METHOD FOR REDUCING INTERFERENCE IN THE INTERLEUKIN-33 ASSAY AND METHOD FOR QUANTIFYING INTERLEUKIN-33 IN A SERUM SAMPLE

Description

/ 28 METHOD FOR REDUCING INTERFERENCE IN THE INTERLEUKIN-33 ASSAY AND METHOD FOR QUANTIFYING INTERLEUKIN-33 IN A SERUM SAMPLE Separated from BR112020020686-5, filed on 10 / 04 / 2019. CROSS-REFERENCE TO RELATED REQUESTS

[001] This application claims the benefit and priority of the Application for U.S. Provisional Patent No. 62 / 655,887 filed April 11, 2018, which is incorporated by reference in its entirety. TECHNICAL FIELD OF THE INVENTION

[002] Aspects of the invention are generally related to immunoassays for the detection and quantification of cytokines, including, but not limited to, interleukin 33 (IL-33). FUNDAMENTALS OF THE INVENTION

[003] Interleukin 33 (IL-33) is a cytokine that is upregulated in response to signals of damage or insult, such as cigarette smoke. It is released by tissue barriers after necrosis and propagates an immune response to the presence of damaged tissue. This “alarm” signal amplifies inflammatory responses to innate and adaptive immunity. Published data indicate that commercial kits for detecting soluble IL-33 generate unreliable results, likely due to interference from endogenous binding partners, such as the soluble ST2 receptor (Nygaard, U., et al., SpringerPlus, 5:33 (2016); Ketelaar, ME, Clin Exp Allergy, 46(6):884-7 (2016); Rivière, E. et al., Ann Rheum Dis, 75(3):633-5 (2016)).

[004] The performance of ligand binding assays in complex matrices, such as serum, can be affected by specific endogenous components that interfere with the assay (Zhong, ZD, et al., AAPS J, 19: 1564 (2017)). Interference in an immunoassay can lead to incorrect interpretation of a patient's results by the laboratory and to the wrong course of treatment administered by the physician (Tate, J., et al., Clin Biochem Rev. Petition 870260029618, dated 03 / 30 / 2026, page 14 / 57 / 28 2004, 25(2): 105-120).

[005] Others have attempted to develop improved immunoassays. Doucet, J. et al. reported an improved IL-13 assay that included an acid dissociation step (Doucet, J. et al., Disease Markers, 35(5):465-474 (2013)). The immunoassay described by Doucet is specific for IL-13 and did not detect IL-33. Furthermore, Doucet's immunoassay did not require sample neutralization prior to detection and did not use a blocking agent to prevent cytokine complex reformation during the assay.

[006] Therefore, there is a need for new assays and methods for detecting IL-33. SUMMARY OF THE INVENTION

[007] Methods and compositions for detecting and quantifying cytokines are provided. The disclosed assays reduced assay interference compared to commercially available assays and / or a control assay. Interference may be cytokine-dependent, cytokine-independent, or both. One embodiment provides an IL-33 immunoassay that reduces assay interference caused by endogenous soluble IL-33 binding molecules present in the sample. Exemplary soluble IL-33 binding molecules include, but are not limited to, anti-IL-33 antibodies, soluble ST2 receptor, and serum components. In some embodiments, a blocking agent is added to the sample to reduce, inhibit, or block IL-33 complexes in the sample from reforming after acid dissociation of IL-33 complexes in the sample. In one embodiment, the blocking agent and the detection reagent do not compete for IL-33 binding.

[008] Another embodiment provides methods for determining the concentration of total IL-33 in human serum samples using an electrochemiluminescence immunoassay. The assay includes acid pretreatment of serum samples to dissociate soluble ligand:drug complexes present in the samples and improve the detection of IL-33 in the presence Petition 870260029618, dated 03 / 30 / 2026, page 15 / 57 3 / 28 of the drug, thus providing a quantitative measurement of total IL-33 levels. In this embodiment, the procedure employs a streptavidin-coated plate, with a biotinylated anti-human IL-33 antibody as the capture reagent, and uses recombinant IL-33 as a standard. Standards, controls, and samples are diluted in acetic acid and neutralized using a Tris base solution containing the detection reagent, for example, a ruthenium-labeled anti-human IL-33 antibody. An antibody that binds to the anti-IL-33 antibody and an anti-ST2 receptor antibody are also added to the Tris solution to minimize interference from the drug (i.e., antibody that binds antibodies to IL-33) or endogenous soluble ST2 receptor. Neutralized standards, controls, and samples are then added to the plate. The IL-33 captured on the plate is measured by a chemiluminescent signal generated by the ruthenium marker when voltage is applied to the plate by the plate reader.The resulting electrochemiluminescent signal (i.e., counts) is proportional to the amount of IL-33 present in the serum samples.

[009] In one embodiment, the method has a sensitivity of 12.5 pg / mL and an ST2 tolerance of > 50 ng / mL. BRIEF DESCRIPTION OF THE FIGURES

[0010] Figure 1 is a diagram of an exemplary test scheme according to an embodiment of the invention. 4-' - - ruthenium marker, - biotin, - streptavidin

[0011] Figure 2A is a line graph of percent recovery versus rhST2 concentration (ng / ml). () Acid-Treated C-AD; (·) Acid-Free C-AD; (□) Acid-Treated CAD; (O) Acid-Free CAD.

[0012] Figure 2B is a bar graph showing the detection of endogenous IL-33 in the serum of three individuals (Individual 1, Individual 2, and Individual 3) analyzed under the indicated conditions: Acid-Free CAD, Acid-Free CAD, Acid CAD, and Acid CAD.

[0013] Figure 3 is a bar graph showing specificity. Petition 870260029618, dated 03 / 30 / 2026, page 16 / 57 / 28 endogenous IL-33 in the serum of Individual 1 and Individual 3 using a modified assay, addition of rST2 and addition of an anti-IL-33 monoclonal antibody.

[0014] Figure 4 is a graph of IL-33 concentration (pg / mL) for Individual 1 and Individual 3 after multiple measurements, showing that the assay is accurate.

[0015] Figure 5 is a line graph showing the tolerance of the method for detecting IL-33 in the presence of an anti-IL-33 mAb analyzed under the indicated conditions: C-AD Acid Treated and CAD- Acid Treated.

[0016] Figure 6 is a graph of endogenous IL-33 (pg / mL) in the serum of normal subjects and subjects with asthma, COPD, and atopic dermatitis. 25 subjects, each from 4 different populations, were acquired from a commercial vendor and were screened for endogenous IL-33 using the C-AD assay with acid treatment.

[0017] Figure 7A is a graph showing human IL-33 (pg / mL) from treated mice.

[0018] Figure 7B is a graph showing human IL-33 (pg / mL) from control mice. DETAILED DESCRIPTION OF THE INVENTION I. Definitions

[0019] It should be appreciated that this disclosure is not limited to the compositions and methods described in this document, as well as the experimental conditions described, since these may vary. It should also be understood that the terminology used in this document is intended to describe specific embodiments only, and is not intended to limit the scope of the present invention, which will be limited only by the appended claims.

[0020] Unless otherwise defined, all technical and scientific terms used in this document have the same meaning as commonly understood by one skilled in the art to which this disclosure pertains. Although any similar compositions, methods and materials Petition 870260029618, dated 03 / 30 / 2026, p. 17 / 57 / 28 or equivalents to those described in this document may be used in the practice or testing of the present invention. All publications mentioned herein are incorporated herein by reference in their entirety.

[0021] The term binding molecule, as used in this document, is intended to refer to molecules that specifically interact with and bind to a specific target. The target may comprise a biological or small (chemical) molecule. The target molecule may define an antigen or an antigenic fragment. Examples of a binding molecule include, but are not limited to, antibodies (including monoclonal antibodies, bispecific antibodies, as well as antibody fragments), fusion proteins, and other antigen-binding molecules known to those skilled in the art.

[0022] The term antibody, as used in this document, is an example of a binding molecule and refers to an immunoglobulin that typically comprises four polypeptide chains, two heavy chains (H) and two light chains (L) linked by disulfide bonds. Each heavy chain comprises a variable heavy chain region (HCVR or VH) and a constant heavy chain region. The constant heavy chain region comprises three domains, Ch1, Ch2, and Ch3. Each light chain contains a variable light chain region (LCVR or VL) and a constant light chain region. The constant light chain region comprises one domain (CL1). The Vh and Vl regions can be further subdivided into regions of hypervariability, designated complementarity-determining regions (CDRs), interspersed with regions that are more conserved, called framework regions (FRs).Each VH and VL is composed of three CDR and four FR, arranged from the amino terminal to the carboxyl terminal in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0023] The term antigen-binding portion of an antibody (or simply antibody portion or antibody fragment), such as Petition 870260029618, dated 03 / 30 / 2026, page 18 / 57 / 28 used in this document, refers to one or more fragments of an antibody that retain the ability to bind specifically to an antigen (e.g., hIL-33). It has been demonstrated that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed by the term antigen-binding portion of an antibody include (i) a Fab fragment, a monovalent fragment comprising the V1, Vh, Cl1, and Ch1 domains; (ii) an F(ab')2 fragment, a bivalent fragment comprising two F(ab)' fragments linked by a disulfide bridge in the hinge region; (iii) an Fc fragment comprising the Vh and Ch1 domains; (iv) an Fv fragment comprising the Vl and Vh domains of a single arm of an antibody, (v) a dAb fragment (Ward, ES, et al., Nature 241: 544-546 (1989)), comprising a VH domain; and (vi) a CDR.Furthermore, although the two Fv fragment domains, Vl and Vh, are encoded by separate genes, they can be joined, using recombinant methods, by a synthetic linker that allows them to be made as a single contiguous chain in which the Vl and Vh regions pair up to form monovalent molecules (known as single-chain Fv (scFv); see, for example, Bird, RE, et al., Science 242: 423-426 (1988); and Huston, JS, et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988)). These single-chain antibodies are also intended to be encompassed by the term antigen-binding portion of an antibody. Other forms of single-chain antibodies, such as diabodies, are also included (see, for example, Holliger, P., et al., Proc. Natl. Acad Sci. USA 90:6444-6448 (1993)).

[0024] A CDR or complementarity-determining region is a region of hypervariability interspersed between regions that are more conserved, called framework regions (FRs). FRs can be identical to human germline sequences or can be modified naturally or artificially. Petition 870260029618, dated 03 / 30 / 2026, page 19 / 57 / 28

[0025] The term epitope is an antigenic determinant that interacts with a specific antigen-binding site in the variable region of an antibody molecule known as a paratope. A single antigen may have more than one epitope. Epitopes can be conformational or linear. A conformational epitope is produced by spatially juxtaposed amino acids from different segments of the linear polypeptide chain. A linear epitope is one produced by adjacent amino acid residues in a polypeptide chain. Under certain circumstances, an epitope may include saccharide moieties, phosphoryl groups, or sulfonyl groups in the antigen.

[0026] The term assay interference refers to endogenous components in the assay that block or inhibit analyte detection, such as IL-33 or an anti-IL-33 antibody. Substances that alter the measurable concentration of the analyte or alter antibody binding can result in immunoassay interference. Assay interference can be analyte-dependent or analyte-independent. Analyte-independent interferences refer to common interferences from hemolysis, lipemia, and the effects of anticoagulant and sample storage, and are independent of analyte concentration. Analyte-dependent interferences in immunoassays refer to the interaction between sample constituents with one or more reagent antibodies. These include compounds with chemical differences but structural similarities that cross-react with the antibody.Endogenous interfering substances that are naturally occurring, polyreactive antibodies or autoantibodies (heterophiles), human anti-animal antibodies or anti-drug antibodies (ADAs), along with other unsuspected binding proteins that are unique to the individual, can interfere with the reaction between the analyte and reagent antibodies in an immunoassay. Interference can be caused by soluble analyte binding targets, endogenous analyte ligands, including but not limited to soluble analyte receptors, soluble analyte ligands, and receptors eliminated from the analyte. Petition 870260029618, dated 03 / 30 / 2026, p. 20 / 57 / 28 analyte or serum factors, such as rheumatoid factor and biotin. Interference may be caused by antibodies to the analyte.

[0027] The term ST2 refers to the receptor for IL-33, which is also referred to as interleukin-1-like receptor-1 (IL-1RL1). ST2 can be membrane-bound or soluble.

[0028] The term immunoassay refers to a detection assay that incorporates a binding moiety that binds immunospecifically directly or indirectly to an analyte. Typically, the binding moiety is an antibody. The antibody may be labeled with a detectable marker.

[0029] The term IL-33 complex refers to the non-covalent association of IL-33 with a component of blood, plasma, or serum. Typically, the IL-33 complex contains IL-33 non-covalently bound to a protein, for example, a protein pharmaceutical, an anti-drug antibody, an IL-33 ligand, or a combination thereof. II. IL-33 Detection and Quantification Assays and Methods of Using Them

[0030] Assays and methods for the detection and quantification of IL-33 are provided. The disclosed assays and methods can be used to detect and quantify IL-33 in a sample. In one embodiment, the sample is obtained from a subject treated or being treated with an IL-33 antagonist or with IL-33.

[0031] The disclosed assays reduced assay interference compared to commercially available assays and / or a control assay. Interference may be IL-33-dependent, IL-33-independent, or both. In one embodiment, the method reduces or inhibits assay interference caused by endogenous soluble IL-33 binding molecules present in the sample. Exemplary soluble IL-33 binding molecules include, but are not limited to, anti-IL-33 antibodies, soluble ST2, and serum components. In some embodiments, a blocking agent is added to Petition 870260029618, dated 03 / 30 / 2026, page 21 / 57 / 28 sample to reduce, inhibit or block IL-33 complexes in the sample against reformation after acid denaturation of IL-33 complexes in the sample. In one embodiment, the blocking agent and the detection reagent do not compete for binding to IL-33.

[0032] In one embodiment, the method has a sensitivity of 12.5 pg / mL and an ST2 tolerance of > 50 ng / mL. A. Interleukin 33

[0033] In one embodiment, IL-33 or human interleukin-33 or human IL-33 refers to unprocessed IL-33 of full length, 270 amino acids (see, for example, UniProtKB accession number O95760 and US Patent Application Publication No. 20180155436, both of which are incorporated by reference in their entirety), or a biologically active fragment thereof, as well as any form of IL-33 that results from processing in the cell. The term also encompasses naturally occurring variants of IL-33, for example, processing variants (see, for example, Hong, et al., (2011), J. Biol. Chem. 286(22):20078-20086), or allelic variants, or any other isoform of IL-33, such as the oxidized or reduced forms of IL-33 described in WO2016 / 156440.The activity of IL-33 that can be neutralized, inhibited, blocked, revoked, attenuated, reduced, or interfered with by an antibody or its antigen-binding fragment, or by an IL-33 trap, includes, but is not limited to, inhibition of IL-33 receptor-mediated signaling or inhibition of IL-33-mediated inflammation. B. Methods for Detecting IL-33

[0034] One embodiment provides a method for detecting IL-33 in a sample by acidifying the sample to a pH sufficient to dissociate IL-33 complexes in the sample and neutralizing the acidified sample with a buffered base solution. Capture and detection reagents are also added to the sample to detect IL-33. In certain embodiments, an agent Petition 870260029618, dated 03 / 30 / 2026, p. 22 / 57 / 28: A blocker is added to the sample to inhibit, reduce, or block components in the IL-33 binding sample.

[0035] Another embodiment provides a method for reducing assay interference by acidifying a sample to a pH sufficient to dissociate IL-33 from IL-33 complexes in the sample and subsequently neutralizing the acidified sample with a buffered basic solution containing a detection reagent. Optionally, a blocking agent that inhibits IL-33 complexes from reforming in the sample may be added to the sample in a separate step or may be present in the buffered basic solution. The method involves adding a capture reagent to the sample and detecting the detection agent, wherein the amount of detection reagent detected correlates with the amount of IL-33 in the sample. The capture agent may be added as a separate step or may be present in the acidification step or in the buffered basic solution. Typically, the IL-33 complexes present in the sample contain IL-33 non-covalently associated with soluble ST2.

[0036] Another embodiment provides a method for quantifying interleukin-33 in a serum sample by acidifying the serum sample to a pH sufficient to dissociate IL-33 complexes in the sample and subsequently neutralizing the acidified sample with a buffered basic solution containing (a) a human anti-IL-33 antibody labeled with a detectable marker and, optionally, (b) a human anti-ST2 antibody. The method involves adding the sample to a streptavidin-coated solid support containing a biotinylated human anti-IL-33 antibody and detecting the detectable marker on the avidin-coated solid support, wherein the amount of detectable marker detected correlates with the amount of IL-33 in the sample.

[0037] Another embodiment provides a method for determining the total IL-33 concentration in human serum samples using a Petition 870260029618, dated 03 / 30 / 2026, p. 23 / 57 / 28 electrochemiluminescence immunoassay. The assay includes acid pretreatment of serum samples to dissociate soluble ligand:drug complexes present in the samples and improve the detection of IL-33 in the presence of drug, thus providing a quantitative measurement of total IL-33 levels. Ligand:drug complexes include, but are not limited to, IL-33 non-covalently bound to an anti-IL-33 antibody. An example of an anti-IL-33 antibody is disclosed in U.S. Patents Nos. 10,000,564 and 9,453,072, which are incorporated by reference in their entirety. In this embodiment, the procedure employs a streptavidin-coated plate, with a biotinylated human anti-IL-33 antibody as the capture reagent, and uses recombinant IL-33 as a standard.Standards, controls, and samples are diluted in acetic acid and neutralized using a Tris base solution containing the detection reagent, for example, a ruthenium-labeled anti-human IL-33 antibody. An antibody that binds to an anti-IL-33 antibody and an anti-ST2 receptor antibody are also added to the Tris solution to minimize interference from the drug or endogenous soluble ST2 receptor. Neutralized standards, controls, and samples are then added to the plate. IL-33 captured on the plate is measured by a chemiluminescent signal generated by the ruthenium label when voltage is applied to the plate by the plate reader. The resulting electrochemiluminescent signal (i.e., counts) is proportional to the amount of IL-33 present in the serum samples.

[0038] In one embodiment, the amount of marker detected in the sample is compared to a reference standard calibrated with known concentrations of IL-33 and corresponding amounts of marker detected for those concentrations. The amount of IL-33 in the sample can be determined by comparing the amount of marker detected in the sample with the reference standard and matching the amount of marker detected with the concentration shown in the reference standard for that amount. Petition 870260029618, dated 03 / 30 / 2026, page 24 / 57 / 28 of marker detected. 1. Biological Samples

[0039] The sample used in the disclosed methods is typically a biological sample, such as a biological fluid containing the analyte to be detected, for example IL-33 or a complex thereof. Biological fluids include, but are not limited to, blood, plasma, serum, and saliva. In a preferred embodiment, the biological sample is a human biological sample.

[0040] In certain embodiments, the sample is obtained from a subject with or suspected of having an inflammatory condition, disease, or disorder. Representative inflammatory conditions, diseases, and disorders include, but are not limited to, Crohn's disease, colitis, ulcerative colitis, atopic dermatitis, asthma, allergic rhinitis, allergic conjunctivitis, eosinophilic esophagitis, nasal polyps, or a combination thereof.

[0041] The analyte to be detected is typically a serum component taken from a subject, preferably a human subject. Representative analytes to be detected and / or quantified in the sample include, but are not limited to, cytokines, protein pharmaceuticals and metabolites or fragments thereof.

[0042] Representative cytokines include interleukins. Interleukins include IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-2,2,2, IL-2, IL-2, IL-2 IL- 25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34, IL-35 and IL-36. In one embodiment, the analyte is IL-33.

[0043] Representative protein pharmaceutical products include, further not limited to recombinant proteins, antibodies and fusion proteins. These antibodies may be polyreactive or autoantibodies (heterophilic), human anti-animal antibodies or anti-drug antibodies. In one embodiment, the protein pharmaceutical is an anti-IL-33 antibody. Petition 870260029618, dated 30 / 03 / 2026, p. 25 / 57 / 28 2. IL-33 ligands

[0044] IL-33 ligands are molecules that bind non-covalently to IL-33 and include, but are not limited to, endogenous components in the sample such as antibodies or soluble analyte binding targets, endogenous IL-33 ligands including, but not limited to, soluble IL-33 receptors, soluble IL-33 ligands and analyte-eliminated receptors. In one embodiment, the IL-33 ligand is the soluble ST2 receptor or soluble IL-1 receptor accessory protein (IL1RAcP).

[0045] ST2 (also known as IL1RL1, DER4, T1, and FIT-1) is a member of the Toll-like receptor / IL-1 superfamily. Members of this superfamily are defined by a common intracellular domain, the Toll-like receptor / Interleukin-1 (TIR) ​​domain. This ~160 amino acid domain is composed of a central five-stranded β-sheet surrounded by five α-helices located at the cytosolic end of the protein. The Toll-like receptor / IL-1 superfamily can be divided into three subfamilies based on their extracellular domains: the IL-1 receptor-like subfamily, the Toll receptor subfamily, and a family composed of their adaptor proteins. 3. Acid Denaturation of IL-33 Complexes

[0046] The sample in the disclosed methods can be acidified for 1 to 60 minutes. In one embodiment, the sample is acidified for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60 minutes.

[0047] Acids that can be used in the acidification step include, but are not limited to, acetic acid, hydrochloric acid, and sulfuric acid.

[0048] The pH sufficient to dissociate IL-33 complexes can be from 3.0 to 5.0. In one embodiment, the pH is 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, Petition 870260029618, dated 03 / 30 / 2026, p. 26 / 57 / 28 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9 or 5.0.

[0049] In one embodiment, a denaturing agent may be added to the sample to dissociate IL-33 complexes including, but not limited to, urea. 4. Blocking agent

[0050] The blocking agent used in the disclosed methods is an agent that reduces or prevents the reformation of IL-33 complexes in the sample after the IL-33 complexes have been dissociated in the acidification step. When the sample is taken from a subject, for example, a human subject, the sample normally contains IL-33 complexes to be detected or quantified. The blocking agent may be an antibody that binds immunospecifically to a component of an IL-33 complex other than IL-33. In one embodiment, the blocking agent is an anti-ST2 antibody.

[0051] In one embodiment, the blocking agent is an antibody that binds to an IL-33 drug, for example, an antibody that binds to an anti-IL-33 antibody. 5. Detection Reagent

[0052] The detection reagent used in the disclosed methods may be an IL-33 antagonist or inhibitor, for example, an anti-IL33 antibody, preferably a human anti-IL-33 antibody or an IL-33 trap. The antibody may be monoclonal, polyclonal, or humanized.

[0053] In some embodiments, the detection reagent is labeled with a detectable marker. Detectable markers are known in the art and include, but are not limited to, a rare transition metal particle, a fluorophore, a chromophore, a quantum dot, noble metal nanoparticles, a radioactive fraction, an enzyme, a biotin / avidin marker, and a chemiluminescent marker. In one embodiment, the detectable marker is ruthenium. a. Anti-IL-33 Antagonists Petition 870260029618, dated 03 / 30 / 2026, p. 27 / 57 / 28

[0054] In certain embodiments, the IL-33 antagonists or inhibitors that may be used in the disclosed assays and methods are anti-IL-33 antibodies or antibody antigen-binding fragments that bind specifically to human IL-33. In one embodiment, the anti-IL33 antibodies described herein for use in the disclosed methods and assays are disclosed in U.S. Patents Nos. 10,000,564 and 9,453,072, which are incorporated by reference in their entirety.

[0055] The anti-IL-33 antibodies used in the disclosed assays and methods specifically bind to IL-33. The term specifically binds, or similar, means that an antibody or its antigen-binding fragment forms a complex with an antigen that is relatively stable under physiological conditions. Methods for determining whether an antibody specifically binds to an antigen are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, and the like. For example, an antibody that “specifically binds” to IL-33 as used in this document includes antibodies that bind to IL-33 or a biologically active portion thereof with a Kd less than about 1.000 nM, less than 500 nM, less than about 300 nM, less than about 200 nM, less than about 100 nM, less than about 90 nM, less than 80 nM, less than about 70 nM, less than about 60 nM, less than about 50 nM, less than about 40 nM, less than 30 nM, less than 20 nM, less than about 10 nM, less than about 5 nM, less than about 4 nM, less than about 3 nM, less than about 2 nM, less than about 1 nM, or less than about 0.5 nM, as measured in a surface plasmon resonance assay. An isolated antibody that binds specifically to human IL-33 may, however, cross-react with other antigens, such as IL-33 molecules from other (non-human) species.

[0056] In some modalities, the IL-33 antagonist is a Petition 870260029618, dated 03 / 30 / 2026, page 28 / 57 / 28 anti-IL-33 antibody, or antigen-binding fragment, comprising a heavy chain variable region (HCVR), light chain variable region (LCVR) and / or complementarity-determining regions (CDRs) comprising any of the amino acid sequences of anti-IL-33 antibodies as set forth in U.S. Patents Nos. 10,000,564 and 9,453,072. In certain embodiments, the IL-33 antagonist is an anti-IL-33 antibody with the binding characteristics of the reference antibody described in U.S. Patents Nos. 10,000,564 and 9,453,072.

[0057] Other anti-IL-33 antibodies and their antigen-binding fragments that may be used in the methods described in this document are disclosed in EP1725261 and U.S. Patent No. 9,970,944, U.S. Patent No. 8,187,596, WO2011031600, WO2015099175 and U.S. Patent No. 9,758,578, WO2015106080 and U.S. Patent No. 10,059,764 (ANB020), US2016 / 0168242, WO2016 / 077381 and U.S. Patent No. 10,093,730, WO2016 / 077366 and US2018 / 0171405, or WO2016 / 156440 and US2018 / 0207265, each of which is incorporated herein by reference in its entirety. b. IL-33 Traps

[0058] In some embodiments, the IL33 antagonists or inhibitors that can be used in the disclosed methods and assays are receptor-based IL-33 traps. Exemplary IL-33 traps include at least one IL-33 binding domain containing an IL-33 binding portion of an IL-33 receptor protein, for example ST2. In certain embodiments, an IL-33 trap further includes an extracellular portion of an IL33 coreceptor, for example, IL-1 receptor accessory protein, or IL-1RAcP. The IL33 trap may also contain at least one multimerization component, which functions to connect the various components of the trap to each other.

[0059] In one embodiment, the IL-33 traps described in this document for use in the disclosed methods and tests are disclosed in U.S. Patent No. 9,637,535 and WO2014 / 152195, which are each incorporated herein. Petition 870260029618, dated 03 / 30 / 2026, p. 29 / 57 / 28 document by reference in its entirety.

[0060] Generally, an IL-33 trap includes a first IL-33 binding domain (D1) linked to a multimerization domain (M). In certain embodiments, IL-33 antagonists include a second IL-33 binding domain (D2) linked to D1 and / or M. According to certain embodiments, D1 comprises an IL-33 binding portion of an ST2 protein. An IL-33 binding portion of an ST2 protein may include or consist of all or part of the extracellular domain of an ST2 protein. In certain embodiments, an ST2 protein is a human ST2 protein. A human ST2 protein, as used in this document, refers to an ST2 protein as shown in amino acids 1-556 of accession number NP_057316.3, which is incorporated by reference in its entirety. According to certain embodiments, D2 includes an extracellular portion of an IL-1RAcP protein.

[0061] The individual components of IL-33 traps can be arranged relative to each other in various ways that result in functional antagonist molecules capable of binding to IL-33. For example, D1 and / or D2 can be linked to the N-terminus of M. In other embodiments, D1 and / or D2 are linked to the C-terminus of M. Still in other embodiments, D1 is linked to the N-terminus of D2, and D2 is linked to the N-terminus of M, resulting in an in-line fusion, from the N-terminus to the C-terminus, of an antagonist molecule represented by the formula D1-D2-M.

[0062] In certain embodiments, IL-33 antagonists have two multimerization domains, M1 and M2, where M1 and M2 are identical to each other. For example, M1 may be an Fc domain with a particular amino acid sequence and M2 is an Fc domain with the same amino acid sequence as M1. c. Other IL-33 Antagonists

[0063] Other agents that can act as IL-33 antagonists and Petition 870260029618, dated 03 / 30 / 2026, page 30 / 57 / 28 that may be used in the disclosed methods and assays include immunoadhesins, peptibodies, soluble ST2 or its derivatives; IL-33 receptor antibodies (e.g., anti-ST2 antibodies, e.g., AMG-282 (Amgen) or STLM15 (Janssen) or any of the anti-ST2 antibodies described in WO2012 / 113813 and U.S. Patent No. 9,309,319, WO2013 / 173761 and U.S. Patent No. 9,982,054, WO2013 / 165894 and U.S. Patent No. 9,951,137, U.S. Patent No. 8,444,987, or U.S. Patent No. 7,452,980), which are incorporated herein by reference in their entirety. Other IL33 antagonists for use in the disclosed methods and assays include ST2-Fc proteins, such as those described in WO2013 / 173761 and U.S. Patent No. 9,982,054, or WO2013 / 165894 and U.S. Patent No. 9,951,137, each of which is incorporated herein by reference in its entirety. 6. Capture Reagent

[0064] In one embodiment, the capture reagent used in the disclosed methods and assays is an IL-33 antagonist as described above, for example, an antibody, preferably an anti-IL-33 antibody, preferably a human anti-IL-33 antibody. An exemplary anti-IL-33 antibody is disclosed in U.S. Patent No. 10,000,564, which is incorporated by reference in its entirety. Other anti-IL-33 antibodies that may be used are described above. In one embodiment, the capture reagent is biotinylated.

[0065] In some embodiments, the capture reagent is fixed or bound to a solid support. The solid support may be a microplate, for example, a streptavidin-coated microplate.

[0066] In some embodiments, the capture reagent is the IL-33 antagonists described above. C. Complementary Diagnosis

[0067] In one embodiment, the disclosed methods and assays for detecting and quantifying IL-33 are used as a diagnostic method. Petition 870260029618, dated 03 / 30 / 2026, page 31 / 57 / 28 supplementary in conjunction with treatments designed to reduce circulating IL-33 levels in a subject being treated or to raise circulating IL-33 levels in a subject being treated. Treatments may include administration of an IL-33 antagonist or administration of IL-33. Exemplary IL-33 antagonists are those described above and include, but are not limited to, anti-IL-33 antibodies, anti-IL-33 fusion proteins, sST decoy receptors, or a combination thereof.

[0068] One embodiment provides a method for monitoring the treatment of an IL-33-related disease in a subject receiving treatments, including the administration of an IL-33 antagonist as described above or the administration of IL-33.Representative diseases, disorders, or pathologies treated with an IL-33 inhibitor or IL-33 include, but are not limited to, asthma, eosinophilic or non-eosinophilic asthma, atopic dermatitis, chronic obstructive pulmonary disease (COPD), inflammatory bowel disease, Crohn's disease, colitis, ulcerative colitis, multiple sclerosis, arthritis, allergic rhinitis, atopic dermatitis, eosinophilic esophagitis, psoriasis, nasal polyps, Alzheimer's disease, atherosclerosis, myocardial infarction, ischemic stroke, and fibrotic conditions, including, but not limited to, Dupuytren's disease, adhesive capsulitis, periarticular fibrosis, keloid or hypertrophic scars, endometriosis, abdominal adhesions, perineural fibrosis, Ledderhose disease, Peyronie's disease, peritendinous adhesions, and periarticular fibrosis.

[0069] The methods and assays described in this document may be used to evaluate, detect, or quantify circulating IL-33 levels in a subject before and after treatment with one or more pharmaceutical compositions containing an IL-33 antagonist as described above (for example, particularly with a drug related to a mechanism of action involving IL-33), with anti-inflammatory therapy, or by immunoabsorbance therapy, or where IL-33 is evaluated after such treatment and Petition 870260029618, dated 03 / 30 / 2026, page 32 / 57 / 28: The concentration or quantity of IL-33 is compared to a predetermined level or against the level measured in the subject before treatment. An unfavorable concentration or quantity of IL-33 observed after treatment confirms that the subject will not benefit from receiving additional or continued treatment, while a favorable concentration or quantity of IL-33 observed after treatment confirms that the subject will benefit from receiving additional or continued treatment.

[0070] In one embodiment, an unfavorable IL-33 concentration detected by the disclosed methods and assays in a sample from a subject receiving treatment incorporating an IL-33 inhibitor is an IL-33 level equal to or greater than the level measured in the subject before treatment. In another embodiment, a favorable IL-33 concentration detected by the disclosed methods and assays in a sample from a subject receiving treatment incorporating an IL-33 inhibitor is an IL-33 level that is lower than the IL-33 level measured in a sample from the subject before treatment.

[0071] In another embodiment, an unfavorable IL-33 concentration detected using the disclosed methods and assays in a sample from a subject receiving treatment incorporating IL-33 is a level that is lower than the IL-33 level measured in a sample from the subject before treatment. In another embodiment, a favorable IL-33 concentration detected using the disclosed methods and assays in a sample from a subject receiving treatment incorporating IL-33 is an IL-33 level that is lower than the IL-33 level measured in a sample from the subject before treatment.

[0072] Confirmation of circulating IL-33 levels aids in the management of clinical studies and in providing better patient care. III. Kits Petition 870260029618, dated 03 / 30 / 2026, page 33 / 57 / 28

[0073] A kit for testing a test sample for the presence, quantity, or concentration of IL-33 (or a fragment thereof) in a test sample is also provided. The kit includes at least one component for analyzing the test sample for IL-33 (or a fragment thereof) and instructions for analyzing the test sample for IL-33 (or a fragment thereof).

[0074] In one embodiment, the kit includes a detection reagent as described above, a capture reagent as described above, a solid support, other reagents, and written instructions for performing the assay. The detection reagent and the capture reagent may be the same or different IL-33 antagonists described above, such as a monoclonal antibody (or a fragment, a variant, or a fragment of a variant thereof), a fusion protein, an IL-33 trap, or an apatamer optionally immobilized on a solid phase.

[0075] The detection reagent is typically labeled with a detectable marker, such as a chemiluminescent marker. The detection reagent may incorporate a detectable marker, as described in this document, such as a fluorophore, a radioactive fraction, an enzyme, a biotin / avidin marker, a chromophore, a chemiluminescent marker or the like, or the kit may include reagents to perform the detectable labeling. Antibodies, calibrators and / or controls may be supplied in separate containers or pre-dispensed in an appropriate assay format, for example, in microtiter plates. In one embodiment, the IL-33 detection reagent is ruthenium-labeled.

[0076] In some embodiments, the kit contains a biotin-labeled capture reagent and a streptavidin-coated solid support, for example, a microtiter plate or electrochemiluminescence platform. In some embodiments, the capture reagent and the microtiter plate or electrochemiluminescence platform are Petition 870260029618, dated 03 / 30 / 2026, page 34 / 57 / 28 supplied in separate containers. In other embodiments, the kit contains a microtiter plate or electrochemiluminescence platform coated with the capture reagent. The kit also contains acid solutions and buffers as described above for sample treatment.

[0077] The kit may include a calibrator or control, for example, isolated or purified IL33. The kit may include at least one container (e.g., tube, microtiter plates or strips, or electrochemiluminescence platform) for performing the assay and / or a buffer, such as an assay buffer or a washing buffer, either of which may be supplied as a concentrated solution, a substrate solution for the detectable marker (e.g., an enzyme marker), or a stopping solution. Preferably, the kit includes all components, i.e., reagents, standards, buffers, diluents, etc., that are necessary to perform the assay. Instructions may be on paper or computer-readable.

[0078] Optionally, the kit includes quality control components (e.g., sensitivity panels, calibrators, and positive controls). The preparation of quality control reagents is well known in the art and is described in insert sheets for a variety of immunodiagnostic products. The members of the sensitivity panel are optionally used to establish the performance characteristics of the assay and are optionally useful indicators of the integrity of the immunoassay kit reagents and the standardization of the assays.

[0079] The kit may also optionally include other reagents needed to conduct a diagnostic assay or facilitate quality control assessments, such as buffers, salts, enzymes, enzyme cofactors, enzyme substrates, detection reagents, and the like. Other components, such as buffers and solutions for the isolation and / or treatment of a test sample (e.g., pretreatment reagents), may also be included. Petition 870260029618, dated 03 / 30 / 2026, page 35 / 57 / 28 may be included in the kit. The kit may additionally include one or more other controls. One or more of the kit components may be lyophilized, and in this case, the kit may also include reagents suitable for the reconstitution of the lyophilized components.

[0080] The various components of the kit are optionally supplied in suitable containers as needed, for example, a microtiter plate. The kit may also include containers for holding or storing a sample (e.g., a container or cartridge for a urine sample). When appropriate, the kit may optionally also contain reaction vessels, mixing vessels, and other components that facilitate the preparation of reagents or the test sample. The kit may also include one or more instruments to assist in obtaining a test sample, such as a syringe, pipette, forceps, measuring spoon, or similar. Examples Example 1: Essay Format

[0081] Figure 1 is a diagram of an exemplary assay for detecting IL-33. This method for quantifying IL-33 is a sandwich immunoassay on an electrochemiluminescence platform. The procedure employs a streptavidin-coated microplate and uses a biotinylated anti-IL-33 antibody as a capture reagent. The method includes acid pretreatment of serum samples to dissociate ligand:soluble binding partner complexes present in the samples and improve the detection of IL-33 in the presence of binding partners, e.g., anti-IL-33 therapeutic drug and / or soluble ST2, thus providing a quantitative measure of total IL-33 levels. The acidified samples were then neutralized in a basic solution containing a labeled anti-IL-33 detection antibody, which in one embodiment can compete with the soluble ST2 receptor and the drug for binding to IL-33.

[0082] Examples of acids that can be used include, but Petition 870260029618, dated 03 / 30 / 2026, page 36 / 57 / 28 are not limited to acetic acid, sulfuric acid, and hydrochloric acid. The sample can be neutralized using a buffered basic solution, for example, a Tris solution.

[0083] An example of an IL-33 antibody that can be used is described in U.S. Patent No. 10,000,564, which is incorporated by reference in its entirety. Other anti-IL-33 antibodies that can be used in the disclosed methods include, but are not limited to, those described in U.S. Patent No. 9,758,578 and U.S. Patent Application Publication 2018 / 0037644.

[0084] In one embodiment, standards, controls, and samples are diluted in acetic acid and neutralized using a Tris base solution containing the detection reagent, for example, a ruthenium-labeled anti-human IL-33 antibody. An anti-IL-33 antibody and an anti-ST2 antibody are also added to the Tris solution to minimize interference from the drug or endogenous binding partners. Neutralized standards, controls, and samples are then added to the plate. IL-33 captured on the plate is measured by a chemiluminescent signal generated by the ruthenium label when voltage is applied to the plate by the plate reader. The resulting electrochemiluminescent signal (i.e., counts) is proportional to the amount of IL-33 present in the serum samples. Example II: Minimizing Endogenous Binding Partner Interference Materials and methods

[0085] Tolerance to recombinant human ST2 was tested using 4 different assay conditions (described below), with the first step (capture) for all conditions being the addition of biotinylated anti-IL-33 mAb to a streptavidin plate.

[0086] For the stepwise progression assay (Capture-Analyte-Detection, CAD), the samples (containing the analyte, IL-33) were first diluted in an acidic solution and allowed to incubate. After the Petition 870260029618, dated 03 / 30 / 2026, page 37 / 57 / 28. After washing the plate, the acidified samples were subsequently diluted in a basic neutralization buffer before addition to the assay plate. After sample incubation, the assay plate is washed and a labeled detection mAb solution is added. After incubation of the detection reagent, the plate is washed, buffer is added, and the plate is read. This assay was also performed without an acidification step, where the samples were diluted in assay buffer before sample addition to the assay plate.

[0087] For the assay performed with the simultaneous addition of analyte and detector (C-AD), samples were first diluted in an acidic solution and allowed to incubate. After washing the plate, the acidified samples were then further diluted in a basic neutralization buffer containing the labeled detection mAb before addition to the assay plate. After sample incubation, the plate is washed, buffer is added, and the plate is read. This assay was also performed without an acidification step, where samples were first diluted in assay buffer before a subsequent dilution in assay buffer containing the labeled detection mAb, prior to adding the sample to the assay plate.

[0088] Recombinant IL-33 at 36 pg / mL was tested under 4 different assay conditions, as described above, in the presence of increasing concentrations of recombinant soluble ST2, as shown in Fig 2A. Results

[0089] Acid treatment is required to detect recombinant human IL-33 in the presence of recombinant ST2. Adding an acid treatment step to the CAD assay improves ST2 tolerance to ~12 ng / mL, and ST2 tolerance can be improved to >50 ng / mL using the acid-treated CAD assay format (Figure 2A). Acid treatment is also required to improve the detection of endogenous IL-33 in human serum. The detection of endogenous IL-33 is improved using the CAD assay format compared to the format Petition 870260029618, dated 03 / 30 / 2026, page 38 / 57 / 28 of CAD assay due to reduction in interference from endogenous binding partners assay (Figure 2B). Example III: Precision and Specificity: Endogenous IL-33 Materials and methods

[0090] The specificity of the assay for IL-33 was confirmed by analysis of 2 different individual serum samples in the C-AD assay with acid treatment as described in Example II, tested with and without the addition of excess recombinant human ST2 or 200 pg / mL of an anti-IL-33 antibody in the basic neutralization solution containing the detection reagent.

[0091] Two individuals were tested using the C-AD assay with acid treatment, as described in Example II. The data represent 4 separate replicate determinations performed on 5 separate days. Results

[0092] The assay is specific for endogenous IL-33, since the addition of binding partners, for example, recombinant ST2 or an anti-IL-33 mAb, to the neutralization solution inhibits the detection of IL-33. Figure 3 is a bar graph showing the specificity for endogenous IL-33.

[0093] The assay demonstrates acceptable precision with a percent coefficient of variation (%CV) of 7.1 and 12.3 for individuals 1 and 3, respectively. Figure 4 is a graph showing the assay precision in quantifying endogenous IL-33 in human serum. Example IV: Detection of IL-33 in the Presence of an Anti-IL-33 mAb (Tolerance to Anti-IL-33 mAb) Materials and methods

[0094] Figure 5 is a line graph showing the tolerance of the method for detecting IL-33 in the presence of an anti-IL-33 mAb. Recombinant IL-33 at 90 pg / mL was tested in the C-AD method with treatment with Petition 870260029618, dated 03 / 30 / 2026, p. 39 / 57 / 28 acid described in example II, in the presence of increasing concentrations of an anti-IL-33 mAb, as indicated in Fig 5. The assay was modified by adding excess antidrug mAb and anti-ST2 mAb to the neutralization solution. Results

[0095] Acid treatment is important for detecting human IL-33 in the presence of anti-IL-33 mAbs. Adding the detection reagent to the neutralization solution, along with the addition of antidrug mAbs and anti-ST2 mAbs, improves the tolerance of the anti-IL-33 mAb in the assay to >500 pg / mL. Example V: Endogenous Levels of IL-33 in Normal and Diseased Individuals Materials and Methods

[0096] 25 individuals, each from 4 different populations, were acquired from a commercial vendor and screened for endogenous IL-33 using the C-AD assay with acid treatment as described in Example II. Results

[0097] Figure 6 is a graph of endogenous IL-33 (pg / mL) in the serum of normal individuals and individuals with asthma, COPD, and atopic dermatitis. All individuals tested resulted in an IL-33 concentration below 31 pg / mL. Example VI: Humanized Mouse Data Materials and methods

[0098] Humanized IL-33 mice were treated with house mites (HDM) for a period of 15 weeks as a model of human respiratory disease. An example of a humanized IL-33 mouse is disclosed in U.S. Patent Publication No. 20170311580, which is incorporated by reference in its entirety. Samples were tested in a modified version of the C-AD assay with acid treatment, as described in Example IV. Petition 870260029618, dated 03 / 30 / 2026, page 40 / 57 / 28 Results

[0099] Figures 7A and 7B show human IL-33 (pg / mL) in treated and control mice. Controls treated with saline, HDM, and isotypes showed similar levels of human IL-33 at the end of treatment. Mice dosed with anti-IL-33 monoclonal antibody showed a marked increase in serum IL-33 levels.

[00100] A total human IL-33 assay was developed that has a sensitivity of 12.5 pg / mL in pure serum. Collectively, the data show that an acid treatment step is important for the removal of endogenous IL-33 binding partners. The addition of the detection antibody to the neutralization solution improves tolerance to recombinant / endogenous ST2. The assay demonstrated specificity for endogenous human IL-33. Mice treated with HDM dosed with an anti-IL-33 antibody showed an increase in human IL-33 due to the bound target taking over the drug half-life, while controls with HDM and Isotypes appeared to remain at baseline.

[00101] Although in the above descriptive report this invention has been described in relation to certain embodiments thereof, and many details have been defined for illustrative purposes, it will be evident to those skilled in the art that the invention is susceptible to additional embodiments and that some of the details described in this document may vary considerably without departing from the basic principles of the invention.

[00102] All references cited in this document are incorporated by reference in their entirety. The present invention can be embodied in other specific ways without departing from the spirit or essential attributes thereof and, consequently, reference should be made to the appended claims, instead of to the descriptive report above, to understand the scope of the invention. Petition 870260029618, dated 03 / 30 / 2026, p. 41 / 57

Claims

1 / 3 CLAIMS 1. A method for reducing interference in the interleukin-33 assay, characterized in that it comprises the steps of: acidifying a sample to a pH sufficient to dissociate IL-33 from IL-33 complexes in the sample; subsequently neutralizing the acidified sample with a buffered basic solution comprising a detection reagent and a blocking agent that inhibits the formation of the IL-33 complex from reformation after acid dissociation of IL-33 complexes in the sample; adding a capture reagent to the sample, wherein the capture reagent is a human anti-IL-33 antibody; and detecting the detection agent, wherein the amount of detection reagent detected correlates with the amount of IL-33 in the sample.

2. Method according to claim 1, characterized in that the sample comprises blood, serum or plasma.

3. Method according to claim 2, characterized in that the sample is from a subject to whom an IL-33 pharmaceutical product has been administered.

4. Method according to claim 3, characterized in that the IL-33 pharmaceutical product comprises an anti-IL-33 antibody.

5. Method according to claim 1, characterized in that the IL-33 complex comprises IL-33 non-covalently bound to a protein.

6. Method according to claim 5, characterized in that the protein is an endogenous serum protein.

7. Method according to claim 5, characterized in that the protein is ST2 or an IL-33 binding fragment thereof.

8. Method according to claim 5, characterized by the fact that the protein is an anti-IL-33 antibody or an IL-33 binding fragment thereof.

9. Method according to any one of claims 1 to 8, characterized in that the sample is acidified to a pH of 3 to 5.

10. Method according to any one of claims 1 to 9, characterized in that the detection reagent comprises an anti-IL-33 antibody conjugated to a detectable marker.

11. Method according to any one of claims 1 to 10, characterized in that the sample is acidified with acetic acid.

12. Method according to any one of claims 1 to 11, characterized in that the blocking agent is an anti-ST2 antibody.

13. Method according to any one of claims 1 to 12, characterized in that the capture reagent is added to the sample during the acidification step.

14. Method according to any one of claims 1 to 13, characterized in that the blocking agent is added to the sample after the neutralization step.

15. Method according to any one of claims 1 to 14, characterized in that IL-33 has a sensitivity of 12.5 pg / mL.

16. Method according to any one of claims 1 to 15, characterized in that the IL-33 method has an ST2 tolerance of > 50 ng / mL.

17. A method according to any one of claims 1 to 16, characterized in that the sample is obtained from a subject diagnosed with, or suspected of having, an inflammatory disease or disorder.

18. Method for quantifying interleukin-33 in a serum sample, characterized in that it comprises the steps of: Petition 870260029618, dated 03 / 30 / 2026, page 43 / 57 3 / 3 acidifying the serum sample to a pH sufficient to dissociate IL-33 complexes in the sample; neutralizing the acidified sample with a buffered basic solution comprising (a) a human anti-IL-33 antibody labeled with a detectable marker and, optionally, (b) a human anti-ST2 antibody; adding the sample to an avidin-coated solid support comprising a biotinylated human anti-IL-33 antibody; and detecting the detectable marker on the avidin-coated solid support, wherein the amount of detectable marker detected correlates with the amount of IL-33 in the sample.

19. Method according to claim 18, characterized in that the solid support is a streptavidin-coated electrochemiluminescence platform.

20. Method according to claim 18 or 19, characterized in that IL-33 has a sensitivity of 12.5 pg / mL and an ST2 tolerance of > 50 ng / mL. Petition 870260029618, dated 03 / 30 / 2026, pp. 44 / 57

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