Sample preparation, adsorbent, device and method based on non-antibody high affinity

Abstract

In various aspects, the present disclosure relates to an adsorbent for separating at least one target protein from a liquid sample, the adsorbent comprising a solid support comprising at least one attached high affinity reagent having an attachment with affinity to the at least one target protein. Other aspects of the present disclosure include kits containing such sorbents and methods of processing samples using the kits.

Description

[0001] This application is a divisional application. The application date of the original application is March 21, 2019, the application number is 201980020840.5 (PCT / US2019 / 023340), and the name of the invention is "Sample preparation, adsorbent, device and method based on non-antibody high affinity".

[0002] Related Applications

[0003] This application claims the benefit of U.S. Provisional Application No. 62 / 646,277, filed on March 21, 2018, the entire disclosure of which is incorporated by reference. Technical Field

[0004] The present disclosure relates to high affinity adsorbents, devices and methods of using the same, for example in conjunction with processes for protein depletion and / or for enrichment of low abundance proteins with enhanced recovery. Background Art

[0005] Identifying and quantifying low-abundance proteins (LAPs) from plasma, serum, or whole blood samples presents a challenge to researchers. This is primarily due to the presence of high-abundance proteins (HAPs). In this regard, 10-12 HAPs account for approximately 95% of the total protein mass of plasma or serum, with human serum albumin being the most prevalent HAP, typically accounting for more than half of the total protein mass of plasma or serum. The remaining proteins constitute LAPs, and many biomarkers fall into this category. The presence of HAPs increases the noise level, which in turn reduces the signal-to-noise ratio when performing the analysis.

[0006] The two key methods for identifying and quantifying LAPs involve affinity-based enrichment of LAPs and / or affinity-based depletion of HAPs. Disadvantages of existing products on the market include (a) the inability to remove LAPs bound to carrier proteins such as human serum albumin (which is known to act as a carrier protein for LAPs), (b) the inability to specifically capture low-abundance proteins, and (c) the need for protein precipitation and reconstitution after depletion of HAPs, a step that results in the loss of LAPs. Summary of the invention

[0007] In various aspects, the present disclosure relates to a method for sample processing, the method comprising: (a) adding a sample fluid containing a sample, the sample containing or potentially containing at least one target protein, to an adsorbent, the adsorbent comprising a solid support having at least one attached high-affinity reagent, the high-affinity reagent having a specific high affinity for the target protein, thereby obtaining an adsorbent with bound target proteins; and (b) adding a washing solution comprising a surfactant and a salt to the adsorbent, thereby removing substances for which the at least one high-affinity reagent does not have a specific affinity (including substances indirectly bound to the adsorbent by being attached to at least one target protein).

[0008] In various embodiments, the target protein is a protein of interest, and the protein of interest is isolated on an adsorbent for further processing.

[0009] In various embodiments, the target protein is not the protein of interest (eg, where the protein is a high abundance protein, etc.), and the wash solution can be collected for further downstream processing of the protein of interest.

[0010] In various embodiments that may be used in conjunction with any of the aforementioned aspects and embodiments, the sample may be selected from a whole blood sample, a plasma sample, and a serum sample.

[0011] In various embodiments that can be used in conjunction with any of the foregoing aspects and embodiments, the sample fluid can be prepared by combining the sample with a pretreatment solution that disrupts protein binding. For example, the pretreatment solution can include an acidic or alkaline aqueous solution, for example, the pretreatment solution can be selected from H3PO4 aqueous solution, NH4OH aqueous solution, ethylenediaminetetraacetic acid (EDTA) aqueous solution, formic acid aqueous solution, acetic acid aqueous solution, trichloroacetic acid (TCA) aqueous solution, and trifluoroacetic acid (TFA) aqueous solution.

[0012] In various embodiments that may be used in conjunction with any of the preceding aspects and embodiments, the wash solution can be a high stringency wash solution.

[0013] In various embodiments that may be used in conjunction with any of the aforementioned aspects and embodiments, the salt may be a chaotropic salt.

[0014] In various embodiments that may be used in combination with any of the foregoing aspects and embodiments, the salt may be selected from LiCl, KCl, NaCl, CsCl, LiI, KI, NaI, ScI, LiSCN, KSCN, NaSCN, CsSCN, Na2CO3, NaHCO3, NH4HCO3, Na2NO3, NaClO4, tetramethylammonium chloride, tetraethylammonium chloride, and trimethylammonium chloride.

[0015] In various embodiments that can be used in conjunction with any of the foregoing aspects and embodiments, the surfactant can be an acid-labile surfactant. Examples of acid-labile surfactants include sodium 3-[(2-methyl-2-undecyl-1,3-dioxolan-4-yl)methoxy]-1-propanesulfonate, sodium 3-(4-(1,1-bis(hexyloxy)ethyl)pyridinium-1-yl)propane-1-sulfonate, and sodium 3-((1-(furan-2-yl)undecyloxy)carbonylamino)propane-1-sulfonate, and the like.

[0016] In various embodiments that can be used in conjunction with any of the aforementioned aspects and embodiments, the surfactant can be an ionic surfactant. For example, the ionic surfactant can be selected from sodium dodecyl sulfate (SDS), sodium deoxycholate, sodium cholate, sodium lauroyl sarcosinate, and the like.

[0017] In various embodiments that can be used in conjunction with any of the foregoing aspects and embodiments, the surfactant can be a nonionic surfactant. For example, the nonionic surfactant can be selected from polysorbate surfactants (including polyoxyethylene sorbitan monolaurate (Tween 20) and polyoxyethylene sorbitan monooleate (Tween 80)), digitonin, maltoside (including n-dodecyl-β-D-maltoside (DDM), n-octyl-β-D-maltoside (DDM)), glucoside (including octyl glucoside and decyl glucoside), 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonic acid inner salt (CHAPS), 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonic acid inner salt hydrate (CHAPSO) and polyethylene glycol p-(1,1,3,3-tetramethylbutyl)-phenyl ether (Triton X-100).

[0018] In various embodiments that may be used in conjunction with any of the aforementioned aspects and embodiments, the target protein can be a high abundance protein or the target protein can be a low abundance protein.

[0019] Examples of highly abundant proteins include albumin, immunoglobulin G (IgG), immunoglobulin A (IgA), immunoglobulin M (IgM), immunoglobulin D (IgD), immunoglobulin D (IgE), alpha-1-antitrypsin, transferrin, haptoglobin, fibrinogen, haptoglobulin, alpha-2-macroglobulin, complement C3, apolipoprotein AI, apolipoprotein A-II, apolipoprotein B, alpha-1-acid glycoprotein, ceruloplasmin, complement C4, complement C1q, prealbumin, plasminogen, transthyretin, and combinations thereof, and the like.

[0020] Examples of low abundance proteins include ceruloplasmin, complement factors C4, C9, C8 and C5, IgD, C1 inhibitor, RBP, iC3b, thyroxine binding globulin, complement proteins, thrombus precursor protein, C-reactive protein, Bb fragment, ferritin, randes, SC5b-9 complex, myoglobin, thyroglobulin, TPA (tissue plasminogen activator), neuron-specific enolase, C-peptide, alpha-fetoprotein, TNF-binding protein, PSA (prostate specific antigen), prostatic acid phosphatase, CEA (carcinoembryonic antigen), myelin basic protein, troponin I, interleukins, MIP-1α, tissue factor, GCSF (granulocyte colony stimulating factor), interferon, and combinations thereof, etc.

[0021] In aspects and embodiments, for example, wherein the protein is a low abundance protein, the method may further comprise adding an elution solution to the adsorbent, thereby removing the low abundance protein from the adsorbent.

[0022] In aspects and embodiments, for example, where the protein is a low abundance protein, the low abundance protein can be digested into fragments when bound to the adsorbent, and the fragments can be eluted. For example, (a) the target protein can be digested by adding a solution comprising a proteolytic enzyme to the adsorbent, or (b) the adsorbent can contain an attached proteolytic enzyme, and the low abundance protein can be digested into fragments by activating the proteolytic enzyme (e.g., the low abundance protein can be digested into fragments by a method that includes chemical cleavage and release of the proteolytic enzyme).

[0023] In aspects and embodiments, for example, where the protein is a low abundance protein, the method may further comprise adding an elution solution to the adsorbent, which results in the release of the complex of the high affinity reagent bound to the low abundance protein from the adsorbent. For example, the high affinity reagent may be attached to the support via an enzymatically cleavable bond, and the elution solution may comprise an enzyme that cleaves the enzymatically cleavable bond.

[0024] In aspects and embodiments, for example, in which the protein is a low abundance protein, (a) the high affinity reagent can be attached to the support via a bond that can be cleaved upon exposure to nucleophilic and / or basic conditions, upon exposure to electrophilic and / or acidic conditions, or upon exposure to reducing conditions, and (b) the adsorbent can be exposed to an elution solution that establishes such conditions, thereby cleaving the complex of the high affinity reagent and the bound target protein.

[0025] In various aspects, the present disclosure relates to an adsorbent for separating at least one target protein from a liquid sample, the adsorbent comprising a solid support comprising at least one attached high affinity reagent having affinity for the at least one target protein.

[0026] In various embodiments, at least one high affinity reagent can be selected for its high affinity for low abundance proteins, high affinity for high abundance proteins, or both. Examples of high abundance proteins include albumin, immunoglobulin G (IgG), immunoglobulin A (IgA), immunoglobulin M (IgM), immunoglobulin D (IgD), immunoglobulin D (IgE), alpha-1-antitrypsin, transferrin, haptoglobin, fibrinogen, haptoglobulin, alpha-2-macroglobulin, complement C3, apolipoprotein AI, apolipoprotein A-II, apolipoprotein B, alpha-1-acid glycoprotein, ceruloplasmin, complement C4, complement C1q, prealbumin, plasminogen, transthyretin, and combinations thereof, and the like. Examples of low abundance proteins include ceruloplasmin, complement factors C4, C9, C8 and C5, IgD, C1 inhibitor, RBP, iC3b, thyroxine binding globulin, complement proteins, thrombus precursor protein, C-reactive protein, Bb fragment, ferritin, randes, SC5b-9 complex, myoglobin, thyroglobulin, TPA (tissue plasminogen activator), neuron-specific enolase, C-peptide, alpha-fetoprotein, TNF-binding protein, PSA (prostate specific antigen), prostatic acid phosphatase, CEA (carcinoembryonic antigen), myelin basic protein, troponin I, interleukins, MIP-1α, tissue factor, GCSF (granulocyte colony stimulating factor), interferon, and combinations thereof, etc.

[0027] In various embodiments that may be used in conjunction with any of the aforementioned aspects and embodiments, the solid support may comprise an inorganic material, an organic material, or a hybrid organic-inorganic material.

[0028] In various embodiments that can be used in conjunction with any one of the foregoing aspects and embodiments, the solid support can comprise a polymeric material. For example, the polymeric material can comprise a copolymer comprising a hydrophobic monomer (e.g., divinylbenzene, styrene, etc.) and a hydrophilic monomer (e.g., vinyl pyrrolidone, N-vinyl caprolactam, etc.).

[0029] In various embodiments that may be used in conjunction with any of the aforementioned aspects and embodiments, the adsorbent may be selected from a strong cation exchange adsorbent, a strong anion exchange adsorbent, a weak cation exchange adsorbent, and a weak anion exchange adsorbent.

[0030] In various embodiments that can be used in conjunction with any of the foregoing aspects and embodiments, at least one high affinity reagent can be attached to the solid support via a chemically cleavable bond. For example, at least one high affinity reagent can be attached to the solid support via a bond that can be cleaved when exposed to nucleophilic and / or alkaline conditions, when exposed to electrophilic and / or acidic conditions, or when exposed to reducing conditions.

[0031] In various embodiments that can be used in conjunction with any one of the aforementioned aspects and embodiments, at least one high-affinity reagent can be connected to a solid support by an enzymatically cleavable bond (e.g., a bond that can be cleaved by a proteolytic enzyme). In certain embodiments of these embodiments, the solid support can include an attached enzyme (e.g., an enzyme that is connected to the support by a chemically cleavable bond), which is suitable for enzymatically cleaving at least one high-affinity reagent from the solid support, thereby allowing the complex of the at least one high-affinity reagent and the target protein to be released from the solid support for further processing.

[0032] In various embodiments that may be used in conjunction with any of the aforementioned aspects and embodiments, the solid support comprises an attached enzyme that may be suitable for digesting a target protein upon activation of the attached enzyme.

[0033] In various embodiments that can be used in conjunction with any of the foregoing aspects and embodiments, the solid support comprises an attached proteolytic enzyme. For example, the proteolytic enzyme can be selected from trypsin, chymotrypsin, Glu-C, Lys-C, Arg-C, Asp-N, BNPS or NCS / urea, clostripain, CNBr, CNBr(methyl-Cys), CNBr(with acid), formic acid, hydroxylamine, iodosobenzoic acid, Lys-N, neutrophil elastase, NBS, NTCB, pancreatic elastase, pepsin A, prolyl endopeptidase, proteinase K, staphylococcal peptidase I, thermolysin, and combinations thereof, and the like.

[0034] In various embodiments that may be used in conjunction with any of the preceding aspects and embodiments, the solid support comprises an attached proteolytic enzyme attached to the solid support via a flexible linker.

[0035] In various embodiments that can be used in combination with any one of the aforementioned aspects and embodiments, the solid support comprises an attached proteolytic enzyme, and the attached proteolytic enzyme is attached to the solid support by a cleavable linking group. For example, the proteolytic enzyme can be attached to the solid support by a linking group that can be enzymatically cleavable. As another example, the proteolytic enzyme can be attached to an adsorbent by a linking group that can be chemically cleavable. For example, the linking group that can be chemically cleavable can be selected from a chemical linking group that is cracked when exposed to nucleophilic and / or alkaline conditions, a chemical linking group that is cracked when exposed to electrophilic and / or acidic conditions, a chemical linking group that is cracked when exposed to reducing conditions, a chemical linking group that is cracked when exposed to oxidizing conditions, a hydroxylamine cleavable linking group, a chemical linking group containing bis-succinimide esters, a chemical linking group containing bisacyl azide, and a chemical linking group containing bismaleimide.

[0036] In various embodiments that may be used in conjunction with any of the aforementioned aspects and embodiments, the proteolytic enzyme can be chemically activated by a change in pH, or the proteolytic enzyme can be thermally activated by heating or cooling.

[0037] In various embodiments that may be used in conjunction with any of the aforementioned aspects and embodiments, the adsorbent may be disposed in a multiwell strip, a multiwell plate, a column, a disposable cartridge, or an ELISA plate.

[0038] In a further aspect, the present disclosure relates to a kit comprising an adsorbent according to any of the preceding aspects and embodiments, and one or more kit components selected from the group consisting of: (a) a wash solution comprising a surfactant and a salt, (b) a pretreatment solution, (c) one or more elution solutions, (d) one or more proteolytic enzyme solutions, (e) a collection plate, (f) a cap pad, (g) calibration and reference standards, and (h) instructions for use.

[0039] In various embodiments that may be used in conjunction with any of the aforementioned aspects and embodiments, the high affinity agent may be an aptamer or an affibody. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 is a schematic diagram of an aptamer sequence, an aptamer sequence in a functional three-dimensional form, and an aptamer bound to a target.

[0041] Figure 2A is a schematic diagram of a high affinity reagent targeting at least a single protein in a sample attached to a solid adsorbent particle according to an embodiment of the present disclosure.

[0042] Figure 2B is a schematic diagram of a high affinity reagent attached to an adsorbent particle that pulls down its target protein as well as non-target proteins that are attached to the target protein through weak chemical interactions. DETAILED DESCRIPTION

[0043] The present disclosure relates to affinity-based methods, and to sorbents, devices, and kits useful for performing affinity-based methods.

[0044] In this regard, in some aspects, the present disclosure relates to an adsorbent that can be used to separate at least one target protein from a liquid sample, the adsorbent comprising a solid support material having attached thereto an affinity reagent, such as an aptamer or an affibody, the affinity reagent having a high affinity for the at least one target protein.

[0045] As used herein, an "aptamer" is a nucleic acid (eg, DNA, RNA, etc.) that selectively binds to a non-nucleic acid target molecule. In some embodiments, the length of an aptamer used herein can range from 15-80 nucleotide bases.

[0046] Figure 1 is a schematic diagram of an aptamer sequence 110s, an activated, functional, three-dimensional form of the aptamer sequence 110f, and a functional, three-dimensional form of the aptamer sequence 110f bound to a target 120, which in the particular embodiment shown is a biomarker. The aptamer can be activated prior to use, for example, by heating (e.g., to 85°C-90°C).

[0047] As a specific example, the human serum albumin aptamer identified as 23G03 (Oligo #372) was purchased from Base Pair Technologies, Houston, Texas, USA, which contains 39 bases (without 3′-6T) or 45 bases (with 3′-6T), has a reported molecular weight of 14,237.4 g / mol (including 3′-6T and biotin) and a Kd of 29.1 nM. In addition to biotin, the 3′ position can also be modified with an amine group and a thiol group. The 5′ end can be modified with an amine, a thiol, fluorescein isothiocyanate (FITC) or cyanine 5 (Cy5). Other aptamers that can be used include, for example, immunoglobulin G (IgG) Fc aptamers, fibrinogen aptamers, and human immunoglobulin M (IgM) mu chain aptamers, etc.

[0048] As used herein, an "affimer" is a stable protein molecule that displays a peptide loop and selectively binds to a specific target molecule. Typically, an affibody comprises a fixed core structure with two variable loops that provide a high affinity binding surface for a specific target protein. In certain embodiments, an affibody can be a low molecular weight (e.g., 12-14 kDa) protein derived from the cysteine ​​protease inhibitor family of cystatins.

[0049] Solid support materials useful for the adsorbents of the present disclosure include any suitable solid support material to which affinity agents (eg, aptamers and / or affibodies) can be attached. Examples of solid support materials include organic materials, inorganic materials, and organic-inorganic hybrid materials.

[0050] Examples of the inorganic material include, for example, a silica-based material, an alumina-based material, a titania-based material, a zirconia-based material, and a carbon-based material.

[0051] In certain beneficial embodiments, the solid support comprises a silica-based material formed by hydrolytic condensation of one or more organosilane compounds, which may, for example, comprise one or more alkoxysilane compounds. Examples of alkoxysilane compounds include, for example, tetraalkoxysilanes (e.g., tetramethoxysilane (TMOS), tetraethoxysilane (TEOS), etc.), alkylalkoxysilanes such as alkyltrialkoxysilanes (e.g., methyltrimethoxysilane, methyltriethoxysilane (MTOS), ethyltriethoxysilane, etc.), and bis(trialkoxysilyl)alkanes (e.g., bis(trimethoxysilyl)methane, bis(trimethoxysilyl)ethane, bis(triethoxysilyl)methane, bis(triethoxysilyl)ethane (BTE), etc.), and combinations thereof.

[0052] In certain embodiments, silica-based materials can be prepared from two monomers: a tetraalkoxysilane, such as TMOS or TEOS, and an alkylalkoxysilane, such as MTOS, or a bis(trialkoxysilyl)alkane, such as BTEE. When BTEE is used as a monomer, the resulting material is sometimes referred to as an ethylene-bridged hybrid (BEH) material and offers various advantages over conventional silica-based materials, including the ability to control surface silanol activity, chemical stability, and mechanical stability.

[0053] In certain beneficial embodiments, the solid carrier comprises an organic material or an organic-inorganic hybrid material in the form of a polymer. In a particular embodiment, the polymer can be a copolymer comprising a hydrophilic monomer (e.g., N-vinyl pyrrolidone, N-vinyl caprolactam, etc.) and a hydrophobic monomer (e.g., divinylbenzene, styrene, etc.).

[0054] In certain embodiments, the adsorbent may be an ion exchange adsorbent, for example, a strong cation exchange adsorbent (e.g., containing a polymer comprising a monomer having one or more strong anionic groups such as a sulfonate group, for example, divinylbenzene having one or more sulfonate groups), a strong anion exchange adsorbent (e.g., containing a polymer comprising a monomer having one or more strong cationic groups such as a quaternary ammonium group, for example, divinylbenzene having one or more quaternary ammonium groups), a weak cation exchange adsorbent (e.g., containing a polymer comprising a monomer having one or more weak anionic groups such as a carboxyl group, for example, divinylbenzene having one or more carboxyl groups), or a weak anion exchange adsorbent (e.g., containing a polymer comprising a monomer having one or more weak cationic groups such as a primary, secondary or tertiary amine group, for example, divinylbenzene having one or more primary, secondary or tertiary amine groups (e.g., one or more piperazine groups)).

[0055] In various aspects, the adsorbent employed is a particulate adsorbent comprising particles of a solid support material, such as those described above having an affinity agent attached thereto.

[0056] In other aspects, the adsorbent can be a monolith, an aerogel, a membrane, a hydrogel, a fiber material, or a microchannel on a sheet having an affinity agent attached thereto.

[0057] The size of the sorbent particles used in connection with the present disclosure can vary widely, and can range, for example, from about 1 um to about 100 um in diameter, among other particle sizes.

[0058] Adsorbent particles used in connection with the present disclosure can be nonporous, superficially porous, or completely porous, including particles having pores having diameters ranging from about 8 nm to about 2500 nm, among other values.

[0059] The adsorbent particles used in conjunction with the present disclosure may be porous or non-porous magnetic particles.

[0060] Affinity agents (eg, aptamers and / or affibodies) can be attached to the solid support material using any suitable binding scheme, including covalent and non-covalent attachments.

[0061] For example, refer to Figure 2A , shows a schematic diagram of an adsorbent particle with an attached high affinity reagent 200 that is capable of targeting at least a single protein in a sample according to an embodiment of the present disclosure. In the illustrated embodiment, an aptamer 210 (i.e., a biotinylated aptamer) with an attached biotin molecule 212 is attached to an adsorbent particle (e.g., a BEH particle 216) with an attached streptavidin molecule 214 (such as described in the Examples below). Numeral 218 represents casein.

[0062] Apparatus according to the present disclosure typically include a high affinity adsorbent as described herein in a packed adsorbent bed.

[0063] Devices according to the present disclosure generally include a housing having a chamber for receiving and holding a high affinity adsorbent. In various embodiments, the housing may be provided with an inlet and an outlet.

[0064] Materials of construction for the housing include inorganic materials, for example, metals such as stainless steel and ceramics such as glass, as well as synthetic polymer materials such as polyethylene, polypropylene and polyetheretherketone (PEEK) and polycarbonate.

[0065] In certain embodiments, the device may include one or more filters for retaining the sorbent in the housing. Exemplary filters may be in the form of, for example, a membrane, a mesh, a frit, or a spherical porous filter.

[0066] In certain embodiments, the solution received in the housing can flow spontaneously (e.g., by capillary action) into the high affinity adsorbent. Alternatively, flow through the adsorbent can be generated by an external force (such as gravity or centrifugation) or by applying a vacuum to the outlet of the housing or a positive pressure to the inlet of the housing.

[0067] Specific examples of shells for use in the present disclosure include, for example, syringes, syringe barrels, columns (e.g., microporous columns, capillary columns, or nanocolumns), multi-well devices such as 4-well to 8-well racks, 4-well to 8-well strips, 48-well to 96-well plates, 96-well to 384-well microelution plates, microelution tip devices (including 4-to-8-tip microelution strips, 96-to-384 microelution tip arrays, single microelution pipette tips), thin layer plates, microtiter plates, rotating tubes, or rotating containers.

[0068] Multiwell formats are often used with robotic fluid dispensing systems. Typical multiwell formats include 48-well, 96-well and 384-well standard plate formats, but other formats are obviously possible.

[0069] In various aspects, the present disclosure relates to a method of performing sample enrichment, the method comprising contacting a sample fluid comprising a sample having at least one target protein with an adsorbent according to the present disclosure.

[0070] The sample fluid can be any fluid that contains or may contain the target protein of interest. In some embodiments, the sample fluid can be in the form of, for example, an aqueous solution or a water-miscible organic solution.

[0071] In some embodiments, the sample fluid is or is derived from a biological sample. Exemplary biological samples include biological fluids, biological tissues, biological matrices, embedded tissue samples, inclusion bodies, cells (e.g., one or more types of cells) and cell culture supernatants. Specific examples of biological samples may include whole blood, plasma, serum, urine, cerebrospinal fluid, synovial fluid, sputum, semen, saliva, tears, gastric juice and other biological fluids, including extracts of tissues (such as liver tissue, muscle tissue, brain tissue, heart tissue, etc.).

[0072] In some embodiments, the sample fluid is or is derived from a reaction mixture, preparative HPLC, chromatography eluate or fraction, or an environmental sample.

[0073] In some embodiments, the sample fluid can be prepared by a method including adding a pretreatment solution that destroys protein binding. For example, in some cases, the sample fluid can include a biological sample combined with a pretreatment solution that destroys protein binding. Examples of pretreatment solutions that destroy protein binding include acidic aqueous solutions and alkaline aqueous solutions. In certain specific embodiments, a pretreatment aqueous solution can be used, which includes ethylenediaminetetraacetic acid (EDTA), formic acid, acetic acid, trichloroacetic acid (TCA), trifluoroacetic acid (TFA), phosphoric acid, NH4OH, and combinations thereof, etc.

[0074] In some embodiments, the target protein may include, for example, one or more highly abundant proteins.

[0075] Examples of highly abundant proteins include albumin, highly abundant immunoglobulins such as immunoglobulin G (IgG), immunoglobulin A (IgA), immunoglobulin M (IgM), immunoglobulin D (IgD), and immunoglobulin E (IgE), alpha-1-antitrypsin, transferrin, haptoglobin, fibrinogen, haptoglobulin, alpha-2-macroglobulin, complement C3, apolipoprotein AI, apolipoprotein A-II, apolipoprotein B, alpha-1-acid glycoprotein, ceruloplasmin, complement C4, complement C1q, prealbumin, plasminogen, transthyretin, and combinations thereof.

[0076] In some embodiments, the target protein may include, for example, one or more low-abundance proteins.

[0077] Examples of low abundance proteins include ceruloplasmin, complement factors such as C4, C9, C8, C5, IgD, C1 inhibitor, RBP, iC3b, thyroxine binding globulin, complement proteins, thrombus precursor protein, C-reactive protein, Bb fragment, ferritin, randes, SC5b-9 complex, myoglobin, thyroglobulin, TPA (tissue plasminogen activator), neuron-specific enolase, C-peptide, alpha-fetoprotein, TNF-binding protein, PSA (prostate specific antigen), prostatic acid phosphatase, CEA (carcinoembryonic antigen), myelin basic protein, troponin I, interleukins, MIP-1α, tissue factor, GCSF (granulocyte colony stimulating factor), interferon, and combinations thereof.

[0078] In certain embodiments involving LAPs, the affinity agent can be attached to a solid support material via a cleavable linker, such that the affinity agent along with any bound protein can be released from the support. Cleavable linkers include chemically labile linkers and enzymatically cleavable linkers.

[0079] Enzymatically cleavable linkers include peptide-based linkers having specific sequences that are cleaved upon exposure to a suitable enzyme (e.g., a protease), particularly an enzyme that is specific for a particular peptide sequence, such as caspase-1, caspase-2, caspase-3, caspase-4, caspase-5, caspase-6, caspase-7, caspase-8, caspase-9, caspase-10, cathepsin B, enterokinase, factor Xa, granzyme B, HRV3C protease, matrix metalloproteinase-2, TEV protease, thrombin, and the like.

[0080] Chemically labile linkers include linkers that cleave upon exposure to nucleophilic and / or basic conditions (e.g., based on cleavable groups such as dialkyldialkoxysilanes, cyanoethyl, sulfones, glycol disuccinate 2-N-acylnitrobenzenesulfonamides, α-thiophene esters, unsaturated vinyl thioethers, activated sulfonamides, malondialdehyde (MDA)-indole derivatives, levulinic esters, hydrazines, acylhydrazones, and alkylthioesters, among other cleavable groups). Chemically unstable linkers also include linkers that cleave when exposed to electrophilic and / or acidic conditions (e.g., linkers based on cleavable groups such as p-methoxybenzyl derivatives, tert-butyl carbamate analogs, dialkyl or diaryl dialkoxysilanes, orthoesters, acetals, aconityl, hydrazones, β-thiopropionates, phosphoramidates, imines, trityl, vinyl ethers, polyketals, 2-(diphenylphosphino)benzoic acid alkyl ester derivatives and 4-(4′-acetylphenoxy)butyric acid, as well as other cleavable groups). Chemically unstable linkers also include linkers that cleave when exposed to reducing conditions (e.g., linkers such as disulfide bonds, diazo linkers, etc.), linkers that can cleave when exposed to oxidative conditions such as those containing diols, and hydroxylamine cleavable linkers such as those containing esters. Chemically unstable homofunctional linkers such as those containing cleavable bis-succinimidyl esters, bisacyl azides, and bismaleimides.

[0081] In various embodiments, the methods described herein may first include contacting a sample fluid containing one or more target proteins with an adsorbent comprising a solid support having an attached affinity reagent (e.g., an aptamer or an affibody) having a specific affinity for the one or more target proteins (such adsorbents are described in more detail above), thereby causing the adsorbent to bind to the one or more target proteins. In this regard, different affinity reagents may be attached to the adsorbent to allow for simultaneous capture of multiple target proteins. The adsorbent may advantageously contain a molar excess of the attached affinity reagent relative to the one or more target proteins in the sample fluid.

[0082] This method is often referred to as a "loading" method, and generally involves passing a sample fluid through a device containing an adsorbent (such devices are described in more detail above). During loading, one or more target proteins along with various unintended non-target substances (including non-target proteins) are adsorbed onto the adsorbent. For example, a sample having Figure 2A The adsorbent particles with attached high affinity reagents 200 shown can be used to bind to target proteins 310 as well as various non-target proteins 312 attached to target proteins 310 through weak chemical interactions, such as Figure 2B As shown schematically in FIG.

[0083] The contact time and / or flow rate during loading can be optimized for appropriate diffusion kinetics and binding of one or more target proteins to the adsorbent. If desired, the sample fluid can be passed through the device multiple times.

[0084] In certain embodiments, for example, in the case where the adsorbent is not easily wetted by water, conditioning and balancing steps may be performed before sample loading. For example, in an exemplary embodiment, among other possibilities, methanol, ethanol, propanol, isopropanol, water, and mixtures thereof may be used for conditioning. For example, in an exemplary embodiment, the balancing solution may have a similar or identical ionic strength to the sample solution to provide a balanced distribution environment in the adsorbent when the sample solution is loaded. Exemplary balancing solutions / solvents may be, but are not limited to, water, aqueous solutions such as buffer solutions (e.g., phosphate buffer solutions), water miscible organic solvent solutions, and the like.

[0085] In certain embodiments, for example, where the adsorbent is sufficiently water wettable, conditioning and equilibration can be omitted prior to sample loading.

[0086] Once the sample is loaded onto the adsorbent, one or more wash solutions can be contacted with the adsorbent to separate non-target species (e.g., species for which the affinity reagent does not have a specific affinity) from the target protein, thereby resulting in elution of the non-target species while allowing the target protein to remain retained on the adsorbent.

[0087] In various embodiments, the one or more wash solutions can include a low stringency wash solution, a high stringency wash solution, or both.

[0088] As used herein, a "high stringency" wash solution is one that is capable of disrupting high affinity interactions (specifically, 10 -8 M Kd or higher interactions, where Kd is the equilibrium dissociation constant). In the case of ligand-protein complex formation, LP can be obtained by a two-state method The corresponding equilibrium dissociation constant is wherein [L], [P], and [LP] represent the molar concentrations of protein (P), ligand (L), and ligand-protein complex (LP), respectively. As used herein, a "low stringency" wash solution is one that disrupts low affinity interactions (specifically, 10 -8 M Kd or higher interactions) in washing solutions.

[0089] In the present disclosure, a high affinity agent having a specific high affinity for a target protein is generally one in which the interaction between the target protein (P) and the ligand (L) (in this case, the high affinity agent) is 10 -8 Reagents with M Kd or higher.

[0090] In various embodiments, the one or more wash solutions may include one or more surfactants and one or more chaotropic salts.

[0091] Examples of chaotropic salts may include, among others, the following: LiCl, KCl, NaCl, CsCl, LiI, KI, NaI, ScI, LiSCN, KSCN, NaSCN, CsSCN, Na2CO3, NaHCO3, NH4HCO3, Na2NO3, NaClO4, tetramethylammonium chloride, tetraethylammonium chloride, and trimethylammonium chloride, among others.

[0092] In various embodiments, the one or more surfactants may include an ionic surfactant or a nonionic surfactant. Examples of ionic surfactants may include the following: sodium dodecyl sulfate (SDS), sodium deoxycholate, sodium cholate, and sodium lauroyl sarcosinate (Sarcosyl), among others. Examples of nonionic surfactants may include, among others, the following: polysorbate surfactants (including polyoxyethylene sorbitan monolaurate (Tween 20) and polyoxyethylene sorbitan monooleate (Tween 80)), digitonin, maltoside (including n-dodecyl-β-D-maltoside (DDM), n-octyl-β-D-maltoside (DDM)), glucoside (including octyl glucoside and decyl glucoside), 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid inner salt (CHAPS), 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid inner salt hydrate (CHAPSO), and polyethylene glycol p-(1,1,3,3-tetramethylbutyl)-phenyl ether (Triton X-100).

[0093] In various embodiments, the one or more surfactants may include one or more acid-labile surfactants. Examples of acid-labile surfactants may include the following, among others: sodium 3-[(2-methyl-2-undecyl-1,3-dioxolan-4-yl)methoxy]-1-propanesulfonate (available as RapiGest TM SF surfactant was purchased from Waters Corporation, Milford, MA, USA), sodium 3-(4-(1,1-bis(hexyloxy)ethyl)pyridinium-1-yl)propane-1-sulfonate (PPS Silent TM Surfactants were purchased from Expedeon Ltd., Over Cambridgeshire, United Kingdom) and sodium 3-((1-(furan-2-yl)undecyloxy)carbonylamino)propane-1-sulfonate (ProteaseMAX TM Purchased from Promega Corporation, Fitchburg, WI, USA).

[0094] HAPs. In some embodiments, the target protein may be one or more HAPs, and the methods described herein may be used to deplete one or more HAPs from a sample fluid, thereby improving the ability to subsequently identify, quantify, or otherwise process various non-target species (including one or more LAPs) in the sample fluid. In some embodiments, a second device containing a high affinity adsorbent specific for capturing LAPs may be used to capture and enrich LAPs from a sample.

[0095] The one or more wash solutions can include a low stringency wash, a high stringency wash, or both.

[0096] For example, low stringency washes can be performed using solutions containing low strength surfactants and moderate salt concentrations to disrupt low Kd protein-protein interactions between one or more target proteins (e.g., HAPs) and other plasma proteins (e.g., LAPs). Alternatively or in addition, high stringency washes can be performed using solutions containing strong surfactants such as SDS and chaotropic salts to disrupt substantially any interactions between the adsorbent and one or more target proteins except for high affinity interactions. Strong surfactants can be used to maintain the proteins in a semi-denatured to fully denatured state if affinity capture of the one or more target proteins is beneficial.

[0097] In these embodiments, the adsorbent with attached target protein(s) (e.g., HAP) can be discarded or regenerated after use by washing with 1 M NaOH and then equilibrating the adsorbent in the solvent used in the loading method, among other possibilities (depending on the type of aptamer-target molecule complex).

[0098] In these embodiments, after the one or more wash solutions are contacted with the adsorbent to separate non-target substances from the one or more target proteins bound to the adsorbent, the one or more wash solutions are collected for further processing, which may include, for example, identifying, quantifying, or otherwise processing the various LAPs in the one or more wash solutions. Prior to doing so, it may be desirable to remove the one or more surfactants and the one or more chaotropic salts from the one or more wash solutions. This may be done, for example, using a suitable solid phase extraction method, such as using HLB adsorbent, PRIMEHLB, MCX or MAX (Waters Corp.), etc.

[0099] Notably, identifying and quantifying LAPs from biological samples such as plasma, serum, or whole blood samples presents a challenge to the investigator, primarily due to the presence of HAPs, one of which (albumin) accounts for more than half of the total protein mass of plasma / serum, and a small number of HAPs (albumin, IgG, transferrin, fibrinogen, IgA, haptoglobin, alpha-1-antitrypsin, alpha-2-macroglobulin, IgM, apolipoprotein AI, alpha-1-acid glycoprotein, complement C3, apolipoprotein A-II, transthyretin) account for approximately 95% of the total protein mass of plasma / serum. Depletion of one or more of these HAPs will improve the investigator's ability to identify, quantify, or otherwise address SAPs. By utilizing a high affinity adsorbent in which a high affinity reagent such as an aptamer or affibody is bound to a solid support, one or more wash solutions can be employed to remove non-target species (e.g., LAP) from HAP bound to the adsorbent, including wash solutions that would otherwise have very high stringency so that the HAP would be removed from the adsorbent along with the non-target species. In this manner, the HAP can be removed from the LAP without the need for precipitation and reconstitution steps, which are commonly used in the art and result in loss of the LAP. Thus, in various embodiments, the methods described herein are used to prepare a sample (e.g., a sample obtained after removal of the HAP during a depletion process) for further analysis and / or processing.

[0100] LAP. In some embodiments, the target protein can be one or more proteins (e.g., one or more LAPs) that have been identified for enrichment. In these embodiments, the methods described herein can be used to enrich those proteins from a sample fluid.

[0101] In this regard, the one or more wash solutions may include a high stringency wash. By utilizing a high affinity adsorbent (wherein a high affinity reagent such as an aptamer or affibody is bound to a solid support), one or more high stringency wash solutions may be employed to remove non-target material (including HAPs) from the adsorbent, which may otherwise result in one or more enriched proteins (e.g., one or more LAPs) being inadvertently removed from the adsorbent along with the non-target material.

[0102] For example, a high stringency wash may be performed with a solution containing a strong surfactant such as SDS or the like to disrupt all but the highest affinity interactions (e.g., less than 10 μg / mL) between the adsorbent-bound affinity reagent and the one or more target proteins. -8 Strong surfactants can also be used to maintain proteins in a semi-denatured to fully denatured state if affinity capture of the target protein or proteins is beneficial.

[0103] In these embodiments where the target protein may be one or more proteins that have been identified for enrichment (e.g., one or more LAPs), after the one or more wash solutions are contacted with the adsorbent to separate non-target substances from the one or more target proteins bound to the adsorbent, the one or more target proteins on the adsorbent may be subjected to further processing. On the other hand, the one or more wash solutions may be discarded.

[0104] In such embodiments, further processing of the target protein bound to the adsorbent may include, for example, quantifying or otherwise processing the various LAPs attached to the adsorbent.

[0105] For example, in some embodiments, one or more target proteins can be subjected to digestion while being retained on an adsorbent. For example, the combined target protein can be digested by one or more proteolytic enzymes, for example one or more endoproteinases such as trypsin, chymotrypsin, Glu-C or Lys-C, which hydrolyze the peptide bonds in the target protein, thereby segmenting the target protein into peptides. Additional proteolytic substances / conditions that can be used for breaking peptide bonds include Arg-C, Asp-N, BNPS or NCS / urea, clostripain, CNBr, CNBr (methyl-Cys), CNBr (with acid), formic acid, Glu-C, hydroxylamine, iodosylbenzoic acid, Lys-N, weak acid hydrolysis, neutrophil elastase, NBS, NTCB, pancreatic elastase, pepsin A, prolyl endopeptidase, proteinase K, staphylococcal peptidase I and thermolysin, etc.

[0106] The proteolytic enzyme may, for example, be added to an adsorbent bed. Alternatively, the proteolytic enzyme may be chemically attached to the adsorbent, in which case a linker may be employed to aid the enzyme in contacting and digesting the bound target protein. The enzyme may also be attached to the support using a cleavable linker, wherein cleavage is chemically triggered, thereby allowing the enzyme to be released for digestion. The proteolytic enzyme attached to the adsorbent may be activated, for example, by increasing the temperature or by establishing a specific pH.

[0107] If added enzyme or cleaved enzyme is used, a sample preparation step may be employed to remove the enzyme and in some cases autolysis products prior to analysis of the target protein.After digestion of the attached LAP, the affinity adsorbent may be regenerated or discarded.

[0108] In some embodiments, prior to digestion, one or more bound target proteins may be subjected to reduction and alkylation. For example, one or more bound target proteins may be subjected to disulfide reduction using a suitable reducing agent such as tris(2-carboxyethyl)phosphine (TCEP) or dithiothreitol (DTT), and free sulfhydryl groups on cysteine ​​residues may be alkylated with an alkylating agent such as iodoacetamide or iodoacetic acid to irreversibly prevent the free sulfhydryl groups from reforming disulfide bonds.

[0109] After digestion, the resulting peptide mixture can be optionally mixed with a buffer (e.g., a buffer with neutralizing properties). The peptide mixture can then be used downstream for protein quantification.

[0110] In some embodiments, for example, where the affinity reagent is attached to the adsorbent via a cleavable bond, the affinity reagent with the attached target protein can be released from the support for further processing.

[0111] For example, affinity reagents can be attached to adsorbents via bonds that are cleaved when exposed to nucleophilic and / or alkaline conditions, when exposed to electrophilic and / or acidic conditions, or when exposed to reducing conditions as discussed above, in which case adsorbents can be exposed to elution solutions that establish such conditions. Affinity reagents can be attached to adsorbents via enzymatically cleavable bonds, for example, bonds can include peptide-based linkers with specific sequences that are cleaved when exposed to suitable enzymes (e.g., proteases), especially enzymes that are specific to specific peptide sequences (such as those discussed previously). Such enzymes can be added to adsorbents, or alternatively, such enzymes can be chemically connected to adsorbents, in which case linkers can be used to help enzymes contact cleavable bonds, and affinity reagents are bound to adsorbents through the cleavable bonds. In some cases, enzymes can be attached to particles via cleavable linkers, in which case linkers can be cleaved to release enzymes, which are then used to cleave affinity reagents. Enzymes attached to adsorbents can be activated, for example, by heating or by establishing specific pH or solvent systems.

[0112] As noted above, in various embodiments, the methods described herein may also include using analytical instruments and / or techniques (e.g., liquid chromatography (LC) (including high performance liquid chromatography (HPLC) and ultra high performance liquid chromatography (UHPLC)), mass spectrometry (MS) (including tandem mass spectrometry (MS / MS), electrospray ionization mass spectrometry (ESI-MS), matrix-assisted laser desorption / ionization mass spectrometry (MALDI-MS), time-of-flight mass spectrometry (TOFMS)), nuclear magnetic resonance, infrared analysis, ultraviolet analysis, or a combination thereof) to analyze a sample (e.g., a sample obtained after removal of HAPs during a depletion process, a sample obtained after removal or digestion of LAPs captured by an adsorbent during an enrichment process, etc.). For example, in some embodiments, a processed sample may be analyzed using liquid chromatography such as HPLC or UHPLC in combination with mass spectrometry such as MALDI-MS or ESI-MS, examples of which include liquid chromatography-mass spectrometry (LC-MS) techniques and liquid chromatography-mass spectrometry / mass spectrometry (LC-MS / MS) techniques. In some cases, the analysis sample may be evaporated to dryness and then reconstituted in another solution before being injected into the liquid chromatography system.

[0113] Kits may also be provided, comprising a high affinity adsorbent as described elsewhere herein and one or more of any of the following: (a) a wash solution comprising a surfactant and a salt as described elsewhere herein, (b) a pretreatment solution as described elsewhere herein, (c) one or more elution solutions as described elsewhere herein, (d) a collection plate or collection cylinder, (e) a cap mat, (f) calibration and reference standards, and (g) instructions for use. The adsorbent may be disposed in a housing, such as a column, a disposable cartridge, a porous strip, a porous plate, or other housing as described elsewhere herein.

[0114] Further aspects of the disclosure are set forth in the following enumerated paragraphs:

[0115] Aspect A1. A method for sample processing, the method comprising: adding a sample fluid containing a sample to an adsorbent, the sample containing or possibly containing at least one target protein, the adsorbent comprising a solid support having at least one attached high-affinity reagent, the high-affinity reagent having a specific high affinity for the target protein, thereby obtaining an adsorbent with a bound target protein; and adding a washing solution comprising a surfactant and a salt to the adsorbent, thereby removing substances for which the at least one high-affinity reagent does not have a specific affinity, including substances that are indirectly bound to the adsorbent by being attached to the target protein.

[0116] Aspect A2. A method according to aspect A1, wherein (a) when the target protein is a protein of interest, the protein of interest is separated on the adsorbent for further processing, or (b) when the target protein is not a protein of interest, the wash solution is collected for further downstream processing of the protein of interest.

[0117] Aspect A3. The method according to any one of Aspects A1-A2, wherein the sample is selected from the group consisting of a whole blood sample, a plasma sample and a serum sample.

[0118] Aspect A4. The method according to any one of aspects A1 to A3, wherein the sample fluid is prepared by mixing the sample with a pretreatment solution that disrupts protein binding.

[0119] Aspect A5. The method according to aspect A4, wherein the pretreatment solution comprises an acidic or alkaline aqueous solution.

[0120] Aspect A6. A method according to aspect A4, wherein the pretreatment solution is selected from an aqueous H3PO4 solution, an aqueous NH4OH solution, an aqueous ethylenediaminetetraacetic acid (EDTA) solution, an aqueous formic acid solution, an aqueous acetic acid solution, an aqueous trichloroacetic acid (TCA) solution and an aqueous trifluoroacetic acid (TFA) solution.

[0121] Aspect A7. The method according to any one of aspects A1 to A6, wherein the wash solution is a high stringency wash solution.

[0122] Aspect A8. The method according to any one of aspects A1 to A7, wherein the salt is a chaotropic salt.

[0123] Aspect A9. The method according to any of Aspects A1 to A7, wherein the salt is selected from LiCl, KCl, NaCl, CsCl, LiI, KI, NaI, ScI, LiSCN, KSCN, NaSCN, CsSCN, Na2CO3, NaHCO3, NH4HCO3, Na2NO3, NaClO4, tetramethylammonium chloride, tetraethylammonium chloride and trimethylammonium chloride.

[0124] Aspect A10. The method according to any one of aspects A1 to A9, wherein the surfactant is an acid-labile surfactant.

[0125] Aspect A11. A method according to aspect A10, wherein the acid-labile surfactant is selected from sodium 3-[(2-methyl-2-undecyl-1,3-dioxolan-4-yl)methoxy]-1-propane sulfonate, sodium 3-(4-(1,1-bis(hexyloxy)ethyl)pyridinium-1-yl)propane-1-sulfonate and sodium 3-((1-(furan-2-yl)undecyloxy)carbonylamino)propane-1-sulfonate.

[0126] Aspect A12. The method according to any one of aspects A1 to A9, wherein the surfactant is an ionic surfactant.

[0127] Aspect A13. The method according to aspect A12, wherein the ionic surfactant is selected from sodium dodecyl sulfate (SDS), sodium deoxycholate, sodium cholate and sodium lauroyl sarcosinate.

[0128] Aspect A14. The method according to any one of aspects A1 to A9, wherein the surfactant is a nonionic surfactant.

[0129] Aspect A15. A method according to aspect A14, wherein the nonionic surfactant is selected from polysorbate surfactants (including polyoxyethylene sorbitan monolaurate (Tween 20) and polyoxyethylene sorbitan monooleate (Tween 80)), digitonin, maltoside (including n-dodecyl-β-D-maltoside (DDM), n-octyl-β-D-maltoside (DDM)), glucoside (including octyl glucoside and decyl glucoside), 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid inner salt (CHAPS), 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid inner salt hydrate (CHAPSO) and polyethylene glycol p-(1,1,3,3-tetramethylbutyl)-phenyl ether (Triton X-100).

[0130] Aspect A16. The method according to any one of aspects A1 to A15, wherein the target protein is a high abundance protein.

[0131] Aspect A17. A method according to Aspect A16, wherein the abundant protein is selected from albumin, immunoglobulin G (IgG), immunoglobulin A (IgA), immunoglobulin M (IgM), immunoglobulin D (IgD), immunoglobulin D (IgE), alpha-1-antitrypsin, transferrin, haptoglobin, fibrinogen, haptoglobulin, alpha-2-macroglobulin, complement C3, apolipoprotein AI, apolipoprotein A-II, apolipoprotein B, alpha-1-acid glycoprotein, ceruloplasmin, complement C4, complement C1q, prealbumin, plasminogen, transthyretin, and combinations thereof.

[0132] Aspect A18. The method according to any one of aspects A1 to A15, wherein the target protein is a low abundance protein.

[0133] Aspect A19. A method according to Aspect A18, wherein the low-abundance protein is selected from plasma ceruloplasmin, complement factors C4, C9, C8 and C5, IgD, C1 inhibitor, RBP, iC3b, thyroxine-binding globulin, complement proteins, thrombus precursor protein, C-reactive protein, Bb fragment, ferritin, randes, SC5b-9 complex, myoglobin, thyroglobulin, TPA (tissue plasminogen activator), neuron-specific enolase, C-peptide, alpha-fetoprotein, TNF-binding protein, PSA (prostate specific antigen), prostatic acid phosphatase, CEA (carcinoembryonic antigen), myelin basic protein, troponin I, interleukin, MIP_1α, tissue factor, GCSF (granulocyte colony stimulating factor), interferon, and combinations thereof.

[0134] Aspect A20. The method according to any one of aspects A18-A19, further comprising adding an elution solution to the adsorbent, thereby removing the low-abundance protein from the adsorbent.

[0135] Aspect A21. The method according to any one of aspects A18-A19, wherein the low abundance protein is digested into fragments when bound to the adsorbent, and the fragments are eluted.

[0136] Aspect A22. The method according to aspect A21, wherein the target protein is digested by adding a solution comprising a proteolytic enzyme to the adsorbent.

[0137] Aspect A23. The method according to aspect A21, wherein the adsorbent comprises an attached proteolytic enzyme, and wherein the low-abundance protein is digested into fragments by activating the proteolytic enzyme.

[0138] Aspect A24. The method according to aspect A21, wherein the adsorbent comprises an attached cleavable enzyme, and wherein the low-abundance protein is digested into fragments by a method comprising chemical cleavage and release of the proteolytic enzyme.

[0139] Aspect A25. A method according to any one of aspects A18-A19, wherein the high affinity reagent is linked to the support via an enzymatically cleavable bond, and wherein the elution solution comprises an enzyme that cleaves the enzymatically cleavable bond.

[0140] Aspect A26. A method according to aspects A18-A19, wherein the high affinity reagent is attached to the support via a bond that is cleaved upon exposure to nucleophilic and / or basic conditions, upon exposure to electrophilic and / or acidic conditions, or upon exposure to reducing conditions, wherein the adsorbent can be exposed to an elution solution that establishes such conditions, thereby cleaving the complex of the high affinity reagent and the bound target protein.

[0141] Aspect A27. The method according to any one of aspects A1 to A26, wherein the high affinity agent is an aptamer or an affibody.

[0142] Aspect B1. An adsorbent for separating at least one target protein from a liquid sample, the adsorbent comprising a solid support comprising an attached aptamer or affibody having affinity for the at least one target protein.

[0143] Aspect B2. The adsorbent according to aspect B1, wherein the solid support comprises silica.

[0144] Aspect B3. The adsorbent according to aspect B1, wherein the solid support comprises a hybrid material.

[0145] Aspect B4. The adsorbent according to any one of aspects B1 to B3, wherein the solid support comprises a polymer material.

[0146] Aspect B5. The adsorbent according to aspect B4, wherein the polymer material comprises a copolymer comprising a hydrophobic monomer and a hydrophilic monomer.

[0147] Aspect B6. The adsorbent according to aspect B5, wherein the hydrophilic monomer is selected from vinyl pyrrolidone and N-vinyl caprolactam.

[0148] Aspect B7. The adsorbent according to any one of aspects B5-B6, wherein the hydrophobic monomer is selected from divinylbenzene and styrene.

[0149] Aspect B8. The adsorbent according to any one of aspects B5 to B7, wherein the adsorbent is selected from a strong cation exchange adsorbent, a strong anion exchange adsorbent, a weak cation exchange adsorbent, and a weak anion exchange adsorbent.

[0150] Aspect B9. The adsorbent according to any one of aspects B1 to B8, wherein the aptamer or affibody is attached to the solid support via a chemically cleavable bond.

[0151] Aspect B10. The adsorbent according to any one of aspects B1 to B8, wherein the aptamer or affibody is attached to the solid support via an enzymatically cleavable bond.

[0152] Aspect B11. An adsorbent according to aspect B10, wherein the solid support comprises an attached enzyme suitable for enzymatically cleaving the aptamer or affibody from the solid support, thereby allowing the complex of the aptamer or affibody and the target protein to be released from the solid support for further processing.

[0153] Aspect B12. The adsorbent according to any one of aspects B1 to B11, wherein the aptamer or affibody is selected for its high affinity for low-abundance proteins, its high affinity for high-abundance proteins, or both.

[0154] Aspect B13. The adsorbent according to any one of aspects B1 to B12, wherein the solid support comprises an attached enzyme, the attached enzyme being suitable for digesting the target protein upon activation of the attached enzyme.

[0155] Aspect B14. The adsorbent according to any one of aspects B1 to B13, wherein the solid support comprises an attached proteolytic enzyme.

[0156] Aspect B15. An adsorbent according to aspect B14, wherein the proteolytic enzyme is selected from trypsin, chymotrypsin, Glu-C, Lys-C, Arg-C, Asp-N, BNPS or NCS / urea, clostripain, CNBr, CNBr(methyl-Cys), CNBr(with acid), formic acid, hydroxylamine, iodosobenzoic acid, Lys-N, neutrophil elastase, NBS, NTCB, pancreatic elastase, pepsin A, prolyl endopeptidase, proteinase K, staphylococcal peptidase I, thermolysin, and combinations thereof.

[0157] Aspect B16. The adsorbent according to any one of aspects B14-B15, wherein the proteolytic enzyme is attached to the solid support via a flexible linker.

[0158] Aspect B17. The adsorbent according to any one of aspects B14-B15, wherein the proteolytic enzyme is attached to the solid support via a cleavable linker.

[0159] Aspect B18. The adsorbent according to any one of aspects B14-B15, wherein the proteolytic enzyme is attached to the solid support via an enzymatically cleavable linker.

[0160] Aspect B19. The adsorbent according to any one of aspects B14-B15, wherein the proteolytic enzyme is attached to the adsorbent via a chemically cleavable linker.

[0161] Aspect B20. An adsorbent according to aspect B19, wherein the chemically cleavable linker is selected from a chemical linker that cleaves when exposed to nucleophilic and / or basic conditions, a chemical linker that cleaves when exposed to electrophilic and / or acidic conditions, a chemical linker that cleaves when exposed to reducing conditions, a chemical linker that can be cleaved when exposed to oxidizing conditions, a hydroxylamine cleavable linker, a chemical linker containing a bis-succinimide ester, a chemical linker containing a bisacyl azide, and a chemical linker containing a bismaleimide.

[0162] Aspect B21. The adsorbent according to any one of aspects B14 to B20, wherein the proteolytic enzyme is chemically activated as a function of pH.

[0163] Aspect B22. The adsorbent according to any one of aspects B14 to B20, wherein the proteolytic enzyme is thermally activated by heating or cooling.

[0164] Aspect B23. The adsorbent according to any one of aspects B1 to B22, wherein the adsorbent is arranged in a multi-well strip, a multi-well plate, a column, a disposable cartridge or an ELISA plate.

[0165] Aspect B24. A kit comprising an adsorbent according to any one of Aspects B1-B23, and one or more kit components selected from the following: (a) a wash solution comprising a surfactant and a salt, (b) a pretreatment solution, (c) one or more elution solutions, (d) one or more proteolytic enzyme solutions, (e) a collection plate, (f) a cap pad, (g) calibration and reference standards, and (h) instructions for use.

[0166] Example

[0167] Example 1. Immobilization of aptamers using reductive amination

[0168] Example 1A. Particle Activation with Aldehyde Silanes

[0169] Inorganic-organic 5 μm BEH300 particles were prepared according to the procedure reported by Wyndham et al. in WO 2008 / 103423. 1.0 g of 5 μm BEH300 (Waters Corporation, Milford MA, USA) with a specific surface area (SSA) of 80 m 2 / g, total pore volume (TPV) 0.8cc / g, average pore diameter (APD) ) was added to a 50 mL round bottom flask equipped with a magnetic stir bar to prepare a slurry, which was mixed with approximately 5 mL of 50 mM ammonium bicarbonate (Sigma Aldrich, St. Louis, MO, USA), pH 9.0 buffer. To this suspension was added 220 μL of triethoxysilylbutyraldehyde (Gelest, Morrisville, PA, USA) pre-dissolved in 5 mL of ethanol, which was added to the flask and the mixture was stirred (300 rpm) at room temperature for 2 hours. The mixture was then transferred to a 50 mL centrifuge tube. The particles were washed three times (10 mg / mL buffer) via centrifugation (4500 rpm) to remove unreacted silane, and washed twice with ethanol and stored at 4°C before use. This method is suitable for preparing aldehyde binding coverages between 1.4 mmol / m 2 - to 3 mmol / m 2 The coverage is easily adjusted by the amount of silane added in the reaction. The carbon content can be determined by combustion analysis and the particle morphology can be determined by SEM.

[0170] Example 1B. Immobilization of Aptamers on Aldehyde-Modified Particles

[0171] Immobilization of the aptamer on aldehyde activated particles was performed using reductive amination.A suspension was prepared by adding 0.6 g of the material from Example 1A in a 50 mL flask equipped with a magnetic bar and slurried with 5 mL of 50 mM ammonium bicarbonate pH 9 buffer. To this suspension was added 16 mg of aptamer previously dissolved in 13 mL, 50 mM ammonium bicarbonate pH 9 buffer, followed by 2 mL of coupling solution (containing 1 M NaCNBH3 (Sigma Aldrich) in 50 mM ammonium bicarbonate pH 9 buffer). The mixture was allowed to stand at room temperature for 2 hours with constant stirring at 300 rpm. The reaction was quenched by adding 2 mL of 1 M ethanolamine (Sigma Aldrich) in buffer and stirring for another 30 minutes at room temperature. The particles were washed four times with 50 mM ammonium bicarbonate pH 9 buffer with shaking for 10 minutes to remove excess aptamer and ethanolamine (Acros Organics, Morris Plains, New Jersey, USA). The particles were washed once with pH 4 trifluoroacetic acid (Sigma Aldrich) and stored in this solvent at 4°C before use.

[0172] Example 1C. Immobilization of a mixture of aptamer and trypsin

[0173] The fixation of aptamer and trypsin is carried out using the reductive amination of aldehyde activated particles.0.6g of the aldehyde activated BEH particles derived from Example 1A are added to a 50mL flask equipped with a magnetic bar, and 5mL of 50mM ammonium bicarbonate pH 9 buffer is used to slurry it.A mixture of 8mg aptamer and 8mg trypsin mixed with benzamidine dissolved in 13mL of 50mM ammonium bicarbonate pH 9 buffer in advance (using acid stable trypsin) is added to the particle solution, and then 2mL coupling solution (comprising 1M NaCNBH3 50mM ammonium bicarbonate pH 9 buffer) is added.The reaction mixture is stirred at room temperature for 2 hours.By adding 2mL 1M ethanolamine (Acros Organics company (Acros Organics)) in the buffer and stirring at room temperature for 30 minutes to quench the reaction.With 50mM ammonium bicarbonate pH 9 buffer by centrifugal washing particles four times, shake 10 minutes to remove excessive aptamer, trypsin and ethanolamine.

[0174] Example 1D. Sequential immobilization of aptamer and trypsin

[0175] Immobilization of the aptamer on the aldehyde activated particles was performed using reductive amination as described in Example 1B, wherein less aptamer allowed trypsin to be immobilized on the same solid support. A suspension was prepared by adding 0.6 g of the material obtained from Example 1A in a 50 mL flask equipped with a magnetic bar, and the suspension was slurried with 5 mL 50 mM ammonium bicarbonate pH 9 buffer. To the suspension was added 8 mg of the aptamer pre-dissolved in 13 mL, 50 mM ammonium bicarbonate pH 9 buffer, followed by 2 mL of coupling solution (1 M NaCNBH3 (Sigma Aldrich) in 50 mM ammonium bicarbonate pH 9 buffer). The mixture was allowed to stand at room temperature for 2 hours while continuously stirring at 300 rpm. The particles were washed four times with 50 mM ammonium bicarbonate pH 9 buffer and shaken for 10 minutes to remove excess aptamer. The particles were redispersed in 5 ml 50 mM ammonium bicarbonate pH 9 buffer and transferred to a 50 mL flask equipped with a magnetic bar for trypsin immobilization. 40mg trypsin and appropriate amount of benzamidine (3g benzamidine corresponding to 1mg trypsin) dissolved in 13mL of 50mM ammonium bicarbonate pH 9 buffer in advance are added to the suspension, and then 2mL coupling solution (comprising 1MNaCNBH3 50mM ammonium bicarbonate pH 9 buffer) is added. The reaction is stirred at room temperature for 2 hours. The reaction is quenched by adding 2mL 1M ethanolamine (Acros Organics company) in the buffer and stirring at room temperature for 30 minutes. The particles are transferred to a 50mL centrifuge tube. The particles are washed four times by centrifugation with 50mM ammonium bicarbonate pH 9 buffer, and shaken for 10 minutes to remove excess trypsin and ethanolamine. Before use, the particles are stored as slurries in water at pH 4 at 4°C.

[0176] Example 2. Immobilization using a streptavidin / biotin activated carrier

[0177] Example 2A. Activation of Aldehyde Support with Streptavidin

[0178] Streptavidin was conjugated to the aldehyde particles of Example 1A by reductive amination using 1.0 g of 3.5 μm BEH600 (Waters Corporation, Milford, MA) with a specific surface area (SSA) of 40 m 2 / g, total pore volume (TPV) 0.6cc / g, average pore diameter (APD) ) to replace BEH300. Prepare suspension by adding 0.6g aldehyde activated particles to a 100mL flask equipped with a magnetic bar, and slurry the suspension with 50mM ammonium bicarbonate pH 9 buffer. In a separate flask, prepare a solution of streptavidin by dissolving 8mg streptavidin in 50mM ammonium bicarbonate (pH 9) at a concentration of 10mg / mL. Add streptavidin solution to the particle suspension, then add 2mL coupling solution (comprising 1M NaCNBH3 50mM ammonium bicarbonate pH 9 buffer), and mix the sample at room temperature for 2 hours. Quench the reaction by adding 2mL 1M ethanolamine (Acros Organics) in the buffer and stirring at room temperature for 30 minutes. Wash streptavidin activated particles with buffer 4 times and centrifuge to remove unreacted streptavidin.

[0179] Any biotinylated linker or protein can be conjugated to these streptavidin activated particles using a streptavidin-biotin protocol, which involves mixing the activated particles with a biotinylation agent in a buffer, such as 50 mM ammonium bicarbonate, pH 9, at room temperature for one hour. The particles are then washed several times with the buffer. Biotinylated linkers may include those with cleavable linkers or biotinylated aptamers.

[0180] Example 2B. Immobilization of mixed enzyme and streptavidin on an activated support

[0181] Use reductive amination to carry out the fixation of acid-stable trypsin on aldehyde activated particles. Prepare suspension by adding 0.6g of the material obtained from Example 1A in a 50mL flask equipped with a magnetic bar, and slurry the suspension with 10mL 50mM ammonium bicarbonate pH 9 buffer. Add 8mg trypsin and an appropriate amount of benzamidine (3g benzamidine corresponds to 1mg trypsin) dissolved in 13mL of 50mM ammonium bicarbonate pH 9 buffer in advance to the suspension, then add 2mL coupling solution (comprising 1M NaCNBH3 50mM ammonium bicarbonate pH 9 buffer). The reaction mixture was stirred at room temperature for 2 hours. The particles were transferred to a 50mL centrifuge tube. Wash the particles four times by centrifugation with 50mM ammonium bicarbonate pH 9 buffer, shake for 10 minutes to remove excessive trypsin. Obtain sample to analyze the amount of immobilized trypsin. The remainder of the sample is redispersed in a suitable buffer, and streptavidin is fixed using the scheme described in Example 2A. The resulting material can be used to bind any biotinylation reagent, including biotinylated aptamers following the protocol described in Example 2A.

[0182] Example 2C. Immobilization of aptamers with a cleavable linker using the streptavidin-biotin system

[0183] Immobilization of the aptamer with a cleavable linker using a streptavidin-biotin complex was performed as follows. Following the protocol described in Example 2A for reacting the streptavidin activated particles with biotin, the streptavidin activated particles from Example 2A were reacted with commercially available NHS-SS-biotin protein (Thermoscientific). A suspension was prepared in a 100 mL flask with 0.6 g of streptavidin-biotin-SS-NHS ester activated particles in 50 mL of 100 mM sodium phosphate buffer (pH 9). In a separate flask, an aptamer solution was prepared by dissolving 8 mg of the aptamer in 10 mL of 100 mM sodium phosphate buffer (pH 9). The aptamer solution was added to the particle suspension and allowed to react for 1 h. The NHS ester end group of biotin reacts with any primary amine on the aptamer or affibody or any protein. The particles were washed four times with buffer, with 10 minutes between washes, and then centrifuged to remove unreacted aptamers. The particles were then rinsed with TFA (pH 4) water and refrigerated.

[0184] Example 2D. Aptamers and cleavable enzymes

[0185] The fixation of the mixture of aptamer and streptavidin on aldehyde-modified particles will now be described. The fixation of aptamer and streptavidin is carried out using the reductive amination of aldehyde-activated particles. The aldehyde-activated BEH particles of Example 1A of 0.6g are added to a 100mL flask equipped with a magnetic bar, and 50mL of 50mM ammonium bicarbonate pH 9 buffers are used to slurry them. A mixture of 8mg aptamers and 8mg streptavidin dissolved in 13mL of 50mM ammonium bicarbonate pH 9 buffers in advance is added to the particle solution, and then 2mL coupling solution (comprising 1MNaCNBH3 50mM ammonium bicarbonate pH 9 buffers) is added. The reaction is stirred at room temperature for 2 hours, and the reaction is quenched by adding 2mL 1M ethanolamine in the buffer and then stirring at room temperature for 30 minutes. The particles are washed four times with 50mM ammonium bicarbonate pH 9 buffers, and shaken for 10 minutes to remove excessive aptamers, streptavidin and ethanolamine. Using the process described in Example 2C, sulfo NHS-SS biotinylation reagent is added to these particles and attached to streptavidin. The cleavable NHS ester on biotin can be used to bind protease (such as trypsin) to the carrier. Use 0.6g of particles with mixed aptamers and sulfo NHS-SS-biotinylation streptavidin and 100mL 50mM sodium phosphate buffer (pH 9) to prepare suspension. Add 8mg trypsin and appropriate amount of benzamidine dissolved in 10mL 100mM sodium phosphate buffer (pH 9) in advance to the particle suspension, and allow it to react at room temperature for 1 hour. Then the particles are transferred to a 50mL centrifuge tube, washed 4 times with buffer and centrifuged to remove unreacted enzyme. Then rinse the particles with TFA (pH 4) water and refrigerate.

[0186] Example 3A. Immobilization of Aptamers on Amine-Modified Particles

[0187] Particle activation with aminosilane will now be described. A suspension of 10.0 g of BEH particles (Waters, Massachusetts, Milford) was prepared by adding the particles to a 250 mL round bottom flask and dispersing the particles in toluene (150 mL, Fisher Scientific, Hampton, NH), and the mixture was dried at 110 ° C for 2 hours under a nitrogen atmosphere with a dean stark water trap. The mixture was then cooled to 40 ° C, followed by the addition of 1 mL of pure aminopropyltrimethoxysilane (Gelest). The reaction mixture was mixed for 10 minutes, then heated at 70 ° C for 2 hours, after which the reaction was cooled down. The aminated particles were washed on 0.5 μm Tyvek and washed twice with 50% ethanol in water and twice with ethanol (Fisher Scientific). The amine-modified particles were dried at 80 ° C for 16 hours under vacuum. The particles were analyzed for CHN for coverage calculation and SEM for particle morphology.

[0188] Example 3B. Attachment of aptamers via cleavable linkers

[0189] Cleavable linkers such as bis[2-(succinimidyloxycarbonyloxy)ethyl]sulfone (BSOCOES) are NHS ester biofunctionalized linkers that can be used to bind aptamers to amine activated particles. A stock solution was prepared by dissolving BSOCOES in DMF for activating amine particles. A suspension was prepared with 0.6 g of amine modified particles in 50 mL of 100 mM sodium phosphate buffer (pH 8). BSOCOES was added to the suspension pre-dissolved in DMF and allowed to react for 1 hour, then 8 mg of pre-dissolved aptamer (10 mg / ml) in 100 mM sodium phosphate buffer was added to the suspension and allowed to react for 1 hour at room temperature. The particles were then washed 4 times with buffer and centrifuged. After washing, the particles were rinsed with pH 4 TFA water and left in the refrigerator.

[0190] Example 4. Depletion of High Abundance Human Serum Albumin (HSA)

[0191] Plasma samples are treated with agents that disrupt protein-protein interactions, such as disodium EDTA, formic acid, TCA, acetic acid, TFA, phosphoric acid, or NH4OH.

[0192] The resulting processed plasma sample (neutralized or not) is added to an adsorbent bed comprising solid support particles (e.g., BEH particles) with a molar excess of a chemically linked HSA affinity reagent, such as an aptamer or affibody targeting HSA. Contact times and flow rates sufficient to achieve appropriate diffusion kinetics and binding of HSA to the affinity reagent are established.

[0193] The following steps may then be used in combination or alone: ​​(a) a low stringency wash with a low strength surfactant and moderate salt concentration to disrupt any low Kd protein-protein interactions between HSA and plasma proteins, and / or (b) a high stringency wash with a solution containing a strong surfactant and a chaotropic salt to disrupt any non-specific binding, thereby disrupting interactions with a Kd higher than the nM Kd interaction between the affinity reagent and the target HSA.

[0194] The eluate from the low stringency wash and / or high stringency wash is collected. The surfactant and salts are then removed and the eluate is concentrated by passing it through an appropriate solid phase extraction (SPE) device, such as the ELISA kit available from Waters Corporation, Milford, Massachusetts, USA. HLB, PRIME HLB, MCX or MAX sorbent device. The resulting clean, concentrated eluate can be used downstream for protein quantitation.

[0195] Example 5. Enrichment of low-abundance thyroglobulin from plasma

[0196] The plasma sample is added directly to an affinity sorbent bed containing solid support particles with a molar excess of a chemically linked thyroglobulin affinity reagent, such as an aptamer or affibody targeting thyroglobulin. Multiple affinity reagents can be attached to the sorbent to enrich multiple targets simultaneously.

[0197] High stringency washes with solutions containing strong surfactants and chaotropic salts are performed to destroy any nonspecific binding, thereby destroying any affinity bonds greater than 10 between the affinity reagent and the target thyroglobulin. -8 The Kd of the MKd interaction is the Kd of the interaction. Discard the wash buffer.

[0198] The bound thyroglobulin is then digested with a proteolytic enzyme (such as trypsin or Lys-C) which can be chemically attached to the adsorbent or added to the adsorbent bed as part of a digestion reagent mixture. In the case where the proteolytic enzyme is chemically attached, a linker of appropriate length can be used to enhance the ability of the enzyme to reach and digest the bound protein. The proteolytic enzyme can be activated, for example, by increasing the temperature or by establishing a specific pH.

[0199] The digested protein mixture is then eluted with a suitable elution solution, such as a buffer, which may have neutralizing properties, and the eluate can then be used for downstream protein quantification.

Claims

1. An adsorbent for separating at least one target protein from a liquid sample, the adsorbent comprising a solid support, the solid support comprising: Adsorbent particles; an aptamer having affinity for at least one target protein attached to the adsorbent particle, wherein the at least one target protein is selected from a low abundance protein and a high abundance protein; and An enzyme attached to the adsorbent particle is adapted to digest the at least one target protein upon activation of the attached enzyme.

2. The adsorbent according to claim 1, wherein the attached enzyme is attached to the adsorbent particles of the solid support via a flexible linker.

3. The adsorbent according to claim 1, wherein the attached enzyme is attached to the adsorbent particles of the solid support via a cleavable linker. The adsorbent according to claim 3 , wherein the cleavable linker is an enzymatically cleavable linker. The adsorbent according to claim 3 , wherein the cleavable linker is a chemically cleavable linker.

6. The adsorbent according to any one of claims 1 to 5, wherein the low abundance protein is selected from the group consisting of ceruloplasmin, complement factors C4, C9, C8 and C5, IgD, C1 inhibitor, RBP, iC3b, thyroxine binding globulin, complement proteins, thrombus precursor protein, C-reactive protein, Bb fragment, ferritin, randes, SC5b-9 complex, myoglobin, thyroglobulin, TPA (tissue plasminogen activator), neuron-specific enolase, C-peptide, alpha-fetoprotein, TNF-binding protein, PSA (prostate specific antigen), prostatic acid phosphatase, CEA (carcinoembryonic antigen), myelin basic protein, troponin I, interleukin, MIP-1α, tissue factor, GCSF (granulocyte colony stimulating factor), interferon, and combinations thereof.

7. The adsorbent according to any one of claims 1 to 6, wherein the high-abundance protein is selected from albumin, immunoglobulin G (IgG), immunoglobulin A (IgA), immunoglobulin M (IgM), immunoglobulin D (IgD), immunoglobulin D (IgE), alpha-1-antitrypsin, transferrin, haptoglobin, fibrinogen, haptoglobulin, alpha-2-macroglobulin, complement C3, apolipoprotein AI, apolipoprotein A-II, apolipoprotein B, alpha-1-acid glycoprotein, ceruloplasmin, complement C4, complement C1q, prealbumin, plasminogen, transthyretin, and combinations thereof.

8. The adsorbent according to any one of claims 1 to 7, wherein the enzyme is selected from trypsin, chymotrypsin, Glu-C, Lys-C, Arg-C, Asp-N, BNPS or NCS / urea, clostripain, CNBr, CNBr(methyl-Cys), CNBr(with acid), formic acid, hydroxylamine, iodosobenzoic acid, Lys-N, neutrophil elastase, NBS, NTCB, pancreatic elastase, pepsin A, prolyl endopeptidase, proteinase K, staphylococcal peptidase, thermolysin, and combinations thereof.

9. The adsorbent according to any one of claims 1 to 8, wherein the enzyme is trypsin.

10. The adsorbent according to any one of claims 1 to 9, wherein the adsorbent is disposed in a multiwell strip, a multiwell plate, a column, a disposable cartridge or an ELISA plate.

11. The adsorbent according to any one of claims 1 to 10, wherein the adsorbent comprises an ethylene bridged hybrid material (BEH).

12. An adsorbent for separating at least one target protein from a liquid sample, the adsorbent comprising a solid support, the solid support comprising: Adsorbent particles; an aptamer having affinity for at least one target protein attached to the adsorbent particle, the at least one target protein being selected from a low abundance protein and a high abundance protein; and An attached enzyme adapted to enzymatically cleave the aptamer from the adsorbent particle of the solid support, thereby allowing the complex of the attached aptamer and the target protein to be released from the solid support for further processing.

13. The adsorbent according to claim 12, wherein the attached enzyme is attached to the adsorbent particles of the solid support via a flexible linker.

14. The adsorbent of claim 12, wherein the attached enzyme is attached to the solid support via a cleavable linker. The adsorbent according to claim 14 , wherein the cleavable linker is an enzymatically cleavable linker. The adsorbent according to claim 14 , wherein the cleavable linker is a chemically cleavable linker.

17. The adsorbent according to any one of claims 12 to 16, wherein the low abundance protein is selected from the group consisting of ceruloplasmin, complement factors C4, C9, C8 and C5, IgD, C1 inhibitor, RBP, iC3b, thyroxine binding globulin, complement proteins, thrombus precursor protein, C-reactive protein, Bb fragment, ferritin, randes, SC5b-9 complex, myoglobin, thyroglobulin, TPA (tissue plasminogen activator), neuron-specific enolase, C-peptide, alpha-fetoprotein, TNF-binding protein, PSA (prostate specific antigen), prostatic acid phosphatase, CEA (carcinoembryonic antigen), myelin basic protein, troponin I, interleukin, MIP-1α, tissue factor, GCSF (granulocyte colony stimulating factor), interferon, and combinations thereof.

18. The adsorbent according to any one of claims 12 to 17, wherein the high abundance protein is selected from the group consisting of albumin, immunoglobulin G (IgG), immunoglobulin A (IgA), immunoglobulin M (IgM), immunoglobulin D (IgD), immunoglobulin D (IgE), alpha-1-antitrypsin, transferrin, haptoglobin, fibrinogen, haptoglobulin, alpha-2-macroglobulin, complement C3, apolipoprotein AI, apolipoprotein A-II, apolipoprotein B, alpha-1-acid glycoprotein, ceruloplasmin, complement C4, complement C1q, prealbumin, plasminogen, transthyretin, and combinations thereof.

19. The adsorbent according to any one of claims 12 to 18, wherein the enzyme is selected from trypsin, chymotrypsin, Glu-C, Lys-C, Arg-C, Asp-N, BNPS or NCS / urea, clostripain, CNBr, CNBr(methyl-Cys), CNBr(with acid), formic acid, hydroxylamine, iodosobenzoic acid, Lys-N, neutrophil elastase, NBS, NTCB, pancreatic elastase, pepsin A, prolyl endopeptidase, proteinase K, staphylococcal peptidase, thermolysin, and combinations thereof.

20. The adsorbent according to any one of claims 12 to 19, wherein the enzyme is trypsin.

21. A kit comprising the adsorbent according to any one of claims 1 to 20, and one or more kit components selected from the group consisting of: (a) a wash solution comprising a surfactant and a salt, (b) a pretreatment solution, (c) one or more elution solutions, (d) one or more proteolytic enzyme solutions, (e) a collection plate, (f) a cap pad, (g) calibration and reference standards, and (h) instructions for use.

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