free metal (e.g., a set of sample barcode isotopes) can be provided in solution and administered to cells (e.g., in the presence of reagents such as alcohol, detergents, and / or DMSO to allow entry of the metal into the cells).

[0175]

[0192] In some cases, the elemental tags used in the assay barcodes and sample barcodes may include elements and / or isotopes not commonly used with element-tagged lyophilized panels or outside the lanthanide family. For example, the assay barcodes and / or sample barcodes may be barcoded using Pd or Te. In some cases, the elemental tag for the assay barcodes and / or sample barcodes may be a cadmium elemental tag containing a cadmium isotope chelated to a polymer (e.g., chelated to a DOTA group on a polymer) or bound to a cadmium-binding protein.

[0176]

[0193] In some cases, kits can be provided that contain barcoded reagents, such as beads, in a single container and multiple separate containers, each containing a unique sample barcode. The barcoded reagents may or may not contain an assay barcode. A user can combine separate volumes of these barcoded reagents with various samples to be assayed. Each sample can then be combined with a unique sample barcode, allowing the sample barcode to bind to barcoded reagents already in or bound to the sample and / or cells or particles in the sample itself. After washing, this sample-barcode-tagged sample contains barcoded reagents, both with or without an assay barcode, that contain the same sample barcode. Thus, this sample barcode can be used to identify cells or particles in this particular sample. The same process can be performed with other samples and other sample barcodes, resulting in a collection of multiple sample-barcode-tagged samples, each tagged with a unique, distinguishable barcode. Optionally, in some cases, sample barcodes can be used without separate barcoding reagents, provided that the sample barcodes themselves can directly target cells or particles in the sample.

[0177]

[0194] In some cases, the association between a particular sample and its sample barcode can be stored, such as in a mapping. This sample barcode information can be accessed by research or analysis software to automatically attribute elemental analyzer data or additional results to the appropriate sample based on the detected presence of the sample barcode.

[0178]

[0195] In some cases, a reporter reagent may be provided with the assay reagent and / or used to detect the presence of an analyte bound to the assay reagent. The reporter reagent may directly bind (specifically or nonspecifically) to the analyte in the sample before or after the analyte is bound by the assay bead. The reporter reagent may include a biomolecule, such as an oligonucleotide (e.g., that hybridizes target RNA or DNA bound to the assay barcoded bead) or an affinity reagent (e.g., an antibody, lectin, or aptamer). Such a reporter biomolecule may be used to help identify the presence of the barcoded reagent (e.g., bead) and / or target particle during investigation. The reporter reagent may include an element tag that is common to all reporter reagents for the same object (e.g., all reporter reagents for beads, or all reporter reagents for a specific target analyte) but is distinguishable from other element tags (e.g., sample barcoded element tags). Each reporter reagent may be associated with a specific object, such as a barcoded reagent (e.g., bead) or target analyte. Reporter reagents can include antibodies or other moieties with targeting functions for their associated targets. Thus, when a reporter reagent is mixed with other reagents (e.g., barcoding reagents) or a sample, the reporter reagent can tag the associated targets with the elemental tag for that reporter reagent. If the elemental tag is detected during investigation by elemental analysis, it can be inferred that the particle being investigated is of the type associated with that particular reporter reagent. For example, if an elemental tag associated with a reporter reagent for a barcoded bead is detected, it can be assumed that the target detected at that time, and therefore the element detected within a specific window before and after that time, is associated with the barcoded bead (e.g., is an isotope associated with an elemental tag within or on the bead). As used herein, a reporter reagent for a cell type can be an antibody from a lyophilized antibody panel.

[0179]

[0196] The reporter biomolecule may include an element tag (e.g., a collection of one or more isotopes on a polymer chain or embedded in or on a bead). In some cases, the reporter reagent (such as a reporter antibody) may be specific for a free analyte associated with a particular assay reagent (e.g., bound by an assay antibody present on the surface of the assay reagent, such as a bead). The reporter antibody and the assay antibody (e.g., an antibody bound to an assay bead) may be the same type of antibody or different types of antibody. In some cases, when the reporter antibody is different from the assay antibody (e.g., when a polyclonal antibody is used), the reporter antibody may be selected so as not to interfere with binding of the assay antibody to the target analyte. In some cases, the reporter reagent and the assay reagent may be made from two different antibodies (e.g., monoclonal antibodies), but this is not necessarily the case.

[0180]

[0197] When using multiple different assay reagents with multiple target analytes, multiple sets of reporter reagents can be made and / or used, each set contains the same element tag (for example, isotopically indistinguishable element tag), but different reporter antibodies are bound to the element tag.Therefore, the different reporter antibodies of the reporter reagent set can target different target analytes, but they all tag these different target analytes with the same element tag associated with every reporter reagent.Alternatively or additionally, reporter reagents can include both high-sensitivity and low-sensitivity element tags, as further described herein.

[0181]

[0198] In one example, a kit can be provided with a set of 10-12 different barcoded reagents, each with a unique assay barcode; a separate set of six different elemental tags can be provided for later combination with the barcoded reagents and / or samples; barcoded reagent reporters (e.g., reporter reagents targeting the barcoded reagents) can be provided with or separate from the barcoded reagents and have their own unique elemental tags; and a set of 30-40 cellular reporters (e.g., reporter reagents each targeting a specific cell type), each with a unique elemental tag. The number of each reagent, barcode, and / or reporter can be adjusted as desired.

[0182]

[0199] In some cases, the barcoded reagents and / or barcodes can be lyophilized and included in a lyophilized panel, such as the lyophilized panels described herein.

[0183]

[0200] In some cases, barcoded reagents can be provided in a preconfigured form by preparing the barcoded reagents with several unique combinations of assay barcodes and sample barcodes. In such cases, each unique barcoded reagent can be stored in a separate container, such as a separate well of a well plate. In one example, a well plate can be established so that all wells along a particular column (or row) share the same assay barcode, while all wells along a particular row (or column) share the same sample barcode. In another example, a well plate can be established so that each filled well contains barcoded reagents with various combinations of a specific unique sample barcode and multiple assay barcodes. Thus, a first well can contain barcoded reagents that all have a first sample barcode but each have a different assay barcode, and a second well can contain barcoded reagents that all have a second barcode but each have a different assay barcode. In some cases, preconfigured barcoded reagents can require the production of thousands of unique bead groups.

[0184]

[0201] In some cases, barcoded reagents (e.g., beads) can be provided in semi-structured form by preparing barcoded reagents with unique assay barcodes and surfaces functionalized to bind to sample barcodes. In such cases, each group of barcoded reagents can be bound to a moiety (e.g., an antibody) with a targeting function related to the assay associated with the assay barcode of that group of barcoded reagents.

[0185]

[0202] When a semi-configured barcoding reagent is provided, the sample barcode can be attached to the barcoding reagent before the barcoding reagent is combined with the sample. In one example, different barcoding reagents can be mixed together and then placed into a set of containers (e.g., wells in a well plate). A unique sample barcode can then be added to each of the containers, and the result can be mixed with a unique sample to perform an assay barcode-identifiable assay on the sample, while simultaneously tagging the sample with the sample barcode.

[0186]

[0203] When a semi-structured barcoding reagent is provided, the sample barcode can be bound to the barcoding reagent after the barcoding reagent is combined with the sample. In one example, the semi-structured barcoding reagents can be provided together or otherwise mixed together. The barcoding reagent can then be added to each of the set of samples. Separately, a unique sample barcode can be mixed with each of the set of samples before or after the barcoding reagent is added. The sample barcode can tag the barcoding reagent and / or cells or particles of the sample.

[0187]

[0204] In one example, the barcoding reagent can include assay barcoded beads functionalized with polydopamine for attachment of a capture antibody. Another molecule (e.g., avidin) can be added along with the capture antibody. After the capture antibody is added to the assay barcoded beads, the beads can be mixed and divided into aliquots for each sample. For sample barcodes, a unique combination of element tags functionalized (e.g., with biotin) to bind to the molecule can be added.

[0188]

[0205] In some cases, a sample barcode can be attached to a reporter, allowing the reporter to function as both a reporter and a sample identifier. For example, an antibody for reporting a specific cell type can also function to identify the sample with which that cell type is associated. In such cases, multiple copies of the antibody and its unique elemental tag can be made, with each copy receiving a unique sample barcode. Each copy can then be provided to each of multiple samples. After washing and interrogation, detection of the unique sample barcode can indicate the presence of a particular sample, and detection of the antibody's unique elemental tag can indicate the presence of its target in that sample. In some cases, multiple antibody panels (e.g., lyophilized antibody panels) can be made, each associated with a particular sample barcode by binding each antibody in the panel to a sample barcode that shares the same unique isotope or the same combination of unique isotopes. In use, each sample-barcoded antibody panel can be combined separately with a different sample, which can then be washed and pooled together before being interrogated using elemental analysis.

[0189]

[0206] In some cases, a sample-specific antibody panel can include antibodies with elemental tags such that the combination of isotopes present in a sample after it has been stained by the sample-specific antibody panel is unique to that sample. In other words, a sample barcode can be incorporated into the antibody panel by distributing that unique combination of isotopes across multiple antibodies in the antibody panel.

[0190]

[0207] In certain embodiments, live cell barcodes (e.g., thiol-reactive tellurium-based barcodes or element-tagged antibodies against widely expressed surface markers) can be used, which can have the added advantage of also barcoding live cells in a sample (e.g., fresh blood). For example, a live cell barcode can include an element-tagged antibody that specifically binds to CD45. This approach can be performed in conjunction with stimulation or another treatment of live cells (e.g., of PBMCs). In some cases, the sample barcode can barcode live cells. In some cases, the sample barcode can be non-damaging to live cells, such as being non-toxic to live cells.

[0191]

[0208] Certain aspects of the present disclosure may be useful for applications other than cytometry. For example, both assay and sample barcodes can be encoded on oligonucleotides attached to antibody-bound beads, and a reporter antibody bearing the oligonucleotide can be used to detect the analyte bound to the bead. The beads can be isolated in droplets, and a reaction product can be formed from the assay / sample barcode oligonucleotides on the beads and the oligonucleotides on the reporter antibody. This reaction product can be detected by sequencing, allowing for the detection of multiple samples and assay barcodes.

[0192]

[0209] Certain aspects of the present disclosure offer advantages not available in existing assays, such as deep immunophenotyping profiling for T cells; a wide range of additional immune lineages due to the ability to capture cell frequencies relative to other leukocytes (such as B cells, NK cells, dendritic cells, and monocytes); maximized information content at the single cell level, especially since every marker is combined, eliminating the need to guess between multiple tubes; the ability to control technical variability using sample barcodes; the ability to dive deep into functions of interest by adding any panels or panel subsets required for those functions; the ability to provide validated antibody cocktails that cover most consumer needs, thereby reducing or eliminating wasted time and effort in validating slightly different cocktails; and the ability to tailor data analysis to specific objectives by achieving rapid insights without operator bias.

[0193]

[0210] In an exemplary use case, many companies are competing to be first to market with their next product or label extension. Their strategy is to conduct early-phase clinical trials in immuno-oncology (I / O) as "basket trials," applying monotherapy or combination therapies to multiple indications (e.g., cancer types) and screening patients for the weakest signs of clinical benefit. In these accelerated trial settings, each sample is particularly valuable, and maximizing the information content of any analytical method, including flow cytometry, is critical. Furthermore, in the immunotherapy class, adverse events are immune-mediated, and comparison of baseline and on-treatment immune phenotypes provides potentially important information. These adverse events are exacerbated when used in combinations that are nearly universally accepted as the future standard in that class, and numerous clinical trials have been initiated to test these combinations. Therefore, aspects of the present disclosure may be particularly useful for such companies.

[0194]

[0211] In an exemplary use case, contract research organizations serve as the execution channel of choice for many corporate clinical trials, including most, if not all, flow cytometry tests. Aspects of the present disclosure may be particularly useful to such research organizations, particularly because of the resulting cost and time efficiencies and long-term stability of the lyophilized panels described herein.

[0195]

[0212] In another exemplary use case, large cancer research centers can serve as Phase I / II clinical trial sites, as well as routinely treat patients with approved drugs. These centers may collect additional information to support new biomarker development and diagnostic testing research. Embodiments of the present disclosure may be particularly useful to such research centers, particularly because of the resulting cost and time efficiencies and long-term stability of the lyophilized panels described herein. In some cases, certain embodiments of the present disclosure can be used as part of laboratory development tests that can be validated and used at those facilities.

[0196]

[0213] These illustrative examples are provided to introduce the reader to the general subject matter described herein and are not intended to limit the scope of the disclosed concepts. The following section describes various additional features and examples with reference to the drawings, in which like numerals indicate like elements and directional descriptions are used to describe the exemplary embodiments, but, as with the exemplary embodiments, should not be used to limit the present disclosure. Elements included in the examples herein may not be drawn to scale.

[0197]

[0214] FIG. 1 is a schematic diagram of an elemental tagging moiety 102 according to certain embodiments of the present disclosure. The elemental tagging moiety 102 in FIG. 1 is an antibody 104 tagged with an elemental tag 106, although any suitable moiety can be used when tagged with an elemental tag 106. The elemental tag 106 can be an isotope or a combination of isotopes. In some cases, the elemental tag 106 can be a polymer chain with multiple metal-containing pendant groups, such as several chelating groups. In some cases, multiple copies of a unique isotope can be included within a single elemental tag 106. In some cases, multiple copies of a unique combination of isotopes can be included within a single elemental tag 106, but this need not be the case.

[0198]

[0215] The elemental tagging moiety 102 can be part of a lyophilized antibody panel disclosed herein. The antibody 104 can be any suitable antibody that has targeting functionality for a target of interest. By appropriately mixing the elemental tagging moiety 102 with a sample, the antibody 104 can bind to any target in the sample, thereby labeling or tagging the sample with an elemental tag 106. After washing to remove unbound elemental tags, the stained sample can then be examined using elemental analysis. Detection of a unique isotope, or unique combination of isotopes, by the elemental analyzer can indicate the presence of the elemental tag 106, and thus the elemental tagging moiety 102, and thus any target to which the elemental tagging moiety 102 is bound.

[0199]

[0216] 2A is a schematic diagram of a barcoded reagent 208 having a distinct sample barcode 212 according to certain embodiments of the present disclosure. The barcoded reagent 208 can be a bead 214, although other barcoded reagents can be used. The bead 214 can include an assay barcode 210 within the bead 214, such as incorporated into the core of the bead 214. The assay barcode 210 can be a type of elemental tag that includes a unique isotope or unique combination of isotopes distinguishable by elemental analysis. In some cases, the bead 214 can include a surface 216 that includes, binds to, or is functionalized to bind various moieties, such as the sample barcode 212, a capture antibody 218, and / or an assay-specific biomolecule 250.

[0200]

[0217] The surface 216 of the bead 214 can bind or be functionalized to bind to an affinity reagent for a particular assay, e.g., a specific capture antibody 218, or a specific assay-specific biomolecule 250, which can be a non-antibody biomolecule. The affinity reagent can bind to a specific target (e.g., a protein or other structure), thereby tagging the target with an assay barcode 210. Thus, after washing to remove unbound barcoded reagent, detection of the assay barcode 210 indicates the presence of a target to which the affinity reagent (e.g., capture antibody 218 or assay-specific biomolecule 250) is bound.

[0201]

[0218] In some cases, the surface 216 of the beads 214 can be functionalized to bind to the sample barcodes 212. The sample barcodes 212 can be elemental tags that contain a unique isotope or unique combination of isotopes that can be identified using elemental analysis. There can be multiple groups of sample barcodes, where all sample barcodes from one group have the same isotope or combination of isotopes, and sample barcodes from different groups are unique and distinguishable from one another using elemental analysis. Thus, any number of different sets of barcoded reagents can be tagged with sample barcodes from the same group, thereby associating each of those barcoded reagents with the sample associated with that group of sample barcodes. Separately, any number of different sets of barcoded reagents can be tagged with sample barcodes from another group, thereby associating that different set of barcoded reagents with another group of sample barcodes.

[0202]

[0219] The sample barcode 212 may be provided separately from the barcoded reagent 208, for example, in a kit that includes multiple different sample barcodes. Thus, when preparing or performing an assay using the barcoded reagent 208, the sample barcode 212 can be mixed with the barcoded reagent 208 before the assay (e.g., by mixing the sample barcode 212 with the beads 214 before mixing both with the sample) or during the assay (e.g., by mixing the sample barcode 212 with the sample and then mixing the beads 214 therein).

[0203]

[0220] 2B is a schematic diagram of the barcoded reagent 208 of FIG. 2A after attachment of a sample barcode 212, according to certain embodiments of the present disclosure. In some cases, attachment of the sample barcode 212 may occur during manufacturing. In such cases, the barcoded reagent 208 can be provided, such as part of a kit, in which the sample barcode 212 is bound to the surface of a bead 214 or otherwise contained within the surface 216 of a bead. In some cases, the sample barcode 212 can be bound to the barcoded reagent 208 prior to attachment of the capture antibody 218 and / or assay-specific biomolecule 250.

[0204]

[0221] In some cases, attachment of the sample barcode 212 can be done as part of preparing to perform the assay (e.g., by mixing the sample barcode 212 with the beads 214 before mixing both with the sample) or as part of performing the assay (e.g., by mixing the sample barcode 212 with the sample and then mixing the beads 214 therein).

[0205]

[0222] 3 is a schematic diagram of a barcoded reagent 308 having an integrated sample barcode 312, according to certain embodiments of the present disclosure. The barcoded reagent 308 can be a bead 314, although other barcoded reagents can be used. The bead 314 can include both an assay barcode 310 and a sample barcode 312 within the bead 314, such as incorporated into the core of the bead 314. The assay barcode 310 and the sample barcode 312 can each be a type of elemental tag that includes a unique, specific isotope or unique combination of isotopes that can be identified by elemental analysis.

[0206]

[0223] In some cases, beads 314 may include surfaces 316 that include, bind to, or are functionalized to bind various attachments, such as capture antibodies 318 and assay-specific biomolecules 350. Surfaces 316 of beads 314 may bind to, or be functionalized to bind to, affinity reagents for a particular assay, e.g., specific capture antibodies 318, or specific assay-specific biomolecules 350, which may be non-antibody biomolecules. The affinity reagents may bind to specific targets (e.g., proteins or other structures), thereby tagging the targets with assay barcodes 310. Thus, after washing to remove unbound barcoded reagents, detection of assay barcodes 310 indicates the presence of targets to which affinity reagents (e.g., capture antibodies 318 or assay-specific biomolecules 350) are bound.

[0207]

[0224] 4 is a schematic diagram of a system for assaying one or more samples 420 using an elemental analyzer 424 and a lyophilized antibody panel 422, according to certain embodiments of the present disclosure. The sample 420 can be any suitable sample, including whole blood or human peripheral blood, although any suitable sample can be used. The sample 420 can be a biological sample. The lyophilized antibody panel 422 can be a lyophilized antibody panel or panel subset disclosed herein. The lyophilized antibody panel 422 can include a plurality of element-tagged antibodies, such as the element-tagged antibodies 104 of FIG. 1.

[0208]

[0225] The lyophilized antibody panel 422 can be mixed with the sample 420 according to an appropriate protocol to resuspend the lyophilized antibody panel 422. In some cases, barcoding reagents 408 can be optionally mixed with the lyophilized antibody panel 422 and the sample 420. Examples of suitable barcoding reagents 408 include sample barcoding reagents and assay barcoding reagents. In some cases, each of the multiple samples 420 can be mixed with a respective set of barcoding reagents 408, where each barcoding reagent in the set includes the same sample barcode, thereby applying a unique sample barcode to each of the multiple samples 420.

[0209]

[0226] After mixing with the lyophilized antibody panel 422, the sample 420 can be investigated using an elemental analyzer 424, such as a mass spectrometer. In some cases, as shown in FIG. 4, the elemental analyzer 424 can include an ICP-MS, which includes an inductively coupled plasma torch 426 and a mass detector 428, although other elemental analyzers can be used. The inductively coupled plasma torch 426 can receive the cells or particles of the stained and / or elementally tagged sample 420 and ionize the sample. The resulting stream of ions can be optionally processed (e.g., filtered) and sent to a mass detector 428. The mass detector 428 can be a time-of-flight detector or other suitable mass detector. The mass detector 428 can determine the presence and amount of various isotopes in the ion stream based on the mass of the ions. The mass detector 428 can output data indicative of the presence and amount of various isotopes over time. Thus, the elemental analyzer 424 can output elemental data, which can include data indicative of the presence and amount of various isotopes over time. In some cases, the elemental data may be saved in a data store as elemental data 482 that may be later accessed for subsequent analysis by the elemental data processor 430. In some cases, the elemental data may be routed to the elemental data processor 430 for immediate analysis. In some cases, the elemental data processor 430 may be incorporated into the elemental analyzer, for example, into the software component of the elemental analyzer, while in some cases, the elemental data processor 430 may be incorporated into a separate computing device.

[0210]

[0227] The elemental analyzer 424 can obtain elemental data regarding the presence and amount of various isotopes over time. In some cases, this elemental data can be processed or segmented into time periods (e.g., time windows) so that any elemental data within a particular time period can be associated with a single cell, bead, or particle detected by the elemental analyzer 424. For example, an elemental analyzer 424 analyzing a sample 420 containing 100 cells can generate elemental data encompassing at least 100 different time periods. Thus, the collection of any isotopes identified within a particular window can be used to elucidate which target or barcode, if detected, is associated with that particular cell, bead, or particle associated with that particular window.

[0211]

[0228] In some cases, mapping data 384 can be stored in a data store, which can be the same or a different data store as the data store in which elemental data 482 is stored. Mapping data 484 can include information for associating an isotope, or combination of isotopes, with a particular target or subject. Mapping data 484 can include information that identifies the expression of the target, and thereby distinguishes between cell types, thereby facilitating automated cell type identification. For example, in the case of element-tagged antibodies targeting CD20, CD45, CD14, CD16, CD8, CD3, and CD4, mapping data 484 can identify the respective isotopes used to tag each of these element-tagged antibodies. 147 Sm, 154 Sm, 160 Gd, 165 Ho, 168 Er, 170 Er and 174 Yb can be identified, thereby mapping each isotope to its respective target. The mapping data 484 can be accessed by the elemental data processor 430.

[0212]

[0229] In some cases, mapping data 484 can include mappings of any number of lyophilized antibody panels. The lyophilized antibody panel being used (e.g., lyophilized antibody panel 422) can be detected manually (e.g., via user selection) or automatically (e.g., via direct sampling of the panel itself or via detection of unique barcodes within a stained sample), thereby informing elemental data processor 430 of which mapping to use. In some cases, elemental data 482 can include additional metadata, such as an identification of which lyophilized antibody panel is being used. In addition to mapping lyophilized antibody panel elemental tags to targets, mapping data 484 can include mappings of other elemental tags to targets, such as mapping of sample barcodes to samples, mapping of assay barcodes to assays, and mapping of reporter barcodes to reported targets. The use of these additional mappings can be determined in the same manner as the mapping of a lyophilized antibody panel is determined.

[0213]

[0230] The elemental data processor 430 can receive the elemental data 482 and the mapping data 484. The elemental data processor 430 can analyze the elemental data 482 using the mapping data 484 to obtain information about the sample, for example, cytometry information about cells or other particles within the sample. In some cases, the elemental data processor 430 can separate results by sample using various sample barcodes. In some cases, the elemental data processor 430 can identify and quantify cell types based on targets in the lyophilized antibody panel 422. In some cases, identifying and quantifying cell types can include applying a gating scheme to the elemental data, as disclosed in further detail herein.

[0214]

[0231] The elemental data processor 430 can output results in any suitable manner, such as those described herein. Exemplary outputs can include storage in a data store, presentation on a display, or input to additional processes. The elemental data processor 430 can output any suitable type of information, such as those identified herein. For example, the elemental data processor 430 can output cell type results (e.g., relative quantification); marker intensities (e.g., a visual output showing the intensity "color" of each cell type in a phenotype tree); marker frequencies, marker intensities, and cell counts; dot plots of any two markers and / or histograms of any markers; or other suitable information.

[0215]

[0232] 5 is a schematic diagram illustrating the analysis and processing of unknown particles 520, 521 according to certain embodiments of the present disclosure. A first unknown particle 520 and a second unknown particle 521 are subjected to elemental analysis 524. A time arrow is depicted to indicate that the first unknown particle 520 is analyzed before the second unknown particle 521. The first and second unknown particles 520, 521 may be particles of a sample, such as sample 420 of FIG. 4, after being mixed with a suitable antibody panel and / or reagents.

[0216]

[0233] The elemental analysis 524 can yield elemental data 582 that can be divided into a first time window 586 and a second time window 587. The elemental data 582 can include information related to isotopes detected by the elemental analyzer during the elemental analysis 524. For example, the elemental data 582 can include information related to the isotopes detected within the first time window 586. 197 Au, 139 La, 197 Au, 106 Pd and 165 Ho and isotopes within the second time window 587 170 Er, 145 Nd, 108 Pd, 165 Ho and 154The isotopes identified within the first time window 586 may be due to the ionization and detection of the first unknown particle 520. The isotopes identified within the second time window 587 may be due to the ionization and detection of the second unknown particle 521.

[0217]

[0234] The elemental data 582 may be subjected to processing 530 to generate output results (e.g., first result 588 and second result 589). Processing 530, which may be performed using an elemental data processor, such as elemental data processor 430 of FIG. 4, may utilize mapping data 584.

[0218]

[0235] The mapping data 584 can include information for identifying targets or barcodes based on the detected elemental tags. As shown in FIG. 5, for illustrative purposes, certain exemplary isotopes are used for a given elemental tag, although other elemental tags and other isotopes can be used. In one example, the cell type mapping data includes several targets (e.g., CD3, CD4, CD61, CD45) and their respective associated elemental tags (e.g., 170 Er, 145 Nd, 165 Ho, 154 The sample barcoding data can include several samples (e.g., first, second, third, fourth) and their respective associated element tags (e.g., 106 Pd, 108 Pd, 130 Te, 126 The assay barcode data can include several assays (e.g., A, B, C, D) and their respective associated element tags (e.g., 139 La and 140 Ce, 139La and 153Eu, 139La and 165 Ho, 139 La and 175 The reporter antibody data can include several targets (e.g., analyte 1, analyte 2, analyte 3, and analyte 4) and their common associated element tags (e.g.,197 In certain embodiments, the reporter antibody may comprise a high-sensitivity element tag (e.g., a gold nanoparticle) and a low-sensitivity element tag (e.g., a polymer chelated to a lanthanide atom).

[0219]

[0236] The isotopes from the first time window 586 are the detected isotopes. 197 Because it contains multiple instances of Au, it can be inferred that the first unknown particle 520 is the target analyte (e.g., one of Analyte 1, Analyte 2, Analyte 3, Analyte 4). This reporter reagent can be used to target only the target analyte that the assay bead is targeted to. Therefore, it can be inferred that the first unknown particle 520 is not a cell, but rather an assay bead (e.g., an assay bead bound to a target analyte). Other detected isotopes within that first time window 586 can be 139 La and 165 Ho, indicating that the first unknown particle 520 is likely to be assay bead C. Furthermore, the detected isotopes within the first time window 586 are 106 Pd, indicating that the first unknown particle 520 is part of the first sample. This information may be provided as part of the first result 588.

[0220]

[0237] The isotopes from the second time window 587 do not include any isotopes associated with the reporter antibody associated with the target of the assay bead. Rather, the isotopes from the second time window 587 170 Er, 145 Nd, 165 Ho and 154 The first time window 586 and the second time window 587 are both shown to contain CD3, CD4, CD61, and CD45, respectively. It can also be inferred that the second unknown particle 521 is a cell. With sufficient additional cell type targeting information, inferences can be made regarding the cell type, as disclosed in further detail herein. The first time window 586 and the second time window 587 are both shown to contain CD3, CD4, CD61, and CD45, respectively. 165Ho, but the absence of reporter antibodies and possibly the presence of other cell type targets (e.g., CD3, CD4, CD45) was detected. 165 This shows that the Ho isotope comes from the cell-type antibody and not from the assay barcode. Finally, within the second time window 587 108 The presence of the Pd isotope indicates that the second unknown particle 521 is part of the second sample. This information can be provided as part of the second result 589.

[0221]

[0238] Thus, even though the first and second unknown particles 520, 521 are analyzed sequentially by the elemental analyzer, processing of the elemental data can still distinguish the particles 520, 521 into their respective particle types and their respective samples.

[0222]

[0239] FIG. 6 is a schematic diagram illustrating three exemplary element tags 632, 634, 636 with charts 638, 640, 642 showing their respective elemental data, according to certain embodiments of the present disclosure. Each element tag 632, 634, 636 may be an element tag used in an element tagging moiety (e.g., element tagging moiety 102 of FIG. 1 ), a sample barcode (e.g., sample barcode 212 of FIG. 2 ), or an assay barcode (e.g., assay barcode 210 of FIG. 2 ). The charts 638, 640, 642 associated with each element tag 632, 634, 636 indicate the actual or relative intensity of expression of the particular element as detected by an elemental analyzer (e.g., elemental analyzer 424 of FIG. 4 ) interrogating a sample containing the respective element tag 632, 634, 636. As used herein, the terms “element A,” “element B,” or “element C” refer to general elements that may be used to tag element tags, such as metals.

[0223]

[0240] Element tag 632 is an example of a single-isotope element tag. Element tag 632 consists of element A 644, with no other elements used to distinguish this element tag from other element tags (e.g., element tag 632 may include other elements, such as carbon and hydrogen, but these other elements are structural and not distinguishing). This element tag 632 may contain one or more instances of element A 644, such as n different copies of an isotope. Upon examination by an elemental analyzer, the elemental data for element tag 632 may indicate the relative expression of element A at a level corresponding to the number of copies n of element A 644 in element tag 632.

[0224]

[0241] Element tag 634 is an example of a single-isotope element tag that is distinguishable from element tag 632. Element tag 634 consists of element B 646. This element tag 634 may contain one or more instances of element B 646, such as y different copies of an isotope. Upon interrogation by an elemental analyzer, the elemental data for element tag 634 may indicate a relative expression of element B at a level corresponding to the number of copies, y, of element B 646 in element tag 634. Thus, element tag 634 is isotopically distinguishable from element tag 632 because different isotopes are detected, and possibly because the different isotopes are detected at different relative expression.

[0225]

[0242] Element tag 637 is an example of a multi-isotopic element tag that is distinguishable from element tags 632 and 634. Element tag 637 is comprised of elements D 645, E 647, and F 649. This element tag 637 may include one or more instances of each of elements D 645, E 647, and F 649. As shown in FIG. 6 , element tag 637 includes y different copies of elements D 645, E 647, and F 649. Upon examination by an elemental analyzer, the elemental data for element tag 637 may indicate the presence (e.g., and possibly the relative expression) of elements D, E, and F within element tag 637. Thus, element tag 637 is isotopically distinguishable from element tags 634 and 636 because a unique combination of isotopes is detected. Unique combinations can be identified based on a list of detected isotopes (e.g., elements D, E, and F), optionally based on the relative expression between detected isotopes within an element tag, and / or optionally based on the actual expression level of detected isotopes compared to other element tags.

[0226]

[0243] Element tag 636 is an example of a multi-isotopic element tag that is distinguishable from element tags 632, 634, and 637. Element tag 636 is comprised of element A 644, element B 646, and element C 648. This element tag 636 may include one or more instances of element A 644, element B 646, and element C 648, e.g., n different copies of element A 644, y different copies of element B 646, and z different copies of element C 648. Upon examination by an elemental analyzer, the elemental data for element tag 636 may indicate the relative expression of elements A, B, and C at levels corresponding to the n, y, and z copies of each element in element tag 636. Thus, element tag 636 is isotopically distinguishable from element tags 634, 636, and 637 because a unique combination of isotopes is detected. Unique combinations may be identified based on a list of detected isotopes (e.g., elements A, B, and C), possibly based on relative expression between detected isotopes within an element tag (e.g., expression of element C is less than expression of element A, which is less than expression of element B), and / or possibly based on actual expression levels of detected isotopes compared to other element tags (e.g., A:n, B:y, C:n may be associated with element tag 636, and another element tag may be associated with A:z, B:n, C:y).

[0227]

[0244] Thus, multiple unique element tags can be created using unique individual isotopes or unique combinations of isotopes. In some cases, a combination of isotopes can refer to a combination that includes a single isotope at a particular expression level, which can be distinguished from a different combination of isotopes that includes the same single isotope at different, distinguishable expression levels.

[0228]

[0245] Each unique isotope, or combination of isotopes, can be thought of as an elemental barcode that can be used to identify any moiety to which it is attached. Thus, when attached to a moiety that has targeting functionality for a particular target, the elemental barcode can be used to identify the target after contacting the elemental tagging moiety with a sample containing the target and washing away the unbound elemental tagging moieties.

[0229]

[0246] Depending on the type of elemental analysis, a certain number of isotopes can be reliably distinguished. For example, an exemplary ICP-MS system can distinguish up to x different mass channels, and therefore x different isotopes (e.g., isotopes 1, 2, 3, 4, 5...x). Therefore, it is possible to reliably distinguish at least 2 of the isotopes based solely on determining the presence of detectable isotopes in the elemental tag. × A unique barcode can be generated for each different combination of these two × It may be desirable to remove certain combinations of isotopes that are likely to be detected in a sample from different combinations of isotopes. For example, considering a set of isotopes or combinations of isotopes used as elemental tags within a lyophilized antibody panel or across a set of potential lyophilized antibody panels, it may be desirable to use completely different isotopes for sample barcoding and / or assay barcoding to avoid the possibility of antibody combinations being detected at or near the same time being mistakenly interpreted as different combinations of isotopes. For example, elemental tag 636 can include element C 648, such that even if elemental tags 632 and 634 are detected at or near the same time, an analytical device or processor will not mistakenly interpret the detection of elements A and B as elemental tag 636 because element C was not detected. In some cases, elemental tag 636 can be further protected from potential misidentification by not using any of the isotopes from elemental tags 632 and 634.

[0230]

[0247] In some cases, assay barcodes and / or sample barcodes are configured to have unique isotopes, but this is not required. In some cases, assay barcodes and / or sample barcodes can include unique combinations of several isotopes from a set of possible isotopes. For example, if a total of six different isotopes are used for sample barcoding, each sample barcode will include a unique combination of three of the six total isotopes, thereby resulting in 20 different unique sample barcodes. For example, if the total number of possible unique isotopes used in barcodes is n and the number of unique isotopes selected for each barcode is k, the total number of possible unique barcodes is:

number

[0231]

[0248] In some cases, the elemental tags used for cell type identification include one or more of a single isotope, e.g., elemental tags 632, 634, because assays often involve detecting multiple tags, all bound to a single cell. In some cases, the elemental tags used to barcode assay beads include unique combinations of isotopes, because the likelihood of detecting multiple assay beads within a single time window is minimal, and therefore the likelihood of detecting overlapping elemental tags is minimal. Using unique combinations of isotopes on assay beads allows for highly unique elemental tags to be achieved using a small number of unique isotopes.

[0232]

[0249] 7 is a schematic diagram of a sample cell 720 tagged with an elemental tagging moiety 702, a sample barcode 712, and a barcoding reagent 708, according to certain embodiments of the present disclosure. The sample cell 720 can be any cell of a sample, such as a blood cell. In some cases, another particle or target, such as a protein found in plasma (e.g., albumin), can be used in place of the sample cell 720. While the sample cell 720 in FIG. 7 is shown as tagged with an elemental tagging moiety 702, a sample barcode 712, and a barcoding reagent 708, in various uses described herein, such a sample can be tagged with any combination of one, two, all three, or more of these entities.

[0233]

[0250] Element tagging moiety 702 can be any suitable moiety (e.g., element tagging moiety 102 of FIG. 1 ), for example, an element tagged antibody of a lyophilized antibody panel. Antibody 704 can bind to element tag 706. Antibody 704 can bind to sample cell 720 via any suitable means, such as binding to an antigen on the surface of sample cell 720. In some cases, the element tagging moiety can infiltrate sample cell 720 (e.g., permeabilized sample cell 720) and bind to an intracellular target.

[0234]

[0251] The element tag 706 associated with an element tagging moiety 702 may be the same across all element tagging moieties 702 with the same antibody 704, however, element tagging moieties with different antibodies (e.g., targeting different targets) may have unique element tags.

[0235]

[0252] The sample barcodes 712 can be elemental tags that bind to the sample cells 720 directly or through an intermediate moiety (e.g., an antibody). The sample barcodes 712 can be designed to bind to all, the vast majority, or most of the cells or particles in the sample. Each sample barcode 712 can have the same isotope, or combination of isotopes, across every sample barcode 712.

[0236]

[0253] FIG. 8 is a schematic diagram illustrating a sample cell 820 tagged with a multi-isotope sample barcode 812, according to certain embodiments of the present disclosure. The sample cell 820 may be the sample cell 720 of FIG. 7. The sample barcode 812 may be bound to the sample cell 820. The sample barcode 812 may include an antibody 852 having a targeting function for the sample cell 820 (e.g., having a targeting function for a cell surface target of an intracellular target). The antibody 852 of the sample barcode 812 may bind to an element tag 832 that includes a unique combination of isotopes associated with the sample barcode 812. For example, the sample barcode 812 may have a virtual barcode of "A, B, C," where A, B, and C are unique isotopes (e.g., 102 Pd, 104 Pd, 105 Pd).

[0237]

[0254] 9 is a schematic diagram illustrating a sample cell 920 tagged with dispersed sample barcode portions 911, 912, 913, according to certain embodiments of the present disclosure. Sample cell 920 may be sample cell 720 of FIG. 7. Dispersed sample barcode portions 911, 912, 913 of sample cell 920 may result in the same overall barcode being applied to sample cell 920 as sample barcode 812 applied to sample cell 820 of FIG. 8.

[0238]

[0255] A sample cell 920 can include multiple dispersed sample barcode portions 911, 912, 913 attached thereto. Each sample barcode portion 911, 912, 913 can include a respective antibody 952, 954, 956 attached to a respective element tag 932, 934, 936, respectively. Each antibody 952, 954, 956 within a dispersed sample barcode system can be the same or different, so long as each antibody has a targeting function associated with the same sample cell 920. For example, antibody 952 can target CD45, antibody 954 can target CD298, and antibody 956 can target b2m. Isotopes throughout the sample barcode (e.g., hypothetical isotopes "A, B, and C") can be distributed across the various barcode portions 911, 912, 913. Thus, elemental tag 932 may contain isotope A, elemental tag 934 may contain isotope B, and elemental tag 936 may contain isotope C. Thus, when sample cell 920 is examined using an elemental analyzer, the same overall sample barcode of "A, B, C" is detected, even though the entire sample barcode is distributed across multiple barcode portions 911, 912, 913.

[0239]

[0256] 10 is a schematic diagram of a barcoded reagent 1008 and a reporter antibody 1019 according to certain embodiments of the present disclosure. The barcoded reagent 1008 can be a bead 1014, although other barcoded reagents can be used. The bead 1014 can include an assay barcode 1010 within the bead 1014, such as incorporated into the core of the bead 1014. The assay barcode 1010 can be a type of elemental tag that includes a unique isotope or unique combination of isotopes identifiable by elemental analysis. In some cases, the bead 1014 can include a surface 1016 that includes, binds to, or is functionalized to bind various attachments, such as a sample barcode 1012, a capture antibody 1018, and an assay-specific biomolecule.

[0240]

[0257] The surface 1016 of the bead 1014 can bind or be functionalized to bind to a specific assay-specific biomolecule, which can be an affinity reagent for a particular assay, e.g., a specific capture antibody 1018, or a non-antibody biomolecule. The affinity reagent can bind to a specific target analyte 1020 (e.g., a protein or other structure), thereby tagging the target analyte 1020 with the assay barcode 1010. Thus, after washing to remove unbound barcoded reagent, detection of the assay barcode 1010 indicates the presence of a target analyte 1020 to which the affinity reagent (e.g., capture antibody 1018 or assay-specific biomolecule 1050) has bound.

[0241]

[0258] In some cases, the surface 1016 of the beads 1014 can be bound to, contain, or functionalized to bind to a sample barcode 1012. The sample barcode 1012 can be an elemental tag containing a unique isotope or a unique combination of isotopes that can be identified using elemental analysis. A particular sample barcode 1012 bound to a bead 1014 can be specific to the sample from which the target analyte 1020 originated. By using multiple groups of sample barcodes with unique isotopes or combinations of isotopes, each original sample can be assayed using its own group of sample barcodes (e.g., mixed with the sample or bound to assay reagents 1008 and then mixed with the sample), and thus detection of a particular sample barcode 1012 can be used to identify which sample the target analyte 1020 originated from.

[0242]

[0259] In some cases, a reporter reagent 1090 can be used. The reporter reagent 1090 can include a reporter antibody 1019 that has targeting functionality for the target analyte 1020. The reporter antibody 1019 can bind to an elemental tag 1032 that includes an isotope or a unique combination of isotopes. Detection of the isotope or unique combination of isotopes associated with the elemental tag 1032 can indicate that the particle detected by the elemental analyzer, and therefore other isotopes detected within a particular time window, is associated with the target analyte 1020 and therefore associated with the assay bead 1014.

[0243]

[0260] Although multiple different reporter reagents 1090 can be used, each with a different type of reporter antibody that has targeting functionality for a variety of different target analytes, the reporter reagents 1090 can all contain the same type of elemental tag (e.g., the same isotope or combination of isotopes). Thus, each reporter reagent 1090 has an identical elemental tag 1032. Because the purpose of the reporter reagent 1090 is simply to determine whether a particular target (e.g., assay bead 1014) is being interrogated by the elemental analyzer, there is no need to distinguish between the different reporter reagents 1090.

[0244]

[0261] In some cases, the reporter antibody 1019 can be the same type of antibody (e.g., a monoclonal antibody) as the capture antibody 1018 of the assay bead 1014. In some cases, the reporter antibody 1019 is a different type of antibody (e.g., a polyclonal antibody) than the capture antibody 1018 of the assay bead 1014, but both the reporter antibody 1019 and the capture antibody 1018 have targeting functionality for the target analyte 1020.

[0245]

[0262] 11 is a schematic diagram illustrating the preparation of a lyophilized antibody panel 1166 according to certain embodiments of the present disclosure. The lyophilized antibody panel 1166 can initially begin as a set 1100 of element-tagged antibody groups 1152, 1154, 1156, 1158. The set 1100 can include any number of antibody groups. Each antibody group can include one or more element-tagged antibodies that share the same antibody and the same element tag. For a panel targeting six different targets, the set 1100 can include six different antibody groups, with each group including multiple individual element-tagged antibodies.

[0246]

[0263] The set 1100 of element-tagged antibodies 1152, 1154, 1156, 1158 can be prepared as needed, for example, as described herein. As an example, each of the element-tagged antibodies 1152, 1154, 1156, 1158 can be individually titrated and diluted with a stabilizer to achieve a desired concentration. The resulting solution can be individually combined with an excipient and mixed together to form an admixture 1164, which can be placed in a container 1162, such as a tube. Optionally, an auxiliary reagent 1160 can be added to the admixture 1164. Such an auxiliary reagent 1160 can include any reagent that can be lyophilized. For example, the auxiliary reagent 1160 can include a lysis reagent (e.g., a red blood cell lysis reagent), a wash buffer, a fixation reagent, and / or a permeabilization reagent. Optionally, the auxiliary reagent 1160 can include a barcoding reagent, such as an assay barcoding reagent or a sample barcoding reagent (e.g., a sample barcode), to the admixture 1164. Optionally, one or more calibration materials (eg, calibration beads) can be added as auxiliary reagents 1160 or materials.

[0247]

[0264] The blend 1164 can be subjected to a freeze-drying process as disclosed herein. For example, the blend 1164 can be subjected to a thermal stage, a vacuum stage, a drying stage, and a holding stage. During the thermal stage, the temperature can be maintained anywhere between -60 and 0°C while the vacuum is maintained in the range of 100 to 500 Torr. During the evacuation stage, the vacuum can be lowered to a range of 100 to 500 mTorr. During the drying stage, the temperature can be operated in the range of -50 to 30°C while the vacuum is maintained in the range of 10 to 150 mTorr. During the holding stage, the temperature can be maintained at a temperature of 10 to 30°C while the vacuum is increased to a range of 100 to 500 mTorr. After the holding stage, the container 1162 can be filled, which can include increasing the pressure within the container, for example, from 200 to 760 Torr, although in some cases, filling can be performed at a pressure below ambient pressure. In some cases, during all stages of freeze-drying, the temperature of the blend 1164 does not rise above the glass transition temperature of the blend 1164. During or after filling, the container 1162 may be filled with an internal atmosphere 1170 of inert gas or dry air and then sealed.

[0248]

[0265] After lyophilization, container 1162 can contain a lyophilized mixture 1168 that makes up a lyophilized panel 1166. This lyophilized panel 1166 can be stored and later resuspended for staining a sample.

[0249]

[0266] 12 is a schematic diagram of a process 1200 for preparing barcoded reagents 1276, 1278 according to certain embodiments of the present disclosure. The process 1200 can begin with an elemental tagging core 1272. The elemental tagging core 1272 can include any suitable elemental tag 1210, such as an elemental tag 1210 including a combination of isotopes selected from La, Ce, Eu, Ho, and Lu. The elemental tagging core 1272 can include the isotope as part of a solid metal core or a polymeric core that chelates or otherwise captures the metal isotope.

[0250]

[0267] A polymer precursor 1273 (e.g., a subunit) can be reacted with an element-tagged core 1272 to produce an element-tagged bead 1274 comprising an element-tagged core 1272 surrounded by a polymer shell 1275. This element-tagged bead 1274 can be reacted with either a capture antibody 1218 or an assay-specific biomolecule 1250.

[0251]

[0268] In some cases, element-tagged beads 1274 having a polymer shell 1275 can be reacted with a capture antibody 1218 having an exposed amine group (e.g., on the Fc region), allowing the capture antibody 1218 to form a covalent bond with the polymer shell 1275, thereby yielding an antibody-based element-tagged barcoding reagent 1276.

[0252]

[0269] In some cases, an element-tagged bead 1274 having a polymer shell 1275 can be reacted with an assay-specific biomolecule 1220 having an exposed amine group, allowing the assay-specific biomolecule 1220 to form a covalent bond with the polymer shell 1275, thereby yielding a biomolecule-based element-tagged barcoded reagent 1278.

[0253]

[0270] In some cases, the antibody-based element tagging barcoding reagent 1276 and / or the biomolecule-based element tagging barcoding reagent 1278 may be further tagged with a sample barcode, for example, using a sample barcode that includes highly reactive functional groups that can bind to the polymer shell 1275, displace the antibody 1218 or biomolecule 1250 from the polymer shell 1275, or bind to additional functional groups present on the barcoding reagent 1276, 1278.

[0254]

[0271] 13 is a flow chart illustrating a process 1300 for staining and analyzing a blood sample according to certain embodiments of the present disclosure. Although the process 1300 is disclosed with respect to analyzing a sample of whole blood, embodiments of the process 1300 can be used to analyze other samples as desired.

[0255]

[0272] In block 1302, a sample of whole blood is provided. The whole blood may be human peripheral blood, whether fresh or frozen. Optionally, in optional block 1304, the whole blood may be tagged with a sample barcode 1304.

[0256]

[0273] At block 1306, PBMCs can be isolated from the sample of whole blood. The PBMCs can be isolated using any suitable technique, such as via centrifugation. Optionally, at block 1308, the PBMCs can be optionally tagged with a sample barcode.

[0257]

[0274] In block 1310, PBMCs can be stained using a lyophilized panel, such as a lyophilized antibody panel or panel subset disclosed herein. In some cases, staining the PBMCs with the lyophilized panel can include recording information about the type of lyophilized panel used, which can be searched to determine the panel's mapping between element tags and targets. Staining the PBMCs can include mixing the lyophilized panel with the PBMCs at a constant temperature for a certain period of time, and then washing unbound antibody from the PBMCs. In some cases, in block 1312, the stained sample can optionally be tagged with a sample barcode.

[0258]

[0275] In optional block 1313, additional cell processing can be performed. The additional cell processing can include additional processing and / or staining steps, such as cell fixation, cell permeabilization, and / or intracellular staining. The additional cell processing in block 1313 can be performed after sample barcoding in block 1312, but does not necessarily have to be.

[0259]

[0276] Plasma may be isolated from the whole blood sample at block 1314. The plasma may be isolated using any suitable technique, such as via centrifugation. Optionally, the plasma may be optionally tagged with a sample barcode at block 1316.

[0260]

[0277] In block 1318, a free analyte bead assay using a reporter antibody is added to the plasma. The free analyte bead assay can include one or more barcoded reagents having an assay barcode and a capture antibody or assay-specific biomolecule to target free analytes in the plasma. Thus, capture antibodies or assay-specific biomolecules that bind to free analytes can tag those free analytes with their respective assay barcodes. The use of a reporter antibody can be optional. The reporter antibody can be used to identify the presence of beads or analytes. The reporter antibody can be selected to bind to all, a majority, or a majority of the beads or analytes. All reporter antibodies for a particular target can share the same elemental tag, such that detection of that elemental tag indicates the presence of that type of target (e.g., bead or analyte). Adding the free analyte bead assay in block 1318 can include mixing the plasma and the free analyte bead assay at a constant temperature for a constant time, and then washing away unbound beads from the plasma. In some cases, the stained plasma can be optionally tagged with a sample barcode in block 1320.

[0261]

[0278] In optional block 1322, PBMC and plasma samples can be pooled together. In some cases, if sample barcoding is applied in blocks 1304, 1308, 1312, 1316, and / or 1320, stained samples (e.g., PBMC and / or plasma) derived from the whole blood of block 1302 can be pooled with stained samples from other whole blood sources (e.g., whole blood from a different patient, or whole blood from the same patient drawn at a different time than the whole blood from block 1302). With sample barcoding, interrogation of the pooled samples can result in data including unique sample barcodes that can be used to separate the data by original sample source.

[0262]

[0279] At block 1324, the stained samples (e.g., PBMCs and / or plasma), whether optionally pooled at block 1322, can be interrogated using an elemental analyzer, such as a mass spectrometer (e.g., ICP-MS). The interrogation at block 1324 can yield elemental data about the samples.

[0263]

[0280] At block 1326, elemental data for the sample can be automatically analyzed. The automated analysis at block 1326 can include accessing a mapping of elemental tags to targets. The automated analysis at block 1326 can include identifying cells or particles of the sample. The automated analysis at block 1326 can include associating the identified cells or particles with a particular sample based on the detected elemental tags associated with the sample barcode. The automated analysis at block 1326 can include associating the identified cells or particles with a particular assay based on the detected assay barcode. The automated analysis at block 1326 can include associating the identified cells or particles with one or more particular markers based on detection of elemental tags associated with the one or more particular markers. In some cases, the automated analysis at block 1326 can include identifying a type of cell or particle based on the associated marker. In some cases, the automated analysis at block 1326 can include generating an output including information associated with the quantification or identification of cells or cell types in the sample. The automated analysis at block 1326 can include generating any suitable output from the elemental data as disclosed herein.

[0264]

[0281] Certain methods and kits may include only a subset of the blocks described in Figure 13. For example, sample barcoding may not be performed, but cells and assay beads from the same sample may still be combined before interrogation. Alternatively, sample barcoding of cells and / or assay beads (e.g., separately or mixed) may be performed at any step before interrogation, and pooling of cells and / or assay beads from different samples may occur at any time after sample barcoding and before interrogation. For example, sample barcodes may be introduced at any step, and sample pooling may occur at any subsequent step. PBMCs in serum and plasma may not need to be separated but instead applied to a lyophilized mixture of element-tagged antibodies and assay barcoded beads, allowing for a more streamlined workflow (although the quality of cell staining may be reduced due to the abundance of free analytes). Sample barcodes may be included in the mixture with the lyophilized mixture or provided separately (to be added to samples before or after staining with the lyophilized panel). In certain embodiments, a tissue sample or cell culture may be provided instead of whole blood in step 1302, and cell isolation in step 1306 is optional. In certain embodiments, step 1318 is the addition of a reporter reagent comprising a biomolecule other than an antibody. In examples where cells rather than assay beads are interrogated, the method may include (in order) steps 1302, 1308, 1310, optionally 1313, 1324, and 1326. In examples where assay beads rather than cells are interrogated, the method may include (in order) steps 1302, 1314, 1316, 1322, and 1324. In some examples, the reporter antibody may be added separately from (before or after) the addition of the bead assay, e.g., after the addition of the bead assay, including wash steps therebetween. In some examples, the cells are permeabilized prior to block 1310, and the lyophilized panel stain comprises antibodies against both extracellular and intracellular targets. In certain examples, cells (e.g., PBMCs) and free analytes (e.g., plasma) are isolated prior to adding the lyophilized panel and bead assay, respectively, and combined prior to sample barcoding and pooling across samples.In some cases, cells are sample barcoded and pooled before being added to the lyophilized panel.

[0265]

[0282] FIG. 14 is a schematic diagram illustrating an exemplary gating strategy 1400 for automatically analyzing elemental data, according to certain embodiments of the present disclosure. The exemplary gating strategy 1400 includes three layers of gating, although any number of layers can be used. Various two-dimensional spaces are used to represent cells or particles detected in a sample investigated using elemental analysis. The higher the presence of an isotope or marker associated with a cell or particle, the further to the right (on the x-axis) or up (on the y-axis) the dot representing the cell or particle will appear in the space. While the space shown in FIG. 14 is labeled with isotopes on the x- and y-axes, it is understood that each isotope can represent a specific marker, such as an antibody or antibody target. Furthermore, isotope compositions can be used instead of individual isotopes along the axes, such as when using isotope compositions to tag specific markers. While these two-dimensional spaces are shown as dot plots for illustrative purposes, the information can be stored and manipulated in any suitable manner to achieve a desired gating strategy.

[0266]

[0283] In a first layer, elemental data from a sample is shown in a two-dimensional space 1402 with isotope A on the x-axis and isotope B on the y-axis. This space 1402 can include two gates 1414, 1416. Objects (e.g., cells or particles) associated with high values ​​of isotope A and isotope B can fall within gate 1414, and objects associated with low values ​​of isotope A and isotope B can fall within gate 1416. In some cases, objects within gate 1414 and / or objects within gate 1416 can be labeled as particular types of cells or objects.

[0267]

[0284] In the second layer, objects within gate 1414 may be plotted on space 1404, and objects from gate 1416 are plotted on space 1410. Space 1404 may plot isotope C on the x-axis and isotope D on the y-axis. Space 1404 may be gated to separate objects with high values ​​for isotope D and low values ​​for isotope C into gate 1418, and objects with low values ​​for isotope D and high values ​​for isotope C into gate 1420. In space 1410, objects from space 1416 may be plotted with isotope E on the x-axis and isotope F on the y-axis. Space 1410 may include a single gate 1440 associated with objects with high values ​​for both isotopes E and F. In some cases, objects within gates 1418, 1420, and / or 1440 may be labeled as specific types of cells or particles.

[0268]

[0285] Objects within gates 1418, 1420, and / or 1440 can pass along a third layer. In the third layer, objects within gate 1418 are plotted on space 1406, objects within gate 1420 are plotted on space 1408, and objects within gate 1440 are plotted on space 1412. In space 1406, objects are plotted with isotope G on the x-axis and isotope H on the y-axis. These objects can be gated by associating gate 1422 with objects having high values ​​for both isotopes G and H, and gate 1424 with objects having high values ​​for isotope G and low values ​​for isotope H. Objects within gate 1422 and / or gate 1424 can be labeled as a particular type of object. In space 1408, objects from space 1420 are plotted with isotope C on the x-axis and isotope B on the y-axis. These objects may be gated by having gate 1426 associated with objects having high values ​​for isotope B and medium values ​​for isotope C, gate 1428 associated with objects having medium high values ​​for isotope B and low values ​​for isotope C, and gate 1430 associated with objects having medium values ​​for isotope B and low values ​​for isotope C. Objects within gates 1426, 1428 and / or 1430 may be labeled as particular types of objects. In space 1412, objects from space 1440 are plotted with isotope F on the x-axis and isotope D on the y-axis. These objects may be gated by having gate 1442 associated with objects having high values ​​for isotopes F and D, gate 1444 associated with objects having medium high values ​​for isotope D and high values ​​for isotope F, gate 1446 associated with objects having medium low values ​​for isotope D and high values ​​for isotope F, and gate 1448 associated with objects having low values ​​for isotopes D and F. Objects within gates 1442, 1444, 1446 and / or 1448 may be labeled as particular types of cells or objects.

[0269]

[0286] Thus, a gating scheme can involve determining a limited region within a multidimensional space (e.g., a two-dimensional space) from which data can be attributed to a particular outcome. The gating scheme can then involve applying subsequent gates to the data within the previously limited region based on a new multidimensional space (e.g., a different two-dimensional space). This process can be repeated as necessary to reach a desired level of differentiation. Thus, successive levels of gating can help narrow down specific cell types or target types based on the presence or absence of various markers. Because unique cell types or target types have different combinations of surface and / or intracellular markers, gating strategies can be defined to identify these various cell or target types based on the presence or absence of various markers.

[0270]

[0287] In some cases, instead of a limited region, cells or particles can be gated according to having marker expression greater or less than a threshold number. For example, an indication of high expression for a given target (e.g., CD45) can be expressed as any expression greater than or equal to a certain threshold. In some cases, different thresholds can be used for the same target at different levels of the gating scheme, or for the same target at the same level of the gating scheme when generated from different preceding gates.

[0271]

[0288] In one example, the mapping data can be used to convert elemental data into target expression. For a sample of cells, the data can be analyzed across a two-dimensional space where target expression of CD45 is on the y-axis and target expression of CD66b is on the x-axis. The region in the upper left corner of the space indicates high expression of CD45 and low or no expression of CD66b. Cells that fall in the upper left corner can be further analyzed across a new space based on target expression of CD56 and CD14. Cells that fall in the lower left corner of the space may show little or no expression of either CD56 or CD14. Cells that fall in that region can be further analyzed across a new space based on target expression of CD19 on the y-axis and CD3 on the x-axis. Cells that fall in the upper left axis may show high expression of CD19 and low or no expression of CD3. Cells that fall in that region can be identified as B cells. This exemplary gating strategy can be represented as CD45+CD66b-; CD56-CD14-; CD19+CD3-. In some cases, the cells within the region can be further analyzed along other dimensions to determine additional characteristics about the cells.

[0272]

[0289] Using the gating strategy notation from the example above, other gating strategies suitable for use with lyophilized antibody panels include the following strategies to identify the following cell types: for CD8 T cells (total; CD161lo / -): CD45+CD66b-; CD19-CD20-; CD14-CD11c-; CD45+CD3+; CD3+TCRgd-; CD4-CD8+; CD8+CD161lo / -; for CD4 T cells (total): CD45+CD66b-; CD19-CD20-; CD14-CD11c-; CD45+CD3+; CD3+TCRgd-; CD4+CD8-. For Tregs: CD45+CD66b-; CD19-CD20-; CD14-CD11c-; CD45+CD3+; CD3+TCRgd-; CD4+CD8-; CD4+CCR4+; CD45RA-CD45RO+; CD25hiCD127lo / -. For γδ T cells: CD4-CD8-: CD45+CD66b-; CD19-CD20-; CD14-CD11c-; CD45+CD3+; CD4-CD8-; CD3+TCRgd+. For total B cells: CD45+CD66b-; CD56-CD14-; CD19+CD3-. For total NK cells: CD45+CD66b-; CD19-CD20-; CD3-CD14-; CD45RA+CD123-; CD45+CD56+. Neutrophils: CD45lo CD66b+; CD294- CD16+. Total monocytes: CD45+ CD66b-; CD19- CD20-; CD3- CD56-; CD11c+ HLA-DR+; CD14+ / - CD11c+. Plasmacytoid dendritic cells: CD45+ CD66b-; CD19- CD20-; CD3- CD14-; HLA-DR+ CD56+ / -; CD123+ CD11c-. CD8 T cells: naive: CD45+ CD66b-; CD19- CD20-; CD14- CD11c-; CD45+ CD3+; CD3+ TCRgd-; CD4- CD8+; CD8+ CD161lo / -; CD8+ CCR7hi; CD45RA+ CD45RO-. For CD4 T cells, naive: CD45+CD66b-; CD19-CD20-; CD14-CD11c-; CD45+CD3+; CD3+TCRgd-; CD4+CD8-; CD4+CCR7hi; CD45RA+CD45RO-.Th1-like: CD45+CD66b-; CD19-CD20-; CD14-CD11c-; CD45+CD3+; CD3+TCRgd-; CD4+CD8-; CD4+CXCR5-; CD4+CCR4-; CD45RA-CD45RO+; CXCR3+CCR6-. MAIT / NKT CD4- cells: CD45+CD66b-; CD19-CD20-; CD14-CD11c-; CD45+CD3+; CD3+CD4-; CD28+CD161hi. Naive B cells: CD45+CD66b-; CD56-CD14-; CD19+CD3-; CD19+CD27+. For primary NK cells: CD45+CD66b-; CD19-CD20-; CD3-CD14-; CD45RA+CD123-; CD45+CD56+; CD56+CD57-. For eosinophils: CD45loCD66b+; CD294+CD16-. For classical monocytes: CD45+CD66b-; CD19-CD20-; CD3-CD56-; CD11c+HLA-DR+; CD14+ / -CD11c+; CD38+CD14hi. For myeloid dendritic cells: CD45+CD66b-; CD19-CD20-; CD3-CD14-; HLA-DR+CD56+ / -; CD123-CD11c+; CD11c+CD38+. For CD8 T cells, central memory: CD45+CD66b-; CD19-CD20-; CD14-CD11c-; CD45+CD3+; CD3+TCRgd-; CD4-CD8+; CD8+CD161lo / -; CD8+CCR7hi; CD45RA-CD45RO+. For CD4 T cells, central memory: CD45+CD66b-; CD19-CD20-; CD14-CD11c-; CD45+CD3+; CD3+TCRgd-; CD4+CD8-; CD4+CCR7hi; CD45RA-CD45RO+. Th2-like: CD45+CD66b-; CD19-CD20-; CD14-CD11c-; CD45+CD3+; CD3+TCRgd-; CD4+CD8-; CD4+CXCR5-; CD45RA-CCR4+; CXCR3-CCR6-. Memory B cells: CD45+CD66b-; CD56-CD14-; CD19+CD3-; CD19+CD27+.For late NK cells: CD45+CD66b-; CD19-CD20-; CD3-CD14-; CD45RA+CD123-; CD45+CD56+; CD56+CD57+. For basophils: CD45+CD66b-; CD19-CD20-; CD3-CD56-; HLA-DR-CD11c-; CD123+CD294+. For intermediate monocytes: CD45+CD66b-; CD19-CD20-; CD3-CD56-; CD11c+HLA-DR+; CD14+ / -CD11c+; CD38lo / -CD14int. For CD8 T cells, effector memory: CD45+CD66b-; CD19-CD20-; CD14-CD11c-; CD45+CD3+; CD3+TCRgd-; CD4-CD8+; CD8+CD161lo / -; CD8+CCR7lo / -; CD8+CD27+. For CD4 T cells, effector memory: CD45+CD66b-; CD19-CD20-; CD14-CD11c-; CD45+CD3+; CD3+TCRgd-; CD4+CD8-; CD4+CCR7lo / -; CD45RA-CD45RO+; CD45RO+CD27+. Th17-like: CD45+CD66b-; CD19-CD20-; CD14-CD11c-; CD45+CD3+; CD3+TCRgd-; CD4+CD8-; CD4+CXCR5-; CD45RA-CCR4+; CXCR3-CCR6+. Plasmablasts: CD45+CD66b-; CD56-CD14-; CD19+CD3-; CD19+CD27+; CD38+CD20-. Non-classical monocytes: CD45+CD66b-; CD19-CD20-; CD3-CD56-; CD11c+HLA-DR+; CD14+ / -CD11c+; CD38-CD14-. For CD8 T cells, terminal effector: CD45+CD66b-; CD19-CD20-; CD14-CD11c-; CD45+CD3+; CD3+TCRgd-; CD4-CD8+; CD8+CD161lo / -; CD8+CCR7lo / -; CD8+CD27-. For CD4 T cells, terminal effector: CD45+CD66b-; CD19-CD20-; CD14-CD11c-; CD45+CD3+; CD3+TCRgd-; CD4+CD8-; CD4+CCR7lo / -; CD45RA-CD45RO+; CD45RO+CD27-.

[0273]

[0290] FIG. 15 is a schematic diagram illustrating a technique 1500 for labeling samples with barcoded reagents and analyzing a set of samples, according to certain embodiments of the present disclosure. A set 1592 of barcoded reagents is provided. The set 1592 of barcoded reagents can include any number of barcoded reagents (e.g., like barcoded reagent 208 of FIG. 2) for any number of assays, optionally element-tagged with assay barcodes. The set 1592 of barcoded reagents can include barcoded reagents with a variety of different capture antibodies and / or assay-specific biomolecules. Three sets 1586, 1588, 1590 of sample barcodes can be provided. While FIG. 15 illustrates the use of three sets 1586, 1588, 1590 of sample barcodes, any number of sets of sample barcodes can be used, which can be predetermined by the number of available sets of sample barcodes and / or the number of different samples to be assayed. Each of the sets of sample barcodes 1586, 1588, 1590 can include several identical sample barcodes, such that the sample barcodes in the first set of sample barcodes 1586 are all identical and all unique compared to the second set of sample barcodes 1588, which are themselves all identical and all unique compared to the third set of sample barcodes 1590, which are themselves all identical. The three sets of sample barcodes 1586, 1588, 1590 and / or the set of barcoded reagents 1592 can be provided as a kit or as part of a kit that includes a lyophilized panel.

[0274]

[0291] The sets of sample barcodes 1586, 1588, 1590 can be individually combined with aliquots of the set of barcoded reagents 1592. Each aliquot of the set of barcoded reagents 1592 can contain a mixture of all different types of barcoded reagents from the set of barcoded reagents 1592. As a result of combining the sets of sample barcodes 1586, 1588, 1590 with the set of barcoded reagents 1592 in individual aliquots, three sets of sample-coded barcoded reagents 1593, 1594, 1595 can be created. The sample barcodes 1586, 1588, 1590 can be bound to the barcoded reagents 1592 or can simply be admixed with the barcoded reagents 1592. Thus, the first set of sample-coded barcoded reagents 1593 can contain barcoded reagents that are all tagged with or admixed with sample barcodes from the first set of sample barcodes 1586. The second set of sample-encoded barcoded reagents 1594 can include barcoded reagents that are all tagged with or mixed with sample barcodes from the second set of sample barcodes 1588. The third set of sample-encoded barcoded reagents 1595 can include barcoded reagents that are all tagged with or mixed with sample barcodes from the third set of sample barcodes 1590. Once the sample barcodes 1586, 1588, 1590 are used to tag the barcoded reagents 1592, excess sample barcodes can be washed from each mixture.

[0275]

[0292] Each of the sets of sample-encoded barcoding reagents 1593, 1594, 1595 can be mixed with a respective sample 1596, 1597, 1598 to tag the samples 1596, 1597, 1598. After washing away unbound barcoding reagents (and optionally unbound sample barcodes), the samples 1596, 1597, 1598 can be combined into a pooled sample 1599.

[0276]

[0293] Elemental analysis can be used to examine the pooled samples 1599, such as using an elemental analyzer (e.g., elemental analyzer 424 of FIG. 4). The resulting elemental data 1582 can be analyzed to detect the presence of elemental tags associated with sample barcodes from the three sets of sample barcodes 1586, 1588, 1590, thereby identifying the individual samples with which the sample barcodes are associated (e.g., samples 1596, 1597, 1598, respectively). An automated analyzer or processor (e.g., elemental data processor 430 of FIG. 4) can automatically separate the elemental data 1582 captured by the elemental analyzer into sample A data 1585 (e.g., elemental data associated with sample A 1596 tagged by a sample barcode from set of sample barcodes 1586), sample B data 1587 (e.g., elemental data associated with sample B 1597 tagged by a sample barcode from set of sample barcodes 1588), and sample C data 1589 (e.g., elemental data associated with sample C 1598 tagged by a sample barcode from set of sample barcodes 1590).

[0277]

[0294] Thus, many samples can be combined and analyzed simultaneously, which can improve overall investigation efficiency and help improve the reliability of data between samples, since all samples were investigated during the same run.

[0278]

[0295] FIG. 16 is a schematic diagram illustrating a technique 1600 for labeling samples and analyzing a set of samples according to certain embodiments of the present disclosure. A set 1692 of barcoded reagents is provided. The set 1692 of barcoded reagents can include any number of barcoded reagents (e.g., like barcoded reagent 208 of FIG. 2) for any number of assays, optionally element-tagged with assay barcodes. The set 1692 of barcoded reagents can include barcoded reagents with a variety of different capture antibodies and / or assay-specific biomolecules. Three sets 1686, 1688, 1690 of sample barcodes can be provided. While FIG. 16 illustrates the use of three sets 1686, 1688, 1690 of sample barcodes, any number of sets of sample barcodes can be used, which can be predetermined by the number of available sets of sample barcodes and / or the number of different samples to be assayed. Each of the sets of sample barcodes 1686, 1688, 1690 can include several identical sample barcodes, such that the sample barcodes in the first set of sample barcodes 1686 are all identical and all unique compared to the second set of sample barcodes 1688, which are themselves all identical and all unique compared to the third set of sample barcodes 1690, which are themselves all identical. The three sets of sample barcodes 1686, 1688, 1690 and / or the set of barcoded reagents 1692 can be provided as a kit or as part of a kit that includes a lyophilized panel.

[0279]

[0296] Sets of sample barcodes 1686, 1688, 1690 can be individually combined with respective samples 1696, 1697, 1698. In some cases, for example, if the sample barcodes are designed to bind to cells or particles in the sample itself, the samples 1696, 1697, 1698 can be washed to remove unbound sample barcodes. The samples 1696, 1697, 1698 can then be pooled together into a pooled sample 1699 that can be combined with set 1692 of barcoding reagents. The unbound barcoding reagents can then be washed, and the pooled sample 1699 can be investigated using elemental analysis, such as using an elemental analyzer (e.g., elemental analyzer 424 of FIG. 4). The resulting elemental data 1682 can be analyzed to detect the presence of elemental tags associated with sample barcodes from three sets of sample barcodes 1686, 1688, 1690, thereby identifying the individual samples with which the sample barcodes are associated (e.g., samples 1696, 1697, 1698, respectively). An automated analyzer or processor (e.g., elemental data processor 430 of FIG. 4) can automatically separate the elemental data 1682 captured by the elemental analyzer into sample A data 1685 (e.g., elemental data associated with sample A 1696 tagged by a sample barcode from set of sample barcodes 1686), sample B data 1687 (e.g., elemental data associated with sample B 1697 tagged by a sample barcode from set of sample barcodes 1688), and sample C data 1689 (e.g., elemental data associated with sample C 1698 tagged by a sample barcode from set of sample barcodes 1690).

[0280]

[0297] Thus, many samples can be combined and assayed together (e.g., assayed using barcoded reagents from barcoded reagent set 1692) and analyzed simultaneously, which can improve assay efficiency, overall interrogation efficiency, and can help improve the reliability of data between samples since all samples were assayed and interrogated during the same run.

[0281]

[0298] In some cases, similar to the technique 1500 described with reference to FIG. 15, each combination of sample barcodes from the three sets of sample barcodes 1686, 1688, 1690 and their respective samples 1696, 1697, 1698 can be individually combined with an aliquot of the set of barcoded reagents 1692 before washing and then pooled into a pooled sample 1699.

[0282]

[0299] FIG. 17 is a schematic diagram illustrating a technique 1700 for preparing a preconfigured set of sample-barcode labeled barcoded reagents according to certain embodiments of the present disclosure. A set 1792 of barcoded reagents is provided. The set 1792 of barcoded reagents can include any number of barcoded reagents (e.g., like barcoded reagent 208 of FIG. 2) for any number of assays, optionally element-tagged with assay barcodes. The set 1792 of barcoded reagents can include barcoded reagents with a variety of different capture antibodies and / or assay-specific biomolecules. Three sets 1786, 1788, 1790 of sample barcodes can be provided. While FIG. 17 illustrates the use of three sets 1786, 1788, 1790 of sample barcodes, any number of sets of sample barcodes can be used, which can be predetermined by the number of available sets of sample barcodes and / or the number of different samples to be assayed. Each of the sets of sample barcodes 1786, 1788, 1790 can include several identical sample barcodes, such that the sample barcodes in the first set of sample barcodes 1786 are all identical and all unique compared to the second set of sample barcodes 1788, which are themselves all identical and all unique compared to the third set of sample barcodes 1790, which are themselves all identical. The three sets of sample barcodes 1786, 1788, 1790 and / or the set of barcoded reagents 1792 can be provided as a kit or as part of a kit that includes a lyophilized panel.

[0283]

[0300] As shown in FIG. 17, barcoded reagent set 1792 includes seven different types of barcoded reagents (e.g., reagents t-z). Each different type of barcoded reagent represents a barcoded reagent having a unique assay barcode and a unique capture antibody or assay-specific biomolecule. Each different type of barcoded reagent in barcoded reagent set 1792 can be distributed across several wells down a respective column of well plate 1785. Thus, each well down a column shares the same type of barcoded reagent, but each well across a row contains a different barcoded reagent.

[0284]

[0301] Each set of sample barcodes 1786, 1788, 1790 may be distributed across a respective column of well plate 1785. Thus, each well across a row shares the same sample barcode, but each well down a column contains a different sample barcode.

[0285]

[0302] As a result of combining a sample barcode and a barcoding reagent within a well of well plate 1785, each well contains a unique combination of sample barcode and barcoding reagent (e.g., t:A; u:B through z:C). The sample barcode can optionally be bound to a barcoding reagent within the same well, or can simply be maintained in the mixture for subsequent binding to a sample cell or particle.

[0286]

[0303] Thus, a set of unique combinations of sample barcodes and barcoded reagents can be generated, and depending on the needs of the user, some or all of the set of unique combinations of sample barcodes and barcoded reagents can be provided to one or more samples to label the samples and, if desired, perform an assay.

[0287]

[0304] The foregoing description of embodiments, including the illustrated embodiment, has been presented only for purposes of illustration and description and is not intended to be exhaustive or to be limited to the precise form disclosed. Numerous modifications, adaptations and uses will be apparent to those skilled in the art.

[0288]

[0305] As used below, any reference to a series of examples should be understood disjunctively as a reference to each of those examples (e.g., "Examples 1-4" should be understood as "Examples 1, 2, 3, or 4").

[0289]

[0306] Example 1 is a panel for elemental analysis comprising a plurality of conjugated antibodies, each of the plurality of conjugated antibodies being tagged with a distinct elemental tag, each distinct elemental tag being distinguishable based on its isotopic composition, and the plurality of conjugated antibodies being in a lyophilized mixture.

[0290]

[0307] Example 2 is the panel of Example 1, wherein the plurality of conjugated antibodies comprises two or more antibodies from the list including: Cd45, CD45RA, CD45RO, Cd123, CD4, CD8a, CD11C, CD57, CXCR3, CD185, CD38, CD56, CD3, CD20, CD66b, HLA-DR, IgD, CD27, CD28, CD127, CD19, CD16, CD161, CD194, CD25, CD294, CD197, CD14, CCR6, and TCR delta gamma.

[0291]

[0308] Example 3 is a panel of Examples 1 or 2 in which the majority of the conjugated antibodies are specific for cell types in human peripheral blood.

[0292]

[0309] Example 4 is a panel of Examples 1-3 in which the majority of the conjugated antibodies are specific for cell surface markers.

[0293]

[0310] Example 5 is the panel of Examples 1-4, where the plurality of conjugated antibodies comprises 10 or more conjugated antibodies in the lyophilized mixture.

[0294]

[0311] Example 6 is a panel of Examples 1-5, where each distinct element tag contains multiple element atoms of one isotope.

[0295]

[0312] Example 7 is the panel of Examples 1-6, where at least two of the conjugated antibodies are tagged with distinct elemental tags having different isotopes of a single element.

[0296]

[0313] Example 8 is the panel of Examples 1-7 further comprising a biomolecule bound to an additional elemental tag, wherein the biomolecule is not an antibody and the additional elemental tag is distinguishable from each distinct elemental tag based on its isotopic composition.

[0297]

[0314] Example 9 is the panel of Examples 1-8, further comprising a non-antibody metal-containing moiety that comprises a metal isotope that is distinguishable from each distinct elemental tag based on its isotopic composition.

[0298]

[0315] Example 10 is the panel of Examples 1-9, where each distinct element tag includes a metal element having an atomic mass greater than 80 amu.

[0299]

[0316] Example 11 is a panel of Examples 1-10, where each distinct element tag comprises a chelated metal.

[0300]

[0317] Example 12 is the panel of Examples 1-11, where each distinct element tag comprises an element that is not endogenous to human peripheral blood.

[0301]

[0318] Example 13 is a panel of Examples 1-12, where the panel has a moisture content of 5% by weight or less.

[0302]

[0319] Example 14 is a panel of Examples 1-12, where the panel has a moisture content of 3% by weight or less.

[0303]

[0320] Example 15 is a panel of Examples 1-12, where the panel has a moisture content of 1% by weight or less.

[0304]

[0321] Example 16 is the panel of Examples 1-12, where the panel has a moisture content of 0.05% by weight or more and 1% by weight or less.

[0305]

[0322] Example 17 is the panel of Examples 1-16 further comprising a lyophilized intercalator, wherein the lyophilized intercalator is included in the lyophilized mixture.

[0306]

[0323] Example 18 is the panel of Examples 1-17, further comprising a lyophilized calibration material, wherein the lyophilized calibration material comprises known amounts of one or more known isotopes, and the lyophilized calibration material is included in a lyophilized mixture.

[0307]

[0324] Example 19 is the panel of Examples 1-18 further comprising an auxiliary reagent, wherein the auxiliary reagent is usable to perform an assay using a plurality of conjugated antibodies, wherein the auxiliary reagent is lyophilized, and wherein the auxiliary reagent is included in the lyophilization mixture.

[0308]

[0325] Example 20 is an assay kit for use with elemental analysis comprising a sealed container and the panel of Examples 1-19, wherein a lyophilized mixture of the panel is stored in the sealed container.

[0309]

[0326] Example 21 is the assay kit of Example 20, wherein the sealed container contains an internal atmosphere of inert gas or dry air.

[0310]

[0327] Example 22 is the assay kit of Example 20 or 21, wherein the plurality of conjugated antibodies comprises a first antibody specific for a cell surface marker and a second antibody specific for an intracellular target.

[0311]

[0328] Example 23 is the assay kit of Examples 20-22, further comprising an additional sealed container and an additional antibody panel comprising at least one additional conjugated antibody tagged with an additional distinct elemental tag distinguishable from each distinct elemental tag based on its isotopic composition, wherein the additional conjugated antibody is lyophilized and stored in the additional sealed container.

[0312]

[0329] Example 24 is the assay kit of Example 23, wherein the plurality of conjugated antibodies comprises antibodies specific for one or more cell surface markers, and an additional conjugated antibody is specific for an intracellular target.

[0313]

[0330] Example 25 is the assay kit of Examples 20-24, further comprising an intercalator comprising an additional distinct element tag distinguishable from each distinct element tag based on its isotopic composition.

[0314]

[0331] Example 26 is the assay kit of Examples 20-25, further comprising a plurality of sample barcoding reagents for labeling a plurality of samples, each of the plurality of sample barcoding reagents comprising a distinct combination of isotopes.

[0315]

[0332] Example 27 is the assay kit of Example 26, further comprising a plurality of containers, wherein each of the plurality of sample-barcoding reagents is contained within a separate container of the plurality of containers.

[0316]

[0333] Example 28 is the assay kit of Examples 26 or 27, wherein each of the plurality of sample-barcoding reagents binds to a majority of cells in the sample.

[0317]

[0334] Example 29 is the assay kit of Examples 26-28, wherein each of the plurality of sample barcoding reagents comprises an element tag functionalized for covalent attachment on or within a cell of the sample.

[0318]

[0335] Example 30 is the assay kit of Examples 26-29, wherein each of the plurality of sample-barcoded reagents specifically binds to a target present across a majority of cells in the sample, or comprises sample-barcoded antibodies that together bind to multiple targets across a majority of cells in the sample.

[0319]

[0336] Example 31 is the assay kit of Example 30, wherein each of the sample barcoded antibodies specifically binds to one or more of CD45, CD298, and b2m.

[0320]

[0337] Example 32 is the assay kit of Examples 30 or 31, wherein the elemental tag of the sample barcoded antibody provides a weaker signal when analyzed using an elemental analyzer than the elemental tags of most of the other antibodies in the panel.

[0321]

[0338] Example 33 is the assay kit of Examples 26-32, wherein the distinct combination of isotopes includes cadmium.

[0322]

[0339] Example 34 is the assay kit of Examples 26-33, where the distinct combination of isotopes includes platinum in cisplatin.

[0323]

[0340] Example 35 is the assay kit of Examples 26-34, wherein each of the plurality of sample-barcoded reagents comprises a set of sample-barcoded antibodies, each sample-barcoded antibody comprising all isotopes of a distinct combination of isotopes.

[0324]

[0341] Example 36 is the assay kit of Examples 26 to 35, wherein each of the plurality of sample barcoding reagents is capable of barcoding a living cell, and each of the plurality of sample barcoding reagents is non-toxic to the living cell.

[0325]

[0342] Example 37 is the assay kit of Examples 20-36, further comprising assay barcoding reagents comprising additional antibodies for detecting different analytes, wherein each assay barcoding reagent comprises a distinct combination of isotopes.

[0326]

[0343] Example 38 is the assay kit of Example 37, wherein each assay barcoded reagent is an assay barcoded bead containing a distinct combination of isotopes.

[0327]

[0344] Example 39 is the assay kit of Example 38, in which the assay barcoding reagents are included in the lyophilized mixture of the panel.

[0328]

[0345] Example 40 is the assay kit of Examples 38 or 39, wherein each assay barcoded bead comprises a unique combination of isotopes present within the assay barcoded bead.

[0329]

[0346] Example 41 is the assay kit of Examples 37-40, wherein the assay barcoding reagents include at least 10 assay barcoding reagents for barcoding at least 10 different analytes, and the assay barcoding reagents are provided premixed.

[0330]

[0347] Example 42 is the assay kit of Examples 37-41, where the different analyte is a free analyte in human peripheral blood.

[0331]

[0348] Example 43 is the assay kit of Examples 37-42, further comprising a combination of reporter antibodies that specifically bind to different analytes, each reporter antibody comprising an elemental tag detectable by elemental analysis.

[0332]

[0349] Example 44 is the assay kit of Example 43, wherein each of the elemental tags of the reporter antibody combination comprises an isotopically identical element detectable by elemental analysis.

[0333]

[0350] Example 45 is the assay kit of Examples 37-44, wherein each assay barcoding reagent is functionalized to attach to a sample barcode that includes an isotopic sample barcoding composition.

[0334]

[0351] Example 46 is the assay kit of Example 45, further comprising a sample barcode comprising an isotopic sample barcode composition.

[0335]

[0352] Example 47 is the assay kit of Examples 45 or 46, wherein the sample barcode is capable of binding to cells of the sample stained with the lyophilized panel.

[0336]

[0353] Example 48 is the assay kit of Examples 20-47, further comprising barcoding reagents, each barcoding reagent comprising an isotopic assay barcoded composition and an isotopic sample barcoded composition, each distinct isotopic assay barcoded composition associated with a distinct analyte, and each distinct isotopic sample barcoded composition associated with a distinct sample.

[0337]

[0354] Example 49 is the assay kit of Example 48, wherein each barcoded reagent is a bead and the isotopic sample barcoded composition is located inside the bead.

[0338]

[0355] Example 50 is the assay kit of Example 48, wherein each barcoded reagent is a bead and the isotopic sample barcoded composition is located on the surface of the bead.

[0339]

[0356] Example 51 is the assay kit of Examples 20 to 50, further comprising an anticoagulant.

[0340]

[0357] Example 52 is the assay kit of Examples 20-51, further comprising a calibration material, wherein the calibration material comprises a known amount of a known isotope.

[0341]

[0358] Example 53 is an assay kit for use with elemental analysis, comprising a plurality of sealed containers and the panel of Examples 1-19, wherein a lyophilized mixture of the panel is distributed across the plurality of sealed containers.

[0342]

[0359] Example 54 is the assay kit of Example 53, further comprising a plurality of sample barcoding reagents for labeling a plurality of samples, each of the plurality of sample barcoding reagents comprising a distinct combination of isotopes, and each of the plurality of sample barcoding reagents contained within a different one of the plurality of sealed containers.

[0343]

[0360] Example 55 is a barcoding system including a barcoding reagent comprising an assay barcode, wherein the assay barcode comprises an isotopic composition associated with a target analyte, the isotopic composition being distinguishable by elemental analysis, the barcoding reagent comprising one of a plurality of sample barcodes or functionalized to bind to at least one of a plurality of sample barcodes, each sample barcode of the plurality of sample barcodes comprising a unique additional isotopic composition distinguishable from the isotopic assay barcode composition by elemental analysis, and each sample barcode of the plurality of sample barcodes can be associated with a separate sample. The barcoding system may optionally further comprise a panel from Example 1 or a related example.

[0344]

[0361] Example 56 is the system of Example 55, where the barcoding reagent is a bead and, optionally, the sample barcode is internal to the bead.

[0345]

[0362] Example 57 is the system of Example 55, in which the barcoding reagents are beads, and the surface of the beads is functionalized to bind multiple sample barcodes.

[0346]

[0363] Example 58 is the system of Examples 55-57, where the barcoded reagent is a bead, and the surface of the bead contains one of a plurality of sample barcodes.

[0347]

[0364] Example 59 is the system of Examples 55-58, wherein the barcoding reagent is functionalized to bind to a plurality of sample barcodes, and optionally, the system further comprises each sample barcode of the plurality of sample barcodes in a separate container.

[0348]

[0365] Example 60 is the system of Examples 55-59, further comprising a sample barcoding reagent comprising at least one of the plurality of sample barcodes, optionally wherein the sample barcoding reagent is capable of binding to both the barcoding reagent and cells of the sample.

[0349]

[0366] Example 61 is the system of Examples 55-60, in which the barcoded reagent is a bead, the assay barcode is present inside the bead, and optionally the interior of the bead comprises a solid metal core, a metal chelating polymer interior, a nanocomposite interior, or a hybrid interior.

[0350]

[0367] Example 62 is a system of Examples 56-58 or 61 in which the beads have a solid metal core and a polymer surface.

[0351]

[0368] Example 63 is the system of Example 62, in which the polymer surface is bound to an antibody that binds to the target analyte.

[0352]

[0369] Example 64 is the system of Example 63, where the target analyte is a free analyte present in blood.

[0353]

[0370] Example 65 is the system of Examples 55-64, further comprising a reporter antibody that specifically binds to the target analyte and comprises an element tag or a combination of high intensity and low intensity element tags.

[0354]

[0371] Example 66 is the system of Example 65, wherein the assay barcoding reagents comprise at least 10 assay barcoding reagents for barcoding at least 10 different analytes, and optionally, each of the plurality of distinct mixtures of assay barcoding reagents comprises an isotopically distinguishable sample barcode. Example 67 is the system of Examples 65 or 66, further comprising a reporter biomolecule that specifically binds to the target analyte of the assay barcoding reagent, wherein the reporter biomolecule comprises an affinity reagent or an oligonucleotide, respectively, and each reporter biomolecule comprises an element tag or a combination of high signal and low signal element tags.

[0355]

[0372] Example 68 is the system of Examples 65-67, wherein at least some of the reporter biomolecules that specifically bind to different target analytes comprise the same element tag.

[0356]

[0373] Example 69 is a method including: providing a plurality of antibodies; conjugating each of the plurality of antibodies to a distinct elemental tag, wherein each distinct elemental tag is distinguishable based on its isotopic composition, and each of the plurality of antibodies is distinguishable by its distinct elemental tag; mixing the plurality of conjugated antibodies together to form an admixture; and lyophilizing the admixture.

[0357]

[0374] Example 70 is the method of Example 69, further comprising spin filtering the plurality of conjugated antibodies.

[0358]

[0375] Example 71 is the method of Example 69 or 70, further comprising: selecting an interrogation scheme for interrogating the sample; and selecting a plurality of antibodies based on the selected interrogation scheme.

[0359]

[0376] Example 72 is the method of Examples 69-71, wherein providing a plurality of antibodies includes providing two or more antibodies from the list including: CD45, CD45RA, CD45RO, Cd123, CD4, CD8a, CD11C, CD57, CXCR3, CD185, CD38, CD56, CD3, CD20, CD66b, HLA-DR, IgD, CD27, CD28, CD127, CD19, CD16, CD161, CD194, CD25, CD294, CD197, CD14, CCR6, and TCR delta gamma.

[0360]

[0377] Example 73 is the method of Examples 69-72, wherein each of the plurality of antibodies is specific to a cell type in human peripheral blood.

[0361]

[0378] Example 74 is the method of Examples 69-73, wherein each of the plurality of antibodies is specific for a cell surface marker.

[0362]

[0379] Example 75 is the method of Examples 69-74, wherein mixing the multiple conjugated antibodies together includes mixing 10 or more antibodies together.

[0363]

[0380] Example 76 is the method of examples 69-75, wherein each distinct element tag includes multiple element atoms of one isotope.

[0364]

[0381] Example 77 is the method of examples 69-76, wherein at least two of the distinct element tags comprise different isotopes of a single element.

[0365]

[0382] Example 78 is the method of Examples 69-77, further comprising providing a biomolecule comprising an additional elemental tag, wherein the additional elemental tag is distinguishable from each separate elemental tag based on its isotopic composition; and wherein the biomolecule is not an antibody, and wherein mixing the multiple conjugated antibodies together further comprises mixing the biomolecule with the multiple conjugated antibodies.

[0366]

[0383] Example 79 is the method of examples 69-78, wherein each distinct element tag comprises a metal element having an atomic mass greater than 80 amu.

[0367]

[0384] Example 80 is the method of Examples 69-79, wherein each distinct element tag comprises a chelated metal.

[0368]

[0385] Example 81 is the method of Examples 69-80, wherein each distinct element tag comprises an element that is not endogenous to human peripheral blood.

[0369]

[0386] Example 82 is the method of Examples 69-81, wherein freeze-drying the blend includes reducing the water content to 5% or less by weight.

[0370]

[0387] Example 83 is the method of Examples 69-82, further comprising mixing an intercalator with the blend prior to lyophilizing the blend, wherein the intercalator comprises an additional elemental tag that is distinguishable from each separate elemental tag based on its isotopic composition.

[0371]

[0388] Example 84 is the method of Examples 69-83, further comprising mixing a calibration material with the admixture before lyophilizing the admixture, wherein the calibration material comprises a known amount of a known isotope.

[0372]

[0389] Example 85 is the method of Examples 69-84, further comprising mixing an auxiliary reagent with the admixture before lyophilizing the admixture, wherein the auxiliary reagent can be used to facilitate performing an assay using multiple conjugated antibodies.

[0373]

[0390] Example 86 is the method of Examples 69-85, wherein lyophilizing the mixture further comprises storing the lyophilized mixture in a sealed container having an internal atmosphere of inert gas or dry air.

[0374]

[0391] Example 87 is the method of Examples 69-86, wherein the plurality of antibodies comprises a first antibody specific for a cell surface marker and a second antibody specific for an intracellular target.

[0375]

[0392] Example 88 is the method of Examples 69-87, further including providing at least one additional antibody; conjugating the at least one additional antibody to at least one additional distinct elemental tag distinguishable from each distinct elemental tag based on its isotopic composition; lyophilizing the at least one additional antibody; and storing the lyophilized at least one additional antibody separately from the lyophilized admixture.

[0376]

[0393] Example 89 is the method of Example 88, wherein the plurality of antibodies includes antibodies specific for one or more cell surface markers, and the additional antibody is specific for an intracellular target.

[0377]

[0394] Example 90 is the method of Examples 69-89, further comprising providing a plurality of sample barcoding reagents for labeling the plurality of samples, wherein each of the plurality of sample barcoding reagents comprises a distinct combination of isotopes.

[0378]

[0395] Example 91 is the method of example 90, further comprising providing a plurality of containers; and storing each of the plurality of sample barcoding reagents in a different container of the plurality of containers.

[0379]

[0396] Example 92 is the method of Example 90 or 91, wherein each of the plurality of sample barcoding reagents binds to a majority of cells in the sample.

[0380]

[0397] Example 93 is the method of Examples 90-92, wherein each of the plurality of sample barcoding reagents comprises an element tag functionalized to covalently or otherwise permanently bind on or within a cell of the sample.

[0381]

[0398] Example 94 is the method of Examples 90-93, wherein each of the plurality of sample-barcoding reagents comprises a sample-barcoding antibody that specifically binds to a target present across a majority of cells in the sample.

[0382]

[0399] Example 95 is the method of Example 94, wherein each of the sample barcoded antibodies specifically binds to one or more of CD45, CD298, and b2m.

[0383]

[0400] Example 96 is the method of Example 94 or 95, wherein each of the sample barcoded antibodies specifically binds to a target present in the sample selected such that the mass signal of the single sample barcoded antibody is weaker than the mass signals of the majority of the multiple conjugated antibodies.

[0384]

[0401] Example 97 is the method of Examples 90-96, wherein the distinct combination of isotopes includes cadmium.

[0385]

[0402] Example 98 is the method of Examples 90-97, wherein the distinct combination of isotopes includes platinum in cisplatin.

[0386]

[0403] Example 99 is the method of Examples 90-98, wherein each of the plurality of sample barcoding reagents comprises a set of sample barcoding antibodies, and each sample barcoding antibody comprises all isotopes of a distinct combination of isotopes.

[0387]

[0404] Example 100 is the method of Examples 90-99, wherein each of the plurality of sample barcoding reagents is capable of barcoding a living cell, and each of the plurality of sample barcoding reagents is non-toxic to the living cell.

[0388]

[0405] Example 101 is the method of Examples 69-100, further comprising providing assay barcoding reagents comprising additional antibodies for detecting different analytes, wherein each assay barcoding reagent comprises a distinct combination of isotopes.

[0389]

[0406] Example 102 is the method of example 101, wherein each assay barcoded reagent is an assay barcoded bead containing a distinct combination of isotopes.

[0390]

[0407] Example 103 is the method of example 102, wherein each assay barcoded bead comprises a unique combination of isotopes present within the assay barcoded bead.

[0391]

[0408] Example 104 is the method of Examples 101-103, wherein the assay barcoding reagents include at least 10 assay barcoding reagents for barcoding at least 10 different analytes, and the assay barcoding reagents are provided in admixture.

[0392]

[0409] Example 105 is the method of Examples 101-104, wherein the different analyte is a free analyte in human peripheral blood.

[0393]

[0410] Example 106 is the method of Examples 101-105, further comprising providing a combination of reporter antibodies that specifically bind to different analytes, wherein each reporter antibody comprises an elemental tag detectable by elemental analysis.

[0394]

[0411] Example 107 is the method of Example 106, wherein each of the elemental tags of the reporter antibody combination comprises an isotopically identical element detectable by elemental analysis.

[0395]

[0412] Example 108 is the method of Examples 101-107, further comprising functionalizing each assay barcoding reagent to attach to a sample barcode comprising an isotopic sample barcoding composition.

[0396]

[0413] Example 109 is the method of Example 108, wherein the sample barcode is bound to cells of the sample being assayed by a lyophilized admixture.

[0397]

[0414] Example 110 is the method of example 108 or 109, further comprising providing a sample barcode comprising an isotopic sample barcoding composition.

[0398]

[0415] Example 111 is the method of Examples 69-110, further comprising providing barcoding reagents, wherein each barcoding reagent comprises an isotopic assay barcoded composition and an isotopic sample barcoded composition, wherein each distinct isotopic assay barcoded composition is associated with a distinct analyte and each distinct isotopic sample barcoded composition is associated with a distinct sample.

[0399]

[0416] Example 112 is the method of example 111, wherein each barcoding reagent is a bead and the isotopic sample barcoding composition is located inside the bead.

[0400]

[0417] Example 113 is the method of Examples 111 or 112, wherein each barcoding reagent is a bead and the isotopic sample barcoding composition is located on the surface of the bead.

[0401]

[0418] Example 114 is the method of Examples 69-113, further comprising providing an anticoagulant.

[0402]

[0419] Example 115 is the method of Examples 69-114, further comprising titrating and diluting each of the plurality of conjugated antibodies to a predetermined concentration prior to combining the plurality of conjugated antibodies.

[0403]

[0420] Example 116 is the method of Examples 69-115, further comprising combining the plurality of conjugated antibodies with an excipient before lyophilizing the mixture.

[0404]

[0421] Example 117 is the method of Example 116, wherein the excipients include a sugar and bovine serum albumin.

[0405]

[0422] Example 118 is the method of Examples 116 or 117, further comprising combining the plurality of conjugated antibodies with a viability stain before lyophilizing the mixture.

[0406]

[0423] Example 119 is the method of Example 118, wherein the viability stain is a rhodium intercalator.

[0407]

[0424] Example 120 is a method including: preparing a sample; providing a lyophilized antibody panel comprising a plurality of conjugated antibodies, wherein each of the plurality of conjugated antibodies is tagged with a distinct elemental tag, and each distinct elemental tag is distinguishable based on its isotopic composition; performing surface staining on cells of the sample using the lyophilized antibody panel; and interrogating the sample using elemental analysis to detect the presence of the distinct elemental tags.

[0408]

[0425] Example 121 is the method of example 120, wherein investigating the sample using elemental analysis includes processing the sample with an inductively coupled plasma mass spectrometer to detect the presence of distinct elemental tags of the lyophilized antibody panel.

[0409]

[0426] Example 122 is the method of Examples 120 or 121, further comprising staining the sample of cells with a viability stain.

[0410]

[0427] Example 123 is the method of Examples 120-122, wherein the viability stain is provided as part of a lyophilized antibody panel.

[0411]

[0428] Example 124 is the method of Example 123, wherein the viability stain is rhodium.

[0412]

[0429] Example 125 is the method of Examples 120-124, further comprising FcR blocking the sample of cells.

[0413]

[0430] Example 126 is the method of Examples 120-125, further comprising fixing the sample after performing the surface staining.

[0414]

[0431] Example 127 is the method of Examples 120-126, further comprising staining the sample with an intercalator, wherein the intercalator comprises an additional distinct element tag that is distinguishable from each distinct element tag based on its isotopic composition.

[0415]

[0432] Example 128 is the method of Example 127, wherein the sample is stained with an intercalator after permeabilizing the sample.

[0416]

[0433] Example 129 is the method of Examples 120-128, further including permeabilizing the sample and performing intracellular staining on the sample of cells using at least one additional antibody, wherein the at least one additional antibody is tagged with an additional distinct elemental tag that is distinguishable from each distinct elemental tag based on its isotopic composition.

[0417]

[0434] Example 130 is the method of Examples 120-129, wherein preparing the sample includes collecting whole blood.

[0418]

[0435] Example 131 is the method of Example 130, wherein preparing the sample includes isolating peripheral blood mononuclear cells from whole blood.

[0419]

[0436] Example 132 is the method of Examples 120-131, further comprising labeling the sample using a sample barcoding reagent, wherein the sample barcoding reagent comprises a distinct combination of isotopes that can be used to distinguish the sample barcoding reagent from additional sample barcoding reagents.

[0420]

[0437] Example 133 is the method of example 132, wherein investigating the sample includes acquiring data by elemental analysis, identifying the sample barcoding reagent by distinct combinations of isotopes in the acquired data, and associating the acquired data with the sample.

[0421]

[0438] Example 134 is the method of example 133, wherein investigating the sample further comprises mixing the sample with an additional sample before obtaining data by elemental analysis.

[0422]

[0439] Example 135 is the method of Examples 120-134, wherein performing the surface staining includes adding a suspension of cells from the sample to the lyophilized antibody panel or resuspending the lyophilized antibody panel; and removing unbound antibody.

[0423]

[0440] Example 136 is the method of Examples 120-135, further comprising providing assay barcoding reagents comprising additional antibodies for detecting different analytes, wherein each assay barcoding reagent comprises a distinct combination of isotopes; and mixing the assay barcoding reagents with the sample and removing unbound antibody before interrogating the sample.

[0424]

[0441] Example 137 is the method of Example 136, wherein the sample comprises plasma and the different analytes are free analytes within the plasma.

[0425]

[0442] Example 138 is the method of Example 136 or 137, wherein providing the assay barcoded reagents and providing the lyophilized antibody panel is carried out by providing an admixture of the lyophilized antibody panel and the assay barcoded reagents.

[0426]

[0443] Example 139 is the method of Examples 136-138, wherein preparing the sample includes collecting whole blood.

[0427]

[0444] Example 140 is the method of Example 139, wherein preparing the sample further comprises isolating peripheral blood mononuclear cells and plasma; performing surface staining comprises mixing a lyophilized antibody panel with the peripheral blood mononuclear cells and removing unbound antibodies; and mixing the assay barcoded reagents with the sample comprises mixing the assay barcoded reagents with the plasma and removing unbound antibodies.

[0428]

[0445] Example 141 is the method of Examples 120-140, wherein examining the sample further includes automatically identifying cell viability.

[0429]

[0446] Example 142 is the method of Examples 120-141, wherein examining the sample further includes automatically identifying the cell population.

[0430]

[0447] Example 143 is the method of Example 142, wherein examining the sample further comprises identifying characteristics of the automatically identified cell populations.

[0431]

[0448] Example 144 is the method of Example 143, wherein investigating the sample further includes comparing the identified features across the identified cell populations, or comparing the identified feature associated with one of the identified cell populations with an identified additional feature associated with the same identified cell population from an additional sample.

[0432]

[0449] Example 145 is the method of Example 143 or 144, wherein identifying the characteristics of the automatically identified cell population includes determining the abundance of one or more targets on or within the cells of the identified cell population.

[0433]

[0450] Example 146 is the method of Examples 143-145, wherein the identified characteristic comprises a proportion of cells in the cell population.

[0434]

[0451] Example 147 is the method of Examples 120-146, wherein investigating the sample further includes generating at least one of a histogram, a 2D dot plot, and a tSNE graph based on the known targets of the lyophilized antibody panel.

[0435]

[0452] Example 148 is the method of Example 147, further comprising automatically accessing a stored mapping of known targets of the lyophilized antibody panel, wherein the stored mapping associates the known targets with associated mass channels.

[0436]

[0453] Example 149 is the method of Examples 136-148, further comprising: labeling the sample with a sample barcoding reagent, wherein the sample barcoding reagent comprises a distinct combination of isotopes that can be used to distinguish the sample barcoding reagent from the additional sample barcoding reagent; providing an additional sample; labeling the additional sample with the additional sample barcoding reagent; performing additional surface staining on additional cells of the additional sample using a lyophilized antibody panel; mixing the assay barcoding reagent with the additional sample and removing unbound antibody; and mixing the sample with the additional sample before interrogating the sample, wherein interrogating the sample comprises interrogating an admixture of the sample and the additional sample.

[0437]

[0454] Example 150 is the method of Example 149, wherein the sample is mixed with additional sample before performing the surface staining.

[0438]

[0455] Example 151 is the method of Example 149 or 150, wherein the assay barcoding reagent is functionalized to bind to the sample barcoding reagent; labeling the sample with the sample barcoding reagent comprises binding the sample barcoding reagent to a first portion of the assay barcoding reagent such that the second portion of the assay barcoding reagent does not include the sample barcoding reagent; mixing the assay barcoding reagent with the additional sample comprises mixing the second portion of the assay barcoding reagent with the additional sample; and mixing the sample with the additional sample occurs after mixing the assay barcoding reagent with the additional sample.

[0439]

[0456] Example 152 is the method of examples 120-151, wherein investigating the sample includes obtaining data associated with the sample using an elemental analyzer, and the method further includes automatically analyzing the data.

[0440]

[0457] Example 153 is the method of example 152, wherein automatically analyzing the data includes applying a cleanup model to the data, and applying the cleanup model includes accessing Gaussian measurements generated by an elemental analyzer associated with ionization of the sample.

[0441]

[0458] Example 154 is the method of Example 152 or 153, wherein automatically analyzing the data includes accessing an element tag assignment model, where the element tag assignment model includes information associating each of the distinct element tags of the lyophilized antibody panel with a cell type; identifying presence information for the distinct element tags of the lyophilized antibody panel; and, for each cell of the sample, determining the cell type using the identified presence information for the distinct element tags and the element tag assignment model.

[0442]

[0459] Example 155 is a barcoding kit for elemental analysis comprising: a plurality of sample barcodes for labeling a plurality of samples, each of the sample barcodes comprising a distinct combination of isotopes distinguishable by elemental analysis, each of the sample barcodes being stored in a separate container; and a set of biomolecules capable of binding to the plurality of samples, the set of biomolecules comprising the plurality of sample barcodes or functionalized to bind to the plurality of sample barcodes.

[0443]

[0460] Example 156 is the barcoding kit of Example 155, wherein each of the sets of biomolecules includes a unique one of the plurality of sample barcodes.

[0444]

[0461] Example 157 is the barcoding kit of Example 155 or 156, wherein each of the set of biomolecules is functionalized to bind to a plurality of sample barcodes, and the set of biomolecules is stored separately from the plurality of sample barcodes.

[0445]

[0462] Example 158 is the barcoding kit of Examples 155-157, wherein the set of biomolecules includes a plurality of beads.

[0446]

[0463] Example 159 is the barcoding kit of Example 158, wherein each bead comprises an exterior surface functionalized to bind multiple sample barcodes.

[0447]

[0464] Example 160 is the barcoding kit of Example 159, wherein each bead comprises an assay barcode within the bead, and each assay barcode comprises an additional combination of isotopes that is distinguishable by elemental analysis from the distinct combination of isotopes of the sample barcode.

[0448]

[0465] Example 161 is a method including: providing a plurality of samples including a first sample and a second sample; providing a plurality of sample barcodes including a first sample barcode and a second sample barcode, wherein each of the sample barcodes includes a distinct combination of isotopes distinguishable by elemental analysis; providing a plurality of biomolecules capable of binding to the plurality of samples, wherein each biomolecule includes one of the plurality of sample barcodes or is functionalized to bind to the plurality of sample barcodes, and wherein the plurality of biomolecules includes a first biomolecule and a second biomolecule; mixing the first biomolecule with the first sample; mixing the second biomolecule with the second sample; removing unbound biomolecules; interrogating the plurality of samples using elemental analysis to obtain elemental data; detecting the presence of each distinct combination of isotopes in the elemental data; and associating the elemental data with one of the plurality of samples using the detected presence of each distinct combination of isotopes.

[0449]

[0466] Example 162 is the method of Example 161, further comprising: mixing the first sample barcode with a mixture comprising the first biomolecule and the first sample; and mixing the second sample barcode with a mixture comprising the second biomolecule and the second sample.

[0450]

[0467] Example 163 is the method of Example 161, further comprising: mixing the first sample barcode with the first biomolecule before mixing the first biomolecule with the first sample; and mixing the second sample barcode with the second biomolecule before mixing the second biomolecule with the second sample.

[0451]

[0468] Example 164 is the method of Examples 161-163, further comprising pooling the first sample and the second sample prior to interrogating the plurality of samples.

[0452]

[0469] Example 165 is the method of Examples 161-164, wherein the plurality of biomolecules comprises a plurality of beads.

[0453]

[0470] Example 166 is the method of example 165, wherein each bead comprises an exterior surface functionalized to bind multiple sample barcodes.

[0454]

[0471] Example 167 is the method of Example 166, wherein each bead comprises an assay barcode within the bead, and each assay barcode comprises an additional combination of isotopes that is distinguishable by elemental analysis from the distinct combinations of isotopes in the sample barcode.

Claims

[Claim 1] Multiple conjugated antibodies 1. A panel for elemental analysis comprising: a plurality of conjugated antibodies, each of which is tagged with a distinct elemental tag, each distinct elemental tag being distinguishable based on its isotopic composition; and wherein the plurality of conjugated antibodies is in a lyophilized mixture.

Images