Reusable cartridge for detecting an analyte in solution - Patent Application 20070122997

Abstract

The present disclosure relates to methods for preparing reusable, desiccant cartridges containing a matrix containing detection molecules, such as antibodies, for the detection or enrichment of analytes in a sample, such as antigens recognized by antibodies. In some embodiments, the cartridge includes a tube or well that holds the matrix. In some embodiments, the matrix or associated cartridge may be stored wet or dry. The present disclosure also relates to cartridges and methods of use thereof.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the benefit of priority of U.S. Provisional Application No. 63 / 116,575, filed on November 20, 2020, which is hereby incorporated by reference in its entirety for any purpose.

[0002] The present disclosure relates to a method for preparing a reusable and dryable cartridge comprising a matrix comprising a detection molecule such as an antibody for the detection or enrichment of an analyte in a sample such as an antigen recognized by the antibody. In some embodiments, the cartridge comprises a tube or well that holds the matrix. In some embodiments, the matrix or associated cartridge may be stored wet or dry. The present disclosure also relates to the cartridge and methods of using it.

Background Art

[0003] To detect a specific analyte in a solution sample, various commercially available matrices can be used. For example, a matrix comprising a material coated with Protein A, Protein G, and / or Protein L can be used to capture the antibody constant regions on the matrix and detect and enrich them compared to other components in the solution. The matrix can be coated directly or via Protein A, Protein G, and / or Protein L on the matrix with an antibody, for example, to detect or enrich an antigen analyte in the sample. For example, the matrix can be coated with an antigen to detect or enrich antibodies against those antigens. A matrix coated with a hapten can also be used to detect an analyte in a solution. The materials forming the matrix, such as resins, beads, etc., are diverse.

[0004] However, many matrices and cartridges that hold such matrices cannot be efficiently reused, for example, when used to detect analytes in solution, particularly in cell lysates, and are discarded after a single use. As a result, the use of such cartridges in routine screening experiments, for example, can be very costly, as it requires a considerable amount of continuous acquisition of protein reagents to coat the cartridges and the regular preparation of fresh cartridges, which is quite time-consuming. For example, certain detection molecules may need to be bound to the matrix before they can be prepared and used, which may require several process steps. Also, some of the necessary reagents, such as commercially available antibodies or antigens, can be very expensive to purchase in the quantities needed to prepare a sufficient amount of coated matrix. Therefore, if the matrix can be reused, a great deal of time and resources can be saved.

[0005] This disclosure describes, among other things, a method for preparing a cartridge containing a matrix that can be reused, for example several times, and can be stored dry between uses for ease of handling, for the detection of analytes in solutions, including in complex solutions such as cell lysates, and for associated reusable cartridges. This disclosure also describes exemplary methods of using the cartridge described herein for the detection of specific analytes in solution samples, such as major histocompatibility complex class I (MHC-I) molecules and other proteins in biological fluids and cell lysates. [Overview of the project]

[0006] This disclosure includes, in particular, a reusable cartridge for detecting an analyte in solution, the cartridge comprising: a) a matrix; b) a detection molecule bound to the matrix, specifically bound to the analyte and optionally crosslinked to the matrix; and c) at least one cryoprotectant, the cartridge being usable at least 10, 20, 50, or 100 times for detecting an analyte in solution, and after each use, the matrix is ​​optionally stored dry with the cryoprotectant. In some embodiments, the cartridge is a tube with both ends open, a tube with one end open, a well, a plate with or without wells, or a chip capable of containing the matrix. In some embodiments, the matrix comprises particles (e.g., beads, grains, chips, or pellets) containing silica or agarose, the particles optionally being magnetic. In some cases, the matrix comprises protein A, protein G, and / or protein L. In some cases, the detection molecule is an antibody. In some cases, the detection molecule is crosslinked to the matrix. In some cases, two or more detection molecules, such as two different antibodies for detecting two different antigen analytes in solution, are bound to the matrix. In some embodiments, the detection molecules are crosslinked to the matrix with a crosslinking agent selected from dimethyl pimerimidate (DMP), cyanate esters, NHS esters, azulactone, carbonyldiimidazole (CDI), maleimide, iodoacetyl, pyridyl disulfide, hydrazide, or carbodiimide. In some embodiments, the analytes detectable by the detection molecules on the matrix are proteins or peptides. For example, in some cases, the analyte is a major histocompatibility complex I (MHC-I) molecule. In some cases, the cartridge can be used at least 10 times for the detection of protein analytes in cell lysates or biological fluids. In some cases, the reusable cartridge is stored in a dry state. In some embodiments, the cartridge can be stored in a dry state after preparation, then stored in a wet state after each use, and then used at least 10 times for the detection of protein analytes in cell lysates or biological fluids.In some embodiments, the cartridge may be used at least 10 times for the detection of protein analytes in cell lysates or biological fluids after being stored dry after each use. In some embodiments, the cartridge may be stored dry after preparation, and then used at least 20 times for the detection of protein analytes in cell lysates or biological fluids after being stored wet after each use. In some cases, the cartridge may be used at least 20 times for the detection of protein analytes in cell lysates or biological fluids after being stored dry after each use. In some embodiments, the cartridge may be stored dry after preparation, and then used at least 50 times for the detection of protein analytes in cell lysates or biological fluids after being stored wet after each use. In some cases, the cartridge may be used at least 50 times for the detection of protein analytes in cell lysates or biological fluids after being stored dry after each use. In some embodiments, the cartridge may be stored dry after preparation, and then used at least 100 times for the detection of protein analytes in cell lysates or biological fluids after being stored wet after each use. In some cases, the cartridge may be used at least 100 times for the detection of protein analytes in cell lysates or biological fluids after being stored dry after each use. In some embodiments, the matrix is ​​stored at an acidic pH. In some embodiments, the matrix is ​​stored at 2–8°C. In some embodiments, the matrix is ​​stored in a humid state. In some embodiments, the matrix is ​​stored at an acidic pH, optionally at 2–8°C. In some cases, the cryoprotectant includes one or more of sucrose, trehalose, ethylene glycol, propylene glycol, glycerol, 2-methyl-2,4-pentanediol (MPD), or dimethyl sulfoxide (DMSO).

[0007] The Disclosure also relates, for example, to a method for preparing a previously used cartridge for reuse, as described above or elsewhere in this Specification, comprising washing the matrix in 1-10% acetic acid, 1% formic acid, or 1% trifluoroacetic acid (TFA) after the analyte has eluted from the cartridge; washing the matrix in a buffer containing a cryoprotectant; and drying the matrix. In some embodiments, the cryoprotectant comprises one or more of sucrose, trehalose, ethylene glycol, propylene glycol, glycerol, 2-methyl-2,4-pentanediol (MPD), or dimethyl sulfoxide (DMSO). In some cases, the matrix is ​​dried by heating the cartridge to at least 30°C and then storing it at room temperature for at least 1 hour. In some cases, the matrix is ​​dried by heating the cartridge to 37°C and then storing it at room temperature for at least 1 hour. In some cases, the matrix is ​​dried and stored in a dry state at 2-8°C until the cartridge is to be reused.

[0008] This disclosure further relates to a method for preparing a reusable cartridge, as described above or separately herein, comprising obtaining a cartridge containing a matrix, contacting the matrix with a detection molecule, and crosslinking the detection molecule to the matrix.

[0009] This disclosure further relates to a method for detecting an analyte in a solution, comprising contacting the matrix of a reusable cartridge, as described above or separately herein, with a solution containing the analyte, and optionally eluting the analyte from the cartridge. In some embodiments, the analyte is a peptide or protein. In some embodiments, the analyte is an MHC-I molecule. In some cases, the solution is a biological fluid or cell lysate. In some cases, the solution is filtered or treated to remove cellular debris and membrane material before contact with the matrix. In some cases, the cartridge has been previously used at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, or 100 times to detect the analyte before use in the method. In some cases, following the elution of the analyte, the cartridge is treated by a previously used cartridge, e.g., a method for preparing the cartridge described above.

[0010] The Disclosure also comprises a kit comprising at least one reusable cartridge as described herein, as described above. In some embodiments, the kit comprises at least one cartridge as described herein, (a) at least one buffer, (b) at least one control cartridge that does not contain a detection molecule or contains a control detection molecule, (c) reagents for preparing the cartridge for storage and reuse, and / or (d) instructions for use.

[0011] It should be understood that the general descriptions above and the detailed descriptions below are illustrative and descriptive only and do not limit the scope of the claims. The accompanying drawings incorporated herein and constituting part of this specification illustrate specific embodiments or aspects and, together with the descriptions, are useful in illustrating the principles described herein. [Brief explanation of the drawing]

[0012] This U.S. provisional application includes at least one drawing drawn in color. If this U.S. provisional application becomes available to the public in the future as a result of the publication of a non-provisional or international application claiming priority to this U.S. provisional application, a copy of this provisional application with the color drawing will be provided by the Patent Office upon request and payment of the necessary fees.

[0013] Figures 1A–E illustrate the use of the reusable cartridge described herein in a method for detecting MHC-I complexes. [Figure 1A] This document provides an overview of the endogenous processing and presentation enrichment workflow for MHC-I-related peptides (MAPs), as well as the steps involved in analysis and quantification based on mass spectrometry. In standard presentation, intracellular proteins are polyubiquitinated within the cell and translocated to the proteasome for degradation. The resulting peptide fragments are transported to the endoplasmic reticulum via TAP proteins. These are then loaded into MHC-I complexes (consisting of MHC-I heavy chains and B2M), further trimmed by ERAP, and transferred to the cell surface as stable MHC-I peptide complexes. The display level of each intrinsic peptide is determined by its abundance in a given cell, degradation rate, and affinity for MHC-I alleles. Enrichment involves applying a lysate containing the MHC-I complexes to a solid support containing an anti-MHC-I antibody, washing away unbound contaminants, and then eluting the MHC-I proteins and associated peptides by acid treatment. Analysis includes peptide isolation and desalting, chromatographic separation, and analysis by mass spectrometry, followed by data processing for identification and quantification steps. [Figure 1B] The table shows small and large capacity cartridges and provides a table of small and large capacity cartridge characteristics, including recombinant protein capacity, cell abundance, and the amount loaded into the customized antibody cartridge. [Figure 1C] This shows unique MHC-I peptides identified using a standard single-use AssayMAP® enrichment workflow, isolated by species and effective cell count. [Figure 1D]This provides an antibody cartridge reuse scheme and a cyclic concentration workflow. The antibody is loaded into a Protein A cartridge, crosslinked, and used to concentrate the MHC-I complex, and then washed before acid elution. The cartridge is then purified by priming with acid and TBS, stored at 4°C, and reused in the same manner. [Figure 1E] Using GRANTA lysates, the number of unique peptides observed after continuous use of custom antibody cartridges is shown, either using cartridges that are always moist or dried and then re-moistened.

[0014] [Figure 2] The gel electrophoresis comparing concentrated analytes with reused, always-moistened or dried and re-moistened cartridges is shown. Lanes 1-3 on the left show titrations of purified HLA protein standards. The numbers on the Y axis indicate the position of the molecular weight ladder band in kilodaltons. Lanes 4-6 show the levels of HLA standards eluted from moist-stored cartridges, and the three lanes on the right, 7-9, show the levels of HLA eluted from dry-stored / re-moistened cartridges. Similar levels in each of lanes 4-9 indicate that dry-stored and re-moistened cartridges can detect or concentrate standard proteins as well as always-moistened cartridges.

[0015] [Figure 3] The Coomassie electrophoresis gel data used to create the calibration curve and estimated volume of recombinant MHC-I complexes on previously used high-volume antibody crosslinked cartridges are shown.

[0016] Figures 4A and 4B show the effect of crosslinking on the enrichment of MHC-I molecules from the cartridge. [Figure 4A] The time indicated for recombinant HLA and B2M protein (subunits of the MHC-I molecule), as well as antibodies that recognize the sample peptide molecule and MHC-I molecule, to elute by liquid chromatography-mass spectrometry (LC-MS) after concentration of the MHC-I molecule on the cartridge, respectively. [Figure 4B] This shows a comparison of LC-MS runs after MHC-I enrichment using cartridges with crosslinked (top) and uncrosslinked (bottom) MHC-I-recognizing antibodies. The results show that crosslinking does not significantly affect MHC-I enrichment (compare the size of the largest peak in the "crosslinked" cartridge to the size of the peak directly below the "uncrosslinked" cartridge). The antibody peak (largest peak in the "uncrosslinked" cartridge) is not visible in the crosslinked elution, indicating antibody retention. The second elution from the column (elution 2) indicates that crosslinking does not result in further nonspecific binding to the cartridge material.

[0017] [Figure 5] The bar graph below the bars in the graph shows the analysis of the viscosity of lysates based on the volume retained on top of the spin filter under various conditions described in the examples below. Under the conditions described (500 μL placed on a 0.45 μm Costar filter and spun at 16,000 g for 1 minute at 4°C), water and lysis buffer showed no retained volume (negative control), while MC38 lysate (50M cells per 1 mL of B buffer, positive control) left at room temperature for 1 day showed retention of over 400 μL. Fresh GRANTA lysate (50M cells per 1 mL of B buffer) showed no retention, but showed a significant increase in viscosity after 4 hours at room temperature (typical unoptimized loading conditions). Lowering the temperature to 4°C reduced aggregation in a manner that was maintained over 18 hours. When the lysate is rapidly frozen in PBS, sucrose (starting at 1M, ending at 200mM), or glycerol (starting at 50% glycerol, ending at 10%), and then thawed, the viscosity remains low even after 4 hours at room temperature (however, 24 hours at room temperature results in a significant increase in viscosity). Combining storage with sucrose and glycerol (resulting in an even more dilute lysate solution) allows for freeze-thawing immediately or after 4 hours at room temperature without apparent storage.

[0018] Figures 6A - 6H show some Venn diagrams showing the overlap in composition between different cartridge concentrates of the same batch of GRANTA lysate (see Example 2). Peptides in each dataset were weighted for abundance using the TIC signal (such that more abundant peptides contributed more to the calculated overlap), but were not corrected for abundance (such that different amounts of signal were conserved in the calculations between LC - MS / MS runs). The numbers represent the number of unique peptides detected in MS / MS runs from solutions containing the MHC - I complex. The comparisons made here include between "wet" and "dry" cartridges, between replicate cartridges of the same type, and include excessive re - use. [Figure 6A] Specifically, the first use (first elution or E1) of a previously wet - stored cartridge (E1W1) is compared to a dry - stored one (E1D1). [Figure 6B] Similar comparisons using different sets of wet - stored and dry - stored cartridges (W2 / D2 and W3 / D3) are shown. [Figure 6C] Similar comparisons using different sets of wet - stored and dry - stored cartridges (W2 / D2 and W3 / D3) are shown. [Figure 6D] Results overlap from the first elution (i.e., first use; E1) of three different cartridges of the same type, all dry - stored (D1, D2, D3), are shown. [Figure 6E] Overlap with the second elution (E2) of three different wet - stored cartridges (W1, W2, or W3) is shown. [Figure 6F] Comparisons of the first, fifth, and ninth uses (E1, E5, and E9) in three different dry - stored cartridges (D1 (Figure 6F), D2 (Figure 6G), D3 (Figure 6H)) are shown. [Figure 6G] Comparisons of the first, fifth, and ninth uses (E1, E5, and E9) in three different dry - stored cartridges (D1 (Figure 6F), D2 (Figure 6G), D3 (Figure 6H)) are shown. [Figure 6H]A comparison of the first, fifth, and ninth uses (E1, E5, and E9) in three different dry storage cartridges (D1 (Figure 6F), D2 (Figure 6G), D3 (Figure 6H)) is shown. [Modes for carrying out the invention]

[0019] definition Unless otherwise defined, scientific and technical terms used in connection with the present invention shall have the meanings generally understood by those skilled in the art.

[0020] In this application, the use of “or” means “and / or” unless otherwise specified. In the context of multiple dependent claims, the use of “or” refers to only one or more preceding independent or dependent claims. Also, terms such as “element” or “component” include both elements and components containing one unit and elements and components containing more than one subunit, unless otherwise specified.

[0021] Where used herein, any concentration range, percentage range, ratio range, or integer range should be understood to include any integers within the listed range, and, where appropriate, fractions thereof (such as one-tenth and one-hundredth of an integer), unless otherwise indicated.

[0022] Units, prefixes, and symbols are shown in the form accepted by the International System of Units (SI). Numerical ranges include the number defining the range. The headings provided herein are not limitations on the various aspects of this disclosure that can be had by referring to the entire specification. Accordingly, the terms defined below are further defined by referring to the entire specification.

[0023] When used in accordance with this disclosure, the following terms should be understood to have the following meanings unless otherwise indicated.

[0024] As used herein, “sample” refers to any test specimen that may contain an analyte for detection or concentration. As used herein, “solution” refers to a sample in liquid form.

[0025] In this specification, “analyte” is used in its broadest sense to refer to a molecule or substance that can be found in a sample or solution and is intended to bind to (i.e., be recognized or detected by) a detection molecule on a matrix, thereby being detected or concentrated. The analyte may, in some cases, be a protein or peptide or another type of biological molecule.

[0026] As used herein, “detection molecule” is any molecule that specifically binds directly or indirectly to the analyte. The detection molecule may be, for example, an antibody, an antigen molecule, a hapten molecule, or any molecule that specifically binds to the analyte and is compatible with the matrix. As used herein, the “detection molecule” bound to the matrix may be a single molecular species or a mixture of two or more different molecular species.

[0027] In some embodiments, the analyte and / or detected molecule may be a protein, polypeptide, or peptide molecule. The terms “polypeptide” and “protein” are used interchangeably and refer to polymers of amino acid residues. Such polymers of amino acid residues may contain, but are not limited to, natural and / or unnatural amino acid residues, and include peptides, oligopeptides, dimers, trimers, and polymers of amino acid residues. The term also includes polymers of amino acids that have modifications such as glycosylation, sialylation, or are complexed with other molecules. As used herein, “peptide” refers to a relatively short polymer of amino acids, such as about 4 to 50 amino acids.

[0028] Proteins can, in some cases, be antibodies. The terms “antibody” or “Ab” as used herein are used in their broadest sense and encompass a variety of antibody structures, including but not limited to monoclonal antibodies ("mAb"), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), antibody conjugates, and antigen-binding fragments, as long as they exhibit the desired antigen-binding activity. As used herein, the term refers to a molecule that contains at least heavy chain complementarity-determining regions (CDRs) 1, 2, and 3, and at least light chain CDRs 1, 2, and 3, and is capable of binding to an antigen. The term also includes antigen-binding fragments. The term “antigen-binding fragment” includes, but is not limited to, antibody fragments capable of binding to antigens, such as Fv, single-chain Fv (scFv), Fab, Fab', and (Fab')2. In contrast, “full-length antibody” refers to an antibody molecule that includes all of its normal variable region and constant region. Antibody conjugates may contain a full-length antibody or antigen-binding fragment directly or indirectly bound to another molecule. As with other proteins and peptides, in some embodiments, the antibody may include various types of post-translational modifications, such as glycosylation.

[0029] In this specification, “detection” is used in its broadest sense, encompassing the selective binding of an analyte in a solution to other molecules in the solution, for example, to determine whether an analyte is present in a solution, and to isolate an analyte from other components in a solution, thereby concentrating the analyte. In some cases, the methods described herein are used to detect the presence of an analyte. In some cases, the methods described herein are used, for example, to “concentrate” or “isolate” an analyte compared to other molecules in a solution, as a result of which further analysis may be performed on the analyte. The terms “concentrate” and “isolate” are used interchangeably in this context herein.

[0030] The term "cartridge" is used broadly to refer to the entire product containing or holding a "matrix." A "cartridge" may have any useful shape or structure and may be made from any useful material, such as glass, plastic, or polymer materials. A "cartridge" may contain any useful material configured or molded to encapsulate or contain a matrix and hold it in place. Generally, a cartridge allows fluid to flow across and / or through the matrix. In some embodiments, the matrix may be placed in a tubular or cylindrical cartridge having openings at both ends (e.g., like a pipette tip, capillary, column, or other structure) to facilitate the transfer of liquid solutions to and from the cartridge during use, such as via an automated liquid dispensing device. In some cases, the cartridge may have a U-shape, or include a well that allows fluid to move from one end of the matrix, or have a plate-like or flat tip-like structure in which the matrix is ​​located in or on top, allowing fluid to flow across its surface.

[0031] The term "matrix" broadly refers to a substance to which detection molecules can bind. Suitable matrices as used herein include chromatographic matrices such as C4, C8, or C18, or various types of beads, particles, or resins such as silica, polymer beads, and / or metal beads. In some cases, the matrix may be coated with proteins such as antibodies, antigens, antibody constant region-binding proteins, or haptens that can recognize detection molecules. For example, proteins A, G, or L may be present on the matrix to recognize antibody detection molecules, or streptavidin may be used on the matrix to recognize biotinylated detection molecules. In a matrix composed of beads or resin particles, the particles may be of any suitable shape or size, as long as they can hold detection molecules such as spherical or nearly spherical beads, or other shapes such as pellets, chips, or tablets. In some embodiments, the matrix particles may be magnetic.

[0032] The detection molecule can be "bound" to the matrix covalently or noncovalently. In some embodiments, a crosslinking reagent can be used to crosslink the detection molecule to the matrix. The binding may also be direct or indirect, i.e., there may or may not be an intermediary molecule between the matrix material and the detection molecule.

[0033] A cartridge may be "reusable" if it can be used to detect an analyte in a first solution, and then, after being washed and re-equilibriumized, can detect an analyte in a second solution without any significant observable difference in its ability to detect the analyte in multiple tests using the same solution.

[0034] The matrices described herein may be stored in a wet or dry state. Storage in a "wet state" means that the matrix is ​​maintained immersed in a liquid solution, such as a buffer solution. Storage in a "dry state" means that the matrix is ​​at least partially exposed to air during storage, or otherwise the liquid is evaporated from the matrix before or during storage.

[0035] As used herein with respect to the binding between the detected molecule and the analyte, the terms “specific binding,” “specifically binding to,” or “specifically recognizing,” or similar terms mean that the intermolecular binding has a higher affinity than the binding between the analyte and the detected molecule would result from nonspecific binding.

[0036] "Mass spectrometry" or "MS" refers to a technique for measuring the mass-to-charge ratio (m / z) of one or more molecules in a sample. As used herein, "tandem MS" or "MS / MS" refers to a process in which a single ion, multiple ions, or a total mass envelope (precursor) is moved to a fragmentation chamber, and the fragmented product is then sent to a mass spectrometer. Depending on the design of the mass spectrometer, the fragmentation event may occur before a single mass spectrometer, between two or more different analytical instruments, or within a single mass spectrometer.

[0037] Cartridges for initial use and reuse, and method for preparing them. This disclosure relates, in particular, to a reusable cartridge for detecting an analyte in solution, wherein the cartridge comprises a matrix arbitrarily held in place by the cartridge or by a structure contained within the cartridge, and a detection molecule that specifically recognizes the analyte is bound to the matrix, for example, by non-covalent bonding to a substance in the matrix. In some cases, the detection molecule can be crosslinked to the matrix, for example, to prevent it from detaching from the matrix during various processes.

[0038] The reusable cartridges described herein may be of any suitable shape for holding a matrix so that the analyte can be detected. In some embodiments, the starting cartridge may include a tube or cylindrical component that acts to hold the matrix. For example, the cartridge may be in the form of a spin or gravity flow column or a pipette tip or chromatography column, allowing fluid to flow across the matrix embedded inside by either centrifugation or gravity flow. Alternatively, the cartridge may include a flexible tube or capillary tube for holding the matrix. In some cases, the matrix may be surrounded by a filter that is coarse enough to allow the solution components to pass freely, but fine enough to hold the matrix material in place. In some embodiments, the cartridge may have a tubular or well-like structure with one end open, or a plate-like structure or a flat tip-like structure for holding the matrix in place. For example, the tube, well, or plate may be open at one end or on one side to allow contact with and removal of the buffer and analyte-containing solutions.

[0039] In some embodiments, a single cartridge includes two or more spaces, cavities, positions, or wells containing a matrix, so that several different detection reactions can be performed using one cartridge. In other embodiments, a single cartridge includes a single matrix component, so that multiple cartridges are required to perform different detection reactions in parallel. In some embodiments, cartridges can be placed in a multi-sample system, such as a 96-sample or 384-sample system, for automated handling and liquid dispensing, for example.

[0040] In some embodiments, the cartridge has a volume capacity of 2 μL to 1 mL, such as 10 μL to 500 μL, 2 μL to 50 μL, 2 μL to 25 μL, 2 μL to 10 μL, 10 μL to 50 μL, 20 μL to 30 μL, 20 μL to 100 μL, 20 μL to 200 μL, or 50 μL to 500 μL, or 100 μL to 1 mL, or 200 μL to 1 mL. In some embodiments, the cartridge has a volume capacity of 5 μL, 10 μL, 20 μL, 25 μL, 50 μL, or 100 μL.

[0041] In some embodiments, the matrix comprises particles of any shape to which detection molecules can be bound, such as grains, beads, chips, spheres, pellets, or tablets. In some embodiments, the particles may be made of a material such as silica or agarose. In some embodiments, the particles may be magnetic, for example, so that they can be moved during the assay as needed, or so that they can be held in place within the cartridge. For example, in some cases, magnetic particles can be moved in and out of the cartridge during the assay. In other embodiments, the matrix is ​​a film or sheet to which detection molecules can be bound. In some embodiments, the matrix can be held in place within the cartridge, for example, via a filter (e.g., in a tube with openings at each end), by gravity (e.g., in a tube with an opening at one end), or by hydrophobic or electrostatic interaction with the cartridge surface (e.g., a matrix sheet or film placed on a plate or chip).

[0042] In some embodiments, detection molecules bind directly to the matrix, for example, by non-covalent bonds. In some embodiments, they bind via covalent bonds to molecules in the matrix. In some embodiments, detection molecules bind indirectly to the matrix via intermediate molecules that themselves are bound to the matrix. For example, in some embodiments, the matrix includes certain types of detection molecules that bind to biotinylated molecules, such as molecules that bind to streptavidin, or molecules that recognize immunoglobulins such as protein A, protein G, or protein L, or two or more combinations of these molecules. For example, by contacting a matrix containing protein A with an antibody detection molecule, it becomes possible to incorporate the antibody detection molecule onto the matrix via binding by protein A on the matrix. In some embodiments, the matrix may contain reagents such as streptavidin or protein A and / or G, but these molecules are not used to specifically recognize a particular analyte, and are merely molecules to which the detection molecule herein is bound, and therefore this term is not used herein as "detection molecule". Thus, for example, in some embodiments, if the matrix contains protein A, the detection molecule may include an antibody or other immunoglobulin or Fc-containing molecule bound to the matrix by protein A. Protein A is used not as the detection molecule itself, but as an intermediate bond between the matrix particles and the detection molecule.

[0043] In some embodiments, the detection molecule added to the matrix may also be crosslinked to the matrix. For example, the detection molecule can be bound to the matrix using crosslinking reagents such as dimethyl pimerimidate (DMP), cyanate esters, NHS esters, azulactone, carbonyl diimidazole (CDI), maleimide, iodoacetyl, pyridyl disulfide, hydrazide, or carbodiimide reagents, or other reagents suitable for immobilizing affinity reagents on the matrix. (For further consideration of how these and further crosslinking reagents may be used to bind molecules to the agarose matrix, see, for example, www.thermofisher.com / us / en / home(slash)life-science(slash)protein-biology(slash)protein-biology-learning-center(slash)protein-biology-resource-library(slash)pierce-protein-methods(slash)covalent-immobilization-affinity-ligands.html.) In other embodiments, crosslinking may not be necessary depending on the expected use of the cartridge and the nature of the detection molecule. In some embodiments, when DMP is used as a crosslinking agent, 1–10 mM DMP, 1–5 mM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, or 10 mM DMP are used to crosslink the detection molecule to the matrix. In some embodiments of this specification, the concentration of DMP used in the crosslinking reaction is about 1 / 4 to 1 / 5 of the concentration typically used in the reaction.

[0044] In some embodiments, the cartridges herein also include at least one cryoprotectant, such as sucrose, trehalose, glycols such as ethylene glycol or propylene glycol, glycerol, 2-methyl-2,4-pentanediol (MPD), or dimethyl sulfoxide (DMSO). For example, the cryoprotectant may be in contact with the matrix to protect the detected molecules and / or other matrix components from damage or denaturation during storage, for example, during storage in a dry state. In some cases, the cryoprotectant includes glucose and / or trehalose. In some cases, the cryoprotectant includes trehalose.

[0045] Accordingly, in some embodiments, the reusable cartridge according to this specification comprises (a) a matrix and b) a detection molecule bound to the matrix, which specifically binds to the analyte and is optionally crosslinked to the matrix. In some embodiments, the reusable cartridge according to this specification comprises (a) a matrix and b) a detection molecule bound to the matrix, which specifically binds to the analyte and is optionally crosslinked to the matrix and (c) at least one cryoprotectant.

[0046] Certain commercially available cartridges and matrices for analyte detection are generally intended for single use and disposal, and are also intended to be kept moist before use. (See, for example, AssayMap® Bravo cartridge, Agilent.) Surprisingly, this disclosure shows that the cartridges described herein can be reused multiple times without significant signal loss to detect analytes in cell culture supernatant or cell lysates, and that the cartridges can be stored dry. In some embodiments, the cartridges are stored dry. In some such cases, the cartridges contain a cryoprotectant and are stored dry. In some cases, the cartridges are stored dry, such as at 2-8°C or 4°C, such as in a refrigerator or cold room. In some cases, dry-stored cartridges are re-moistened before use to detect analytes. In some cases, the cartridge matrix is ​​dried by heating to at least 30°C, e.g., 30–45°C, 30–37°C, 30°C, or 37°C for, for, for at least 30 minutes, 30–60 minutes, or 60–120 minutes, and then cooled at room temperature for at least 1 hour, e.g., at least 2 hours, at least 4 hours, at least 8 hours, at least 12 hours, or overnight, before being optionally stored at a low temperature before reuse. In some embodiments, the cartridge is stored in a moist state, e.g., the matrix is ​​immersed in a buffer solution. In some cases, the cartridge is stored in a moist state, such as 2–8°C or 4°C, such as in a refrigerator or cold room. In some such cases, the cartridge contains a cryoprotectant and is stored in a moist state.

[0047] In some embodiments, the cartridge is stored as described above at a pH of 7.0 or less. In some embodiments, the cartridge is stored at an acidic pH (i.e., a pH less than 7.0). In some embodiments, the cartridge is stored at a pH of 4 to 7, for example, pH 4 to 6, pH 4 to 6.5, pH 4.5 to 6.5, pH 4.8 to 6.8, pH 5 to 7, pH 4 to 5, pH 5 to 6, pH 6 to 6.5, or pH 6 to 7. In some embodiments, the cartridge is stored in a buffer containing EDTA and / or sodium azide.

[0048] In some embodiments of this specification, the cartridge is a cartridge that has been used at least once previously for the detection of an analyte in a solution. For example, in some embodiments, the cartridge is used to detect an analyte in a solution, the detected analyte is eluted and removed from the cartridge, and the cartridge matrix is ​​brought into contact with a buffer containing a cryoprotectant for storage between uses. In some embodiments, the buffer is optionally acidic pH and / or contains EDTA and / or sodium azide. In some embodiments, the cartridge can be reused at least nine times without a significant observable decrease in detection capability. For example, in some cases, the cartridge can be reused at least nine times with the same sample without a significant change in the composition or amount of analyte detected from the sample between the first run and the last run. (See, for example, Figures 6A to 6H.) In some embodiments, the cartridge can be reused at least 10, 20, 50, or 100 times without a significant observable decrease in detection capability. For example, in some embodiments, after at least 10, 20, 50, or 100 uses, a cartridge having a matrix containing antibody detection molecules remains capable of eluting at least 90% of the protein analyte from the cell lysate or biological fluid compared to the analyte eluted in the first use. In some embodiments, a cartridge having a matrix containing antibody detection molecules remains capable of eluting at least 95% of the protein analyte from the cell lysate or biological fluid compared to the analyte eluted in the first use after at least 10, 20, 50, or 100 uses. In some embodiments, after at least 10, 20, 50, or 100 uses, a cartridge having a matrix containing antibody detection molecules remains capable of detecting concentrates of protein analyte in a biological sample at at least 90% of the cartridge's efficiency in its first use. In some embodiments, after at least 10, 20, 50, or 100 uses, a cartridge having a matrix containing antibody detection molecules remains capable of detecting concentrates of protein analyte in a biological sample at at least 95% of the cartridge's efficiency in its first use.In some such cases, the cartridges are stored dry with a cryoprotectant between uses. In some such cases, the cartridges are stored moist with or without a cryoprotectant between uses. In some cases, the cartridges are stored either dry or moist at 2-8°C, such as in a refrigerator or cool room, or at 4°C between uses. In some cases, between uses, they are stored in an acidic pH buffer and / or a buffer containing EDTA and / or sodium azide.

[0049] In some embodiments, the cartridge is used at least twice for the detection of the analyte in the solution. In some embodiments, the cartridge is used at least three times for the detection of the analyte in the solution. In some embodiments, the cartridge is used at least four times for the detection of the analyte in the solution. In some embodiments, the cartridge is used at least five times for the detection of the analyte in the solution. In some embodiments, the cartridge is used at least six times for the detection of the analyte in the solution. In some embodiments, the cartridge is used at least seven times for the detection of the analyte in the solution. In some embodiments, the cartridge is used at least eight times for the detection of the analyte in the solution. In some embodiments, the cartridge is used at least nine times for the detection of the analyte in the solution. In some embodiments, the cartridge is used at least ten times for the detection of the analyte in the solution. In some embodiments, the cartridge is used at least twenty times for the detection of the analyte in the solution. In some embodiments, the cartridge is used at least fifty times for the detection of the analyte in the solution. In some embodiments, the cartridge is used at least 100 times for the detection of the analyte in the solution. In some such cases, the cartridge is stored dry with a cryoprotectant with the previous or after use. In some such cases, the cartridges are stored in a moist state with or without cryoprotectant after each use. In some cases, the cartridges are stored either dry or moist at 2-8°C, such as in a refrigerator or cold room, or at 4°C between uses. In some cases, they are stored moist between uses in an acidic pH buffer and / or a buffer containing EDTA and / or sodium azide. In some cases, they are stored dry between uses in a cryoprotectant, but are dried after being treated with an acidic pH buffer containing EDTA and / or sodium azide.

[0050] If the cartridge is stored in a dry state at 2-8°C or 4°C in a refrigerator or cold room, it may be stored for at least 24 hours, at least 48 hours, at least 1 week, at least 2 weeks, at least 1 month, at least 3 months, or at least 6 months. In some cases, after adding a cryoprotectant, it can be stored in a dry state at 2-8°C or 4°C in a refrigerator or cold room for at least 24 hours, at least 48 hours, at least 1 week, at least 2 weeks, at least 1 month, at least 3 months, or at least 6 months. Furthermore, in some embodiments, it can be stored under the above conditions after pretreatment with an acidic buffer containing EDTA and / or sodium azide before drying. If the cartridge is stored in a wet state, it can be stored in an acidic pH buffer containing EDTA and / or sodium azide for at least 24 hours, at least 48 hours, at least 1 week, at least 2 weeks, at least 1 month, at least 3 months, at least 6 months, or at least 1 year between uses at 2-8°C, for example, in a refrigerator or cold room, or at 4°C. In other cases, the cartridge can be stored moist in such a buffer at room temperature, provided that the cartridge is stored under conditions such that the matrix remains immersed in the buffer, i.e., remains moist. The disclosure also relates to a method for preparing a reusable cartridge for storage between uses. For example, in some methods, after prior analyte detection and elution, the cartridge is washed at least once in a buffer, e.g., 1-10% acetic acid, e.g., 1%, 5%, or 10% acetic acid, 1% formic acid, or 1% trifluoroacetic acid (TFA), and then the matrix is ​​brought into contact with a cryoprotectant, e.g., in a buffer with a pH of 7 or less, or in a buffer with a pH of 4-7, or in an acidic buffer with a pH of 4-6, pH of 4-6.5, pH of 4.5-6.5, pH of 4.8-6.8, pH of 5-7, pH of 4-5, pH of 5-6, pH of 6-6.5, or pH of 6-7. In some embodiments, the cryoprotectant comprises one or more of sucrose, trehalose, ethylene glycol, propylene glycol, glycerol, 2-methyl-2,4-pentanediol (MPD), or dimethyl sulfoxide (DMSO).In some embodiments, the cryoprotectant comprises sucrose and / or trehalose. In some embodiments, the cryoprotectant-containing buffer also comprises EDTA and / or sodium azide. In some embodiments, the cartridge is then dried. In some cases, the cartridge matrix is ​​dried by heating to at least 30°C, e.g., 30–45°C, 30–37°C, 30°C, or 37°C for e.g., at least 30 minutes, 30–60 minutes, or 60–120 minutes, and then cooled at room temperature for at least 1 hour, e.g., at least 2 hours, at least 4 hours, at least 8 hours, at least 12 hours, or overnight, and then optionally stored at a low temperature before reuse.

[0051] In some embodiments, reusable cartridges are prepared by obtaining a cartridge containing a matrix, or by optionally adding a matrix to a cartridge, contacting the matrix with the detection molecules, binding the molecules to the matrix, and optionally crosslinking the detection molecules to the matrix. In some cases, excess unbound detection molecules are then removed. Before use, the cartridge may be equilibrated with a suitable buffer.

[0052] In some embodiments, reusable cartridges are prepared using antibodies as detection molecules. In some such cases, the matrix may contain molecules that specifically recognize a portion of the antibody, such as an Fc domain or other constant region, and ideally allow the antibody to bind to the matrix in a region that does not interfere with its antigen-binding function. For example, in some embodiments, purified or isolated antibodies are exposed to a matrix containing, for example, protein A, protein G, or protein L, or a combination thereof such as protein A and protein G, and excess antibodies are removed to bind to the matrix. In some cases, a crosslinking agent is used to crosslink the antibody detection molecule to protein A, G, and / or L containing the matrix. Similarly, if other detection molecules such as haptens or other binders of the analyte to be detected are used, they may be crosslinked to the matrix.

[0053] In some embodiments, one type of detection molecule, such as an antibody specific to a particular antigen, is bound to the matrix. In other embodiments, two or more types of detection molecules may be bound to the matrix. In some cases, a single matrix may contain detection molecules that recognize multiple analytes, such as two different antibodies targeting two different antigens, or several different antibodies targeting a group of analytes. In other cases, for example, two or more different antibodies that recognize different parts of the same antigen may be bound to the matrix. Thus, in some embodiments, the matrix may be bound with, for example, 2, 3, 4, 5, 10, 20, 100, or 2-5, 5-10, 10-20, 10-50, 10-100, or 50-100 different detection molecules so that the matrix can recognize a series or panel of analytes in solution.

[0054] In some cases, the detection molecule is crosslinked to the matrix, for example, to prevent leaching from the matrix after repeated use. In some embodiments, the crosslinking agent is selected from dimethyl pimerimidate (DMP), cyanate esters, NHS esters, azulactone, carbonyl diimidazole (CDI), maleimide, iodoacetyl, pyridyl disulfide, hydrazide, or carbodiimide. In some embodiments, crosslinking is carried out using DMP. In some embodiments, the crosslinking reaction with DMP is carried out in the cartridge at a DMP concentration of 1 / 4 to 1 / 5 of the normally recommended concentration for such crosslinking reactions. Thus, for example, DMP may be recommended to be used at about 20–25 mM, in which case DMP of less than 10 mM may be used to crosslink protein detection molecules such as antibodies to the matrix. In some cases, 3–10 mM DMP, 3–7 mM DMP, 5–10 mM DMP, or 4–6 mM DMP may be used. In some cases, 5 mM DMP may be used. In some embodiments, reducing the crosslinking agent concentration compared to generally recommended concentrations improved the ability to reuse cartridges without significantly impairing the cartridge matrix's ability to detect analytes.

[0055] Exemplary use of a reusable cartridge for polypeptide detection This disclosure also encompasses methods for using and reusing the cartridges described herein. In some embodiments, the analyte is, for example, a peptide or protein. For example, the analyte may be a peptide or protein specifically recognized by an antibody detection molecule. For example, as described herein, an example of a protein analyte is an MHC-I complex molecule.

[0056] In some cases, analytes such as proteins or other biological molecules may be detected in biological solutions such as body fluids (e.g., blood, plasma, urine, etc.) or cell lysates. For example, the methods herein may allow for the concentration of MHC-I complex molecules from biological fluids or cell lysates, which can then be further analyzed in subsequent steps such as chromatography and mass spectrometry.

[0057] In some embodiments, the solution is filtered before exposure to the cartridge. For example, filtration may help remove large particles that could clog the matrix or interfere with subsequent reuse. In some embodiments, the solution is filtered through a 0.1 μm to 1 μm filter, such as 1 μm, 0.2 μm, 0.22 μm, 0.3 μm, 0.4 μm, 0.45 μm, 0.5 μm, 0.6 μm, 0.7 μm, or 0.8 μm filter, or through a 0.1 μm to 0.5 μm filter, such as a 0.5 μm to 0.5 μm filter, such as 0.1 μm, 0.2 μm, 0.22 μm, 0.3 μm, 0.4 μm, 0.45 μm, or 0.1 μm filter. In some embodiments, when a biological fluid or cell lysate is used for analyte detection, the fluid is cooled, for example, on ice, or to a temperature of, for example, 2 to 15°C, before contact with the cartridge matrix. For example, in some embodiments, the sample is cooled to a temperature of 2–10°C, 4–10°C, or 10–15°C. In some embodiments, a cryoprotectant such as sucrose, glycerol, or trehalose is added to the solution before lowering the temperature. In some embodiments, the solution is rapidly frozen after adding the cryoprotectant. In some embodiments, the biological fluid or cell lysate is diluted with, for example, isotonic buffer before contact with the cartridge matrix. In some embodiments, the total protein concentration in the solution may be determined before contact with the matrix, for example, by a bicinchoninic acid (BCA) assay.

[0058] In some embodiments, once the analyte is eluted from the cartridge of this specification, it may be further analyzed by processes such as electrophoresis, chromatography, structural analysis, or mass spectrometry or chromatography followed by mass spectrometry.

[0059] In some embodiments, the amount of analyte bound to the matrix can also be quantified. For example, the amount of analyte can be determined, for instance, by electrophoresis and densitometry, or by integrating the signal from the analyte in the chromatographic peak.

[0060] In some cases, the method of use involves detecting or concentrating an analyte with a cartridge that has been previously used to detect or concentrate the same or similar analyte. In some cases, the cartridge has been used at least once previously. In some cases, the cartridge has been used at least twice previously. In some cases, the cartridge has been used at least three times previously. In some cases, the cartridge has been used at least four times previously. In some cases, the cartridge has been used at least five times previously. In some cases, the cartridge has been used at least six times previously. In some cases, the cartridge has been used at least seven times previously. In some cases, the cartridge has been used at least eight times previously. In some cases, the cartridge has been used at least nine times previously.

[0061] In some cases, use involves preparing the cartridge for subsequent use by washing the cartridge at least once with a buffer such as 1-10% acetic acid, e.g., 1%, 5%, or 10% acetic acid, 1% formic acid, or 1% trifluoroacetic acid (TFA) after eluting the analyte, and optionally bringing the matrix into contact with the cryoprotectant. The cryoprotectant may be in a buffer such as a buffer with a pH of 7 or less, or in a buffer such as a pH of 4-7, or in an acidic buffer such as a pH of 4-6, pH of 4-6.5, pH of 4.5-6.5, pH of 4.8-6.8, pH of 5-7, pH of 4-5, pH of 5-6, pH of 6-6.5, or pH of 6-7. In some embodiments, the cryoprotectant comprises one or more of sucrose, trehalose, ethylene glycol, propylene glycol, glycerol, 2-methyl-2,4-pentanediol (MPD), or dimethyl sulfoxide (DMSO). In some embodiments, the cryoprotectant comprises sucrose and / or trehalose. In some embodiments, the cryoprotectant comprises trehalose. In some embodiments, the cryoprotectant-containing buffer also comprises EDTA and / or sodium azide. In some embodiments, the cartridge is then dried. In some cases, the cartridge matrix is ​​dried by heating to at least 30°C, e.g., 30–45°C, 30–37°C, 30°C, or 37°C for e.g., at least 30 minutes, 30–60 minutes, or 60–120 minutes, and then cooled at room temperature for at least 1 hour, e.g., at least 2 hours, at least 4 hours, at least 8 hours, at least 12 hours, or overnight, and then optionally stored at a low temperature before reuse.

[0062] kit including cartridge This disclosure also encompasses kits comprising the cartridges of this specification. In some embodiments, the cartridges have not yet been used. In other embodiments, they have been used previously at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, or 100 times. In some embodiments, the kit comprises cartridges stored in a dry state as described above. In some embodiments, the kit comprises cartridges stored in a wet state as described above. In some embodiments, the kit further comprises instructions for use. In some embodiments, the kit further comprises reagents for detecting or concentrating analytes in a sample, e.g., equilibration, washing, and / or elution buffers. In some embodiments, the kit further comprises controls such as a blank cartridge that does not contain the detection molecule or a cartridge having a structurally similar detection molecule that is not intended to recognize the analyte. In some embodiments, the kit may be packaged for transport to another location. In some embodiments, the kit comprising cartridges may be stored in a dry state at room temperature, possibly 2–8°C, for at least 24 hours, 48 ​​hours, at least 1 week, at least 2 weeks, at least 1 month, at least 3 months, or at least 6 months, as described above. In some embodiments, the kit, including the cartridges, may be stored in a humid state at room temperature, or otherwise at 2–8°C, for at least 24 hours, 48 ​​hours, at least 1 week, at least 2 weeks, at least 1 month, at least 3 months, at least 6 months, or at least 1 year, as described above. In some embodiments, the kit may also include reagents used to wash and store the cartridges, such as a cryoprotectant, a buffer containing one or more of EDTA and sodium azide. In some embodiments, the kit includes, for example, packs of 10, 50, 100 (e.g., a 96-well plate containing 96 well cartridges, or a set of about 100 tubular cartridges that fit into a 96-well plate system), 500, or 1000 cartridges.

[0063] The following examples provide further details of the exemplary cartridges described herein and methods for preparing, storing, and using them, but are not intended to limit the scope of the disclosures herein. [Examples]

[0064] Example 1. Preparation and testing of reusable cartridges Cartridge preparation The following materials were obtained to prepare antibody-coated cartridges: six large AssayMAP® Protein A cartridges (Agilent), a Seahorse 1-well μ plate, an Eppendorf PCR plate (deep-well plate), PBS, TBS, and a solution of 25 mM Tris buffer, 1% acetate elution buffer, and anti-human HLA A, B, C antibodies w6 / 32 (1 mg / mL in PBS). The cartridges were assembled in the designated rows in the plate, equilibrated with PBS, contacted with the antibody solution to bind the antibodies to Protein A on the cartridge matrix, washed, and then exposed to a crosslinking reagent. Specifically, the cartridges were placed in the multi-well plate, and using an automated liquid dispenser (Agilent AssayMAP® Bravo), the cartridges were first primed, equilibrated with PBS, and 250 μL of PBS was used at a flow rate of 300 μL / min, followed by 150 μL of PBS at a flow rate of 20 μL / min. Next, the antibody solution was added at a flow rate of 20 μL / min in 1 mL increment, and then the cartridge was washed with PBS to remove excess antibody.

[0065] Next, the cartridges were treated with a crosslinking reagent to crosslink the antibodies to the protein A matrix. The cartridges were first primed and equilibrated with 200 mM triethanolamine (TEA) buffer pH 8.2, then treated with 200 μL of 5 mM DMP at a flow rate of 5 μL / min. The cartridges were then washed first with 250 μL of TBS, then with 250 μL of 25 mM Tris at a flow rate of 20 μL / min, and then in contact with 1% acetic acid elution buffer (50 μL at 10 μL / min). The cartridges were then treated twice with acetic acid, Tris, and then TBS buffer (100 μL, 200 μL, and 200 μL volumes at a flow rate of 20 μL / min, respectively), and placed in 190 μL of TBS, 1 mM EDTA, and 0.025% sodium azide storage buffer to ensure that the liquid remained above the top of the matrix in each cartridge column.

[0066] A specific cartridge was dried as follows, and the other cartridges were stored in storage buffer. A dry buffer solution of 20 mM His-acetic acid, 200 mM trehalose, and pH 6 was prepared. 270 μL of the dry buffer solution was added to the cartridge selected for dry storage. The cartridge was then dried at 37°C for 1 hour, and then stored overnight at room temperature before use.

[0067] Cartridge Test Next, antibody-coated and crosslinked cartridges prepared as described above, stored in a wet state and then in a dry state before use, were tested for binding to analytes in solution within the AssayMap® Bravo system. Specifically, three wet-stored and three dry-stored w6 / 32 antibody cartridges were used for testing. The cartridges were primed and equilibrated with TBS (150 μL at 300 μL / min for priming, then 100 μL at 20 μL / min for equilibration), and the analyte solution was added (50 μL at 10 μL / min). HLA-containing solution was added to each of the wet-stored and dry-stored w6 / 32 cartridges. The cartridges were then washed with 250 μL of TBS at a flow rate of 20 μL / min, then with 250 μL of 25 mM Tris pH 8.0 at 20 μL / min, and subsequently eluted with 50 μL of 1% acetic acid at a flow rate of 10 μL / min. Next, the eluate was loaded onto an acrylamide electrophoresis gel, then heated in a microwave for 1.5 minutes, followed by shaking for 10 minutes, and stained with SimplyBlue® (ThermoFisher Scientific) by rinsing in water for 1 hour.

[0068] Gerlene's analysis is as follows, and the results are shown in Figure 2. [Table 1]

[0069] As seen in Figure 2, HLA elution from the wet and dry storage cartridges (lanes 4-9 on the right side of Figure 2) appears similar, indicating that the cartridges can be used after either wet or dry storage.

[0070] Example 2: Use of cartridges for enriching MHC-I complexes in biological samples Materials and peptide synthesis Unless otherwise specified, all chemicals were purchased from Sigma-Aldrich. Antibodies were purchased from CST (Danvers, Massachusetts) or Abcam (Burlingame, California). dTag13 degrader compounds were purchased from Tocris. General plastic products were purchased from Corning, and AssayMAP® plastic products were purchased from Agilent as specified in the user manual for use with AssayMAP® Bravo. Peptides were purchased from JPT Peptide Technologies (Berlin, Germany), dissolved in 50% ethylene glycol (Sigma), and stored at -20°C.

[0071] Purification of HLA and B2M Recombinant HLA alleles and β2M were overexpressed in E. coli, purified from inclusion bodies, and stored at -80°C under denaturing conditions (6M guanidine HCl, 25mM Tris, pH 8.0). Briefly, the β2M and HLA biomass pellets were resuspended in lysis buffer (PBS + 1% Triton X-114) at a concentration of 5 mL / g. The resuspended pellets were subjected to microfluidization twice at 1000 bar. The resulting suspension was rotated in an ultracentrifuge at 30,000 g for 20 minutes. The pellets were collected, washed with 500 mL of lysis buffer, and centrifuged at 30,000 g for 20 minutes. The pellets were collected and washed a second time as described above. Next, the purified inclusion bodies were dissolved in denaturing buffer (20mM MES, pH 6.0, 6M guanidine HCl) at a concentration of 10 mL / g. The suspension was then stirred overnight at 4°C. The dissolved pellet was centrifuged at 40,000 g for 60 minutes, the supernatant was collected, and filtered through a 0.22 μm filter. The concentration was determined using a BCA assay. The samples were rapidly frozen and stored at -80°C before use for MHC-I complex generation.

[0072] Recombinant MHC-I complex formation In a 5L reaction, the selected peptide (0.01mM), oxidized and reduced glutathione (0.5mM and 4.0mM, respectively), recombinant HLA allele (0.03mg / ml), and β2M (0.01mg / ml) were all combined in refolding buffer (100mM Tris, pH 8.0, 400mM L-arginine, 2mM EDTA). The refolding mixture was stirred at 4°C for 4 days. The refolding solution was filtered through a 0.22μm filter, concentrated, and buffer-exchanged by tangential flow filtration (TFF) (Millipore) to 25mM Tris (pH 7.5). Next, protein components were analyzed by LC / MS to ensure that the HLA was in the appropriate reduced state. The refolded MHC-I complex was purified by ion-exchange chromatography using a 5mL HiTrap® Q HP column in an AKTA Pur FPLC. The column was equilibrated with 25 mM Tris (pH 7.5) at a flow rate of 5 mL / min in a 10 column volume (CV). The MHC-I complex was loaded onto the column at a flow rate of 5 mL / min and eluted using a 1 M NaCl gradient over 30 CVs of 25 mM Tris, pH 7.5, from 0 to 60%. The entire eluted peak fraction was electrophoresed by SDS-PAGE, and the fractions containing β2M and HLA bands were pooled. The pooled fraction was replaced with storage buffer (25 mM Tris, pH 8.0, 150 mM NaCl). Protein concentration was determined by UV absorbance at 280 nm, and the samples were rapidly frozen and stored at -80°C.

[0073] Adhesive and suspension cell culture MC38 cells were obtained as a frozen stock, immediately cultured in a working stock, and frozen in growth medium + 10% DMSO. MC38 cells were grown in RPMI-1640, 10% FBS, 2 mM glutamine, 1× pen-strep, and 25 mM HEPES. Cells were subcultured at 37°C and 5% CO2 for a doubling time of 18 hours. GRANTA-519 (GRANTA) cells were also cultured in a working stock as described above. GRANTA cells were cultured in RPMI-1640, 10% FBS, 2 mM glutamine, and 1× pen-strep. Cells were subcultured in an Infors Minitron at 110 rpm, at 37°C and 5% CO2 for a doubling time of 48 hours.

[0074] Cell processing and recovery Cells were processed and incubated at 37°C and 5% CO2 (adherent MC38 cells) and 110 rpm (suspended GRANTA cells). To harvest adherent MC38 cells, the medium was aspirated and cold Accutase (Innovative Cell Technologies, Inc.) was immediately added to the plate. After allowing the plate to stand at room temperature for 5 minutes, the cells were lifted from the plate using agitation. The Accutase cell mixture was then added to a Falcon tube containing growth medium, the cells were counted using ViCell XR to determine the viable cell concentration per 1 mL, and then 250 million cells were transferred to 50 mL Falcon tubes for each condition. To harvest suspended GRANTA cells, the cells were counted and transferred as described above. All cells were then pelletized by centrifugation, the supernatant was removed, and the pellet was rapidly frozen in liquid nitrogen and then placed at -80°C.

[0075] Cell lysis and preservation The cell pellets were removed from -80°C, rapidly thawed in a 37°C water bath, and then placed on ice. 250 million GRANTA cells were dissolved in 5 mL of undenatured OG surfactant buffer (PBS, 0.25% sodium deoxycholate, 0.2 mM iodoacetamide, 1 mM EDTA, 1% octyl-beta-d glucopyranoside (OG), 1× protease + phosphatase inhibitor (Sigma)), and the lysate was transferred to one 5 mL Eppendorf tube. 250 million MC38 cells were each dissolved in 10 mL of OG buffer and transferred to two 5 mL Eppendorf tubes. The lysates were left on ice for 30 minutes, and then 20,000 g were clarified by spinning down at 4°C for 60 minutes. The clarified lysate was immediately decanted into a 50 mL Falcon tube vacuum filter (0.45 μm, Corning) and filtered under gentle vacuum. The filtered solution was transferred in 4 mL aliquots (either the entire GRANTA 250M cell lysate or half of the MC38 250M cell lysate) into each 15 mL Falcon tube on ice, followed by 1.33 mL of 50% glycerol and 1 M sucrose (final concentrations of 10% glycerol and 200 mM sucrose). The tubes were mixed by inversion until homogeneous, 10 μL was removed for BCA analysis, and 30 μL was removed for Western blotting and in-gel digestion (for comprehensive proteomics). The Falcon tubes were then rapidly frozen and placed at -80°C.

[0076] Cartridge preparation, storage, and reuse Cartridge crosslinking was performed as follows: Briefly, dry Protein A cartridges were primed in PBS, then loaded with 1 mg of antibody at 1 mg / mL, followed by washing with PBS (priming was performed at 300 μL / min for all cartridge sizes; washing and loading of small cartridges were performed at 10 μL and 5 μL / min, respectively; washing and loading of large cartridges were performed at 20 μL / min). The cartridges were then equilibrated in 200 mM triethanolamine (TEA, Sigma), loaded with 5 mM dimethylpimeridate (DMP, Sigma) in TEA, pH 8.2 for 40 minutes at room temperature, and then washed sequentially with TBS, 25 mM Tris, pH 8.0 (Tris buffer), 1% acetic acid, Tris buffer, and finally TBS. The cartridges were then stored at 4°C in an empty cartridge rack filled with TBS, 1 mM EDTA, and 0.025% sodium azide, and then Parafilmed. To prepare the dried cartridge, the cartridge buffer was replaced with 20 mM histidine, 200 mM trehalose, pH 6.0, left at 37°C for 1 hour, and then left at room temperature in the dark for at least 18 hours to complete drying. The dried cartridge was then reconstituted in the same manner as the dried Protein A cartridge.

[0077] The reused cartridges were transferred to AssayMAP® Bravo, the necks of each cartridge were dried with a cotton swab (if transferred from 4°C), then primed with water, primed with 1% acetic acid, and washed with water before reuse.

[0078] MHC concentration using cartridges Diluted frozen cell lysates were removed from a -80°C freezer onto dry ice, rapidly thawed in a 37°C water bath, and then placed on moist ice without mixing. The crosslinked cartridges were removed from the refrigerator and transferred to an AssayMAP® system. The cups were lightly tapped dry using a Qtip, primed with water, primed with 1% acetic acid, and washed with water.

[0079] 500 μL of each solution was transferred to a 0.45 μm Costar spin filter tube, spun down at 16,000 g at 4°C for 1 minute, and then placed on ice. The spin filter tubes were checked for retention of the solution on the filter. If the amount of retained solution exceeded 10-20 μL, it was considered that this could clog the cartridge, and therefore the material was not used for cartridge experiments. The reflux flow-through into each 15 mL Falcon tube was stored on ice. The samples were cooled to 10°C.

[0080] From each 8.3 mL Falcon tube, 1.1 mL per well was transferred to the four wells of a Deepwell® sample plate (3.9 mL of sample remained in the Falcon tube). Using the AssayMAP® Bravo system, the cartridge was primed and equilibrated as follows, and the sample was loaded: cartridge TBS priming (150 μL at 250 μL / min), TBS equilibration (100 μL at 20 μL / min), and sample loading (1 mL at 20 μL / min). The flow-through plate was replaced, and an additional 800 μL of sample was added to each of the four wells. Further sample loading (800 μL at 20 μL / min), followed by TBS washing (250 μL at 25 μL / min), Tris washing (250 μL at 25 μL / min), and 1% acetate elution (60 μL at 10 μL / min). 10 μL was removed from each well, and MHC-I complex enrichment was verified by Coomassie-stained electrophoresis gel. The remaining 4 × 50 μL was combined into a single 1.5 mL tube, rapidly frozen, and stored at -80°C. The cartridge was then primed with 1% acetic acid, washed with Tris buffer and TBS, and stored at 4°C in an empty cartridge rack filled with TBS, 1 mM EDTA, and 0.025% sodium azide, and then Parafilmed.

[0081] Figure 1D shows the workflow for preparing and using cartridges for detecting MHC-I complexes in cell lysates. Initially, the inventors found that once the cartridges became clogged after the first use, their ability to be reused was impaired. However, by using a larger lysate filter to reduce the amount of DMP crosslinking agent (e.g., 25 mM to 5 mM), diluting the filtered lysate 1.33 times with glycerol and sucrose, and lowering the temperature of the lysate during concentration, precipitation was minimized, and the cartridges were found not to clog further during repeated use.

[0082] Specifically, as shown in Figure 5, under the conditions described herein (500 μL placed on a 0.45 μm Costar filter and rotated at 16,000 g for 1 minute at 4°C), water and lysis buffer showed no retained volume (negative control), while MC38 lysate (50M cells per 1 mL of B buffer, positive control) after 1 day at room temperature showed retention of more than 400 μL. Fresh GRANTA lysate (50M cells per 1 mL of B buffer) showed no retention, but storage of the lysate at room temperature (typical unoptimized loading conditions) for 4 hours showed a significant increase in viscosity. Lowering the temperature to 4°C reduced aggregation in a manner that was maintained for 18 hours. When the lysate was rapidly frozen in PBS, sucrose (1 M starting, 200 mM final), or glycerol (50% glycerol starting, 10% final) and then thawed, the viscosity remained low even after 4 hours at room temperature (however, the viscosity increased significantly after 24 hours at room temperature). Combining the storage of sucrose and glycerol (resulting in a further diluted soluble solution) allows for freeze-thaw cycles without apparent retention, both immediately and after 4 hours at room temperature.

[0083] Using this optimized workflow, the inventors demonstrated that the lysate can be concentrated at least nine times in the above assay using a single cartridge with little to no observed reduction in the intrinsic peptide or change in the composition of the detected peptide (see Figure 1E, Figures 6F-6H). This finding holds true even when the antibody crosslinking cartridge is dried after crosslinking, stored in a dry state, and then re-wetted before use.

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Claims

1. 1. A reusable cartridge for detecting an analyte in a solution, said cartridge comprising: a) a matrix; b) a detector molecule bound to the matrix, the detector molecule specifically binding to the analyte, the detector molecule being crosslinked to the matrix with a crosslinker selected from dimethylpimelimidate (DMP), cyanate ester, NHS ester, azlactone, carbonyldiimidazole (CDI), maleimide, iodoacetyl, pyridyl disulfide, hydrazide, or carbodiimide; and c) a cryoprotectant; and Including, After the analytes have eluted from the cartridge, the used cartridge is prepared for reuse by washing the matrix in 1-10% v / v acetic acid, 1% v / v formic acid, or 1% v / v trifluoroacetic acid (TFA), washing the matrix in a buffer containing a cryoprotectant, and storing the matrix in a dry or wet state; and A reusable cartridge, wherein the cartridge can be used at least 10, 20, 50, or 100 times for the detection of the analyte in a solution.

2. 2. The reusable cartridge of claim 1, wherein the cartridge is a tube open at both ends, a tube open at one end, a well, a plate with or without wells, or a chip capable of containing the matrix.

3. 3. The reusable cartridge of claim 1 or 2, wherein the matrix comprises particles comprising either silica or agarose, the particles being beads, grains, chips or pellets.

4. A reusable cartridge as described in claim 3, wherein the particles are magnetic.

5. The reusable cartridge of any one of claims 1 to 4, wherein the matrix comprises protein A, protein G, and / or protein L.

6. The reusable cartridge according to any one of claims 1 to 5, wherein the detection molecule is an antibody.

7. A reusable cartridge according to any one of claims 1 to 6, wherein the analyte detectable by the detection molecule on the matrix is ​​a protein or a peptide.

8. 8. The reusable cartridge of claim 7, wherein the analyte is a major histocompatibility complex I (MHC-I) molecule.

9. 9. The reusable cartridge of any one of claims 1 to 8, wherein the cartridge can be used at least 10 times for the detection of protein analytes in cell lysates or biological fluids.

10. A reusable cartridge described in any one of claims 1 to 9, wherein the matrix is ​​stored in the dry state.

11. 11. The reusable cartridge of claim 10, wherein the cartridge can be used at least 10 times, at least 20 times, at least 50 times, or at least 100 times for the detection of protein analytes in cell lysates or biological fluids after being stored in the dry state after each use.

12. 12. A reusable cartridge according to claim 10 or 11, wherein the matrix is ​​stored at an acidic pH.

13. A reusable cartridge according to any one of claims 10 to 12, wherein the matrix is ​​stored at 2 to 8°C.

14. 14. The reusable cartridge of any one of claims 1 to 13, wherein the cryoprotectant comprises one or more of sucrose, trehalose, ethylene glycol, propylene glycol, glycerol, 2-methyl-2,4-pentanediol (MPD), or dimethyl sulfoxide (DMSO).

15. A reusable cartridge according to any one of claims 1 to 14, wherein the matrix is ​​dried by heating the cartridge to at least 30°C, followed by storage at room temperature for at least 1 hour.

16. 16. The reusable cartridge of claim 15, wherein the matrix is ​​dried by heating the cartridge to 37°C, followed by storage at room temperature for at least 1 hour.

17. 17. A reusable cartridge according to claim 15 or 16, wherein after the matrix has dried, the cartridge is stored in a dry state at 2-8°C until reuse.

18. 15. A method for preparing a reusable cartridge according to any one of claims 1 to 14, comprising obtaining a cartridge comprising a matrix, contacting the matrix with the detection molecule, and cross-linking the detection molecule to the matrix.

19. 15. A method for detecting an analyte in a solution, the method comprising contacting the matrix of a reusable cartridge according to any one of claims 1 to 14 with a solution containing the analyte, and eluting the analyte from the cartridge.

20. 20. The method of claim 19, wherein the analyte is a peptide or a protein.

21. The method of claim 18, wherein the analyte is an MHC-I molecule.

22. The method of any one of claims 19 to 21, wherein the solution is a biological fluid or a cell lysate.

23. 23. The method of claim 22, wherein the solution is filtered or treated to remove cellular debris and membraneous material before contacting the matrix.

24. 22. The method of any one of claims 19 to 21, wherein the cartridge has been previously used to detect the analyte at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, or 100 times prior to use in the method.

25. A method according to any one of claims 19 to 24, wherein following elution of the analytes, the cartridge is treated by a method according to any one of claims 15 to 17.

26. A kit comprising at least one reusable cartridge according to any one of claims 1 to 14.

27. 27. The kit of claim 26, further comprising: (a) at least one buffer; (b) at least one control cartridge that does not contain a detection molecule or that contains a control detection molecule; (c) reagents for preparing the cartridge for storage and reuse; and / or (d) instructions for use.

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