WO2026081529A1 - Use of il-1r2 in identifying immune state of subject - Google Patents

Abstract

The present application provides a method for identifying the immune state of a subject, and a use of a reagent for detecting an IL-1R2 level in a sample from a subject in the preparation of products for identifying the immune state of the subject. The present application further provides an antibody that binds to human IL-1R2 and a use thereof, and a hybridoma cell producing the antibody that binds to human IL-1R2 and a use thereof.

Description

Application of IL-1R2 in identifying the immune status of subjects

Cross-reference to related applications

This application claims priority to Chinese Patent Application No. 202411462291.6, filed on October 18, 2024, the contents of which are incorporated herein by reference in their entirety.

Technical Field

This application relates to the fields of biological detection and antibody preparation. Specifically, this application provides a method for identifying the immune status of a subject, the use of a reagent for detecting the level of IL-1R2 in a sample from the subject in the preparation of a product for identifying the immune status of the subject, an antibody that binds to human IL-1R2 and its use, and hybridoma cells that produce antibodies that bind to human IL-1R2 and their use.

Background Technology

Systemic inflammatory response syndrome (SIRS) triggered by infection leads to sepsis. Sepsis is a life-threatening organ dysfunction caused by a dysregulated host response to infection. Sepsis has a high morbidity and mortality rate and remains a leading cause of death from infections both inside and outside hospitals. A recent statistical report highlights that nearly 50 million people worldwide develop sepsis each year, with onset across all age groups. In 2017, sepsis accounted for nearly 20% of all deaths globally. Furthermore, patients with a history of sepsis have a higher risk of readmission and death. Sepsis poses a serious threat to human health and also imposes a significant socioeconomic burden.

Immune dysfunction is closely related to the occurrence and development of sepsis, and patients may simultaneously exhibit both excessive inflammatory response and immunosuppression. The former can trigger early tissue damage and organ dysfunction, while the latter, when severe and persistent, can further induce various fatal complications, significantly increasing the mortality rate of patients in the middle and late stages of sepsis. In recent years, with the continuous improvement of organ support technology, the mortality rate in the pro-inflammatory phase has decreased, but the mortality rate in the immunosuppressive phase remains high. Therefore, the focus of research on sepsis-related immune dysfunction has shifted from excessive inflammation to immunosuppression. However, for a long time, the exact mechanisms by which severe injury causes immune dysfunction and its role in sepsis have been poorly understood, resulting in a lack of effective immune monitoring and regulation measures in clinical practice. Dynamic and accurate assessment of immune status is a prerequisite for timely identification of immune dysfunction in sepsis patients and determining the timing of immunomodulatory therapy. However, due to insufficient understanding of the precise molecular mechanisms and cellular basis of sepsis-induced immunosuppression, there is a lack of clinical monitoring indicators and assessment systems that can effectively reflect the immune status of sepsis patients.

In the newly released "Expert Consensus on the Diagnosis and Treatment of Sepsis Immunosuppression," experts pointed out that while lymphocyte counts and monocyte-mask antigen DR (mHLA-DR) are widely used to assess changes in immune function in sepsis patients, limitations remain. Although lymphocyte counts are readily available, their specificity is relatively low due to numerous confounding factors. Furthermore, mHLA-DR testing requires flow cytometry, which is complex and costly, and the definition of a warning threshold is still undefined. In recent years, researchers have applied multi-omics approaches to explore immune surveillance, aiming to discover new sepsis-related cell subsets and biomarkers; however, their translational significance and value require further validation through large-scale clinical trials.

Therefore, developing new methods and products for identifying the immune status of patients with sepsis and even systemic inflammatory response syndrome is of great biological and medical significance.

Summary of the Invention

Firstly, this application provides a method for identifying the immune status of a subject, comprising:

Determine the level of interleukin-1 receptor 2 (IL-1R2) in samples from the subject.

In one or more embodiments of this application, determining the level of IL-1R2 in a sample from the object includes contacting the sample with a reagent for detecting the level of IL-1R2.

In one or more embodiments of this application, determining the level of IL-1R2 in a sample from the object includes contacting the sample with a first antibody and a second antibody.

The first antibody comprises a heavy chain variable region containing HCDR1 (as shown in SEQ ID NO:2), HCDR2 (as shown in SEQ ID NO:3), and HCDR3 (as shown in SEQ ID NO:4), and a light chain variable region containing LCDR1 (as shown in SEQ ID NO:5), LCDR2 (as shown in SEQ ID NO:6), and LCDR3 (as shown in SEQ ID NO:7); and/or

The second antibody comprises a heavy chain variable region containing HCDR1 as shown in SEQ ID NO:8, HCDR2 as shown in SEQ ID NO:9, and HCDR3 as shown in SEQ ID NO:10, and a light chain variable region containing LCDR1 as shown in SEQ ID NO:11, LCDR2 as shown in SEQ ID NO:12, and LCDR3 as shown in SEQ ID NO:13;

The amino acid sequences of HCDR and LCDR are defined according to the IMGT numbering scheme.

In one or more embodiments of this application, the contact is simultaneous or successive.

In one or more embodiments of this application, the level of IL-1R2 in a sample is detected by immunological methods.

In one or more embodiments of this application, the level of IL-1R2 is compared with a cut-off value of the IL-1R2 level used to identify the immune status of the subject.

Secondly, this application provides the use of reagents for detecting IL-1R2 levels in samples from an object in the preparation of products for identifying the immune status of the object.

In one or more embodiments of this application, the immune state is an immunosuppressive state.

In one or more embodiments of this application, the object is a mammal.

In one or more embodiments of this application, the subject suffers from systemic inflammatory response syndrome, cancer, dengue fever, or colitis.

In one or more embodiments of this application, the reagent is a protein.

In one or more embodiments of this application, the product for identifying the immune status of the subject is selected from: reagent kits, test strips, test cards, and microfluidic detection devices.

In one or more embodiments of this application, the IL-1R2 is soluble IL-1R2 or membrane-bound protein IL-1R2.

In one or more embodiments of this application, the antibody comprises a heavy chain variable region containing HCDR1, HCDR2, and HCDR3 and a light chain variable region containing LCDR1, LCDR2, and LCDR3, wherein

The amino acid sequence of HCDR1 is shown in SEQ ID NO:2, the amino acid sequence of HCDR2 is shown in SEQ ID NO:3, the amino acid sequence of HCDR3 is shown in SEQ ID NO:4, the amino acid sequence of LCDR1 is shown in SEQ ID NO:5, the amino acid sequence of LCDR2 is shown in SEQ ID NO:6, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:7; or

The amino acid sequence of HCDR1 is shown in SEQ ID NO:8, the amino acid sequence of HCDR2 is shown in SEQ ID NO:9, the amino acid sequence of HCDR3 is shown in SEQ ID NO:10, the amino acid sequence of LCDR1 is shown in SEQ ID NO:11, the amino acid sequence of LCDR2 is shown in SEQ ID NO:12, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:13.

The amino acid sequences of HCDR and LCDR are defined according to the IMGT numbering scheme.

In one or more embodiments of this application, the product for identifying the immune status of the subject comprises a first antibody and/or a second antibody;

The first antibody comprises a heavy chain variable region containing HCDR1 (as shown in SEQ ID NO:2), HCDR2 (as shown in SEQ ID NO:3), and HCDR3 (as shown in SEQ ID NO:4), and a light chain variable region containing LCDR1 (as shown in SEQ ID NO:5), LCDR2 (as shown in SEQ ID NO:6), and LCDR3 (as shown in SEQ ID NO:7); and/or

The second antibody comprises a heavy chain variable region containing HCDR1 as shown in SEQ ID NO:8, HCDR2 as shown in SEQ ID NO:9, and HCDR3 as shown in SEQ ID NO:10, and a light chain variable region containing LCDR1 as shown in SEQ ID NO:11, LCDR2 as shown in SEQ ID NO:12, and LCDR3 as shown in SEQ ID NO:13;

The amino acid sequences of HCDR and LCDR are defined according to the IMGT numbering scheme.

In one or more embodiments of this application, the cut-off value for the IL-1R2 level used to identify the immune status of the subject is any value in the range of 19-29 ng/mL.

In one or more embodiments of this application, the immune status of an object whose IL-1R2 level is greater than the cut-off value is identified as an immunosuppressed state. In one or more embodiments of this application, the immune status of an object whose IL-1R2 level is less than the cut-off value is identified as a non-immunosuppressed state.

In one or more embodiments of this application, the immune status of the subject (e.g., immunosuppressed status) also needs to be identified in conjunction with at least one of the following clinical indicators: absolute lymphocyte count (ALC), monocyte HLA-DR positivity rate, medical history, medication history, age, body mass index, and albumin level.

Thirdly, this application provides an antibody that binds to human IL-1R2, comprising a heavy chain variable region containing HCDR1, HCDR2, and HCDR3 and a light chain variable region containing LCDR1, LCDR2, and LCDR3, wherein...

The amino acid sequence of HCDR1 is shown in SEQ ID NO:2, the amino acid sequence of HCDR2 is shown in SEQ ID NO:3, the amino acid sequence of HCDR3 is shown in SEQ ID NO:4, the amino acid sequence of LCDR1 is shown in SEQ ID NO:5, the amino acid sequence of LCDR2 is shown in SEQ ID NO:6, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:7; or

The amino acid sequence of HCDR1 is shown in SEQ ID NO:8, the amino acid sequence of HCDR2 is shown in SEQ ID NO:9, the amino acid sequence of HCDR3 is shown in SEQ ID NO:10, the amino acid sequence of LCDR1 is shown in SEQ ID NO:11, the amino acid sequence of LCDR2 is shown in SEQ ID NO:12, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:13.

The amino acid sequences of HCDR and LCDR are defined according to the IMGT numbering scheme.

In one or more embodiments of this application, the amino acid sequence of the heavy chain variable region of the antibody has at least 90% identity with SEQ ID NO: 14 or 15; and/or

The amino acid sequence of the variable region of the light chain of the antibody has at least 90% identity with SEQ ID NO:16 or 17.

In one or more embodiments of this application, the antibody is a whole antibody, a Fab fragment, a Fab' fragment, an F(ab') ₂ fragment, an Fv fragment, a single-chain Fv fragment (scFv), an Fd fragment, a single-domain antibody, or a dAb fragment; and/or

The antibody is a monoclonal antibody; and/or

The antibody contains a heavy chain constant region selected from IgG1, IgG2, or IgG4 subtypes; and/or

The antibody contains a light chain constant region selected from the κ or λ subtype; and/or

The antibody binds to human IL-1R2 with the amino acid sequence shown in SEQ ID NO:1.

In one or more embodiments of this application, the antibody is produced by a hybridoma cell line with accession number CCTCC NO:C2024249 or CCTCC NO:C2024250.

Fourthly, this application provides hybridoma cells that produce antibodies that bind to human IL-1R2, which are deposited under accession numbers CCTCC NO:C2024249 or CCTCC NO:C2024250.

Fifthly, this application provides a product for detecting human IL-1R2 levels in a sample, which comprises a first antibody and/or a second antibody;

The first antibody comprises a heavy chain variable region containing HCDR1 (as shown in SEQ ID NO:2), HCDR2 (as shown in SEQ ID NO:3), and HCDR3 (as shown in SEQ ID NO:4), and a light chain variable region containing LCDR1 (as shown in SEQ ID NO:5), LCDR2 (as shown in SEQ ID NO:6), and LCDR3 (as shown in SEQ ID NO:7); and/or

The second antibody comprises a heavy chain variable region containing HCDR1 as shown in SEQ ID NO:8, HCDR2 as shown in SEQ ID NO:9, and HCDR3 as shown in SEQ ID NO:10, and a light chain variable region containing LCDR1 as shown in SEQ ID NO:11, LCDR2 as shown in SEQ ID NO:12, and LCDR3 as shown in SEQ ID NO:13;

The amino acid sequences of HCDR and LCDR are defined according to the IMGT numbering scheme.

In a sixth aspect, this application provides a method for detecting human IL-1R2 levels in a sample, the method comprising contacting the sample with a first antibody and a second antibody; optionally, the contact is simultaneous or sequential.

The first antibody comprises a heavy chain variable region containing HCDR1 (as shown in SEQ ID NO:2), HCDR2 (as shown in SEQ ID NO:3), and HCDR3 (as shown in SEQ ID NO:4), and a light chain variable region containing LCDR1 (as shown in SEQ ID NO:5), LCDR2 (as shown in SEQ ID NO:6), and LCDR3 (as shown in SEQ ID NO:7); and/or

The second antibody comprises a heavy chain variable region containing HCDR1 as shown in SEQ ID NO:8, HCDR2 as shown in SEQ ID NO:9, and HCDR3 as shown in SEQ ID NO:10, and a light chain variable region containing LCDR1 as shown in SEQ ID NO:11, LCDR2 as shown in SEQ ID NO:12, and LCDR3 as shown in SEQ ID NO:13;

The amino acid sequences of HCDR and LCDR are defined according to the IMGT numbering scheme.

In one or more embodiments of this application, the level of human IL-1R2 in a sample is detected by immunological methods.

Seventhly, this application provides the use of the antibody described in the third aspect or the hybridoma cell described in the fourth aspect in the preparation of a product for detecting human IL-1R2 levels in a sample.

In one or more embodiments of this application, the product for detecting human IL-1R2 levels in a sample is selected from: kits, test strips, test cards, and microfluidic detection devices.

In one or more embodiments of this application, the human IL-1R2 is either human soluble IL-1R2 or human membrane-bound protein IL-1R2.

In one or more embodiments of this application, the first antibody or the second antibody is labeled with a detectable marker selected from: enzymes, fluorescent molecules, radioactive isotopes, chemiluminescent molecules, latex particles, gold particles, detectable ligands, and any combination thereof.

In one or more embodiments of this application, the first antibody or the second antibody is attached to a solid support.

In one or more embodiments of this application, the sample is selected from: plasma, blood, urine, serum, lymph, gastric juice, bile, saliva, sweat, cerebrospinal fluid, and any combination thereof.

Attached Figure Description

Figure 1 shows the results of correct expression and purity identification of recombinant human soluble IL-1R2 protein. In Figure 1, A shows the results of Western blotting using a His-tagged antibody to verify correct expression of soluble IL-1R2 in cell culture supernatant; B shows the results of SDS-PAGE to verify the purity of recombinant human soluble IL-1R2 protein.

Figure 2 shows the binding and dissociation curves of different concentrations of monoclonal antibody 2E10 and IL-1R2 protein detected by SPR.

Figure 3 shows the binding and dissociation curves of different concentrations of monoclonal antibody 3D10 and IL-1R2 protein detected by SPR.

Figure 4 shows the binding and dissociation curves of different concentrations of monoclonal antibody R020 and IL-1R2 protein detected by SPR.

Figure 5 shows the binding and dissociation curves of different concentrations of antibody AP73653 and IL-1R2 protein detected by SPR.

Figure 6 shows the ROC curve analysis results of detecting human plasma IL-1R2 levels using monoclonal antibodies 2E10 and 3D10 to identify immunosuppression.

Figure 7 shows the plasma IL-1R2 levels in epigenetic healthy individuals, non-immunosuppressed sepsis patients, and immunosuppressed sepsis patients as detected using monoclonal antibodies 2E10 and 3D10 paired.

Preservation Instructions

1. Cell name: Hybridoma cell line ZIL1R-2E10

Preservation Institution: China Center for Type Culture Collection

Abbreviation for depository institution: CCTCC

Address: Wuhan University, Wuhan, China (No. 299 Bayi Road, Wuchang District, Wuhan, Hubei Province)

Deposit date: August 22, 2024

Collection Center Registration Number: CCTCC NO: C2024249

2. Cell name: Hybridoma cell line ZIL1R-3D10

Preservation Institution: China Center for Type Culture Collection

Abbreviation for depository institution: CCTCC

Address: Wuhan University, Wuhan, China (No. 299 Bayi Road, Wuchang District, Wuhan, Hubei Province)

Deposit date: August 22, 2024

Collection Center Registration Number: CCTCC NO: C2024250

Sequence Description

SEQ ID NO:1 shows the amino acid sequence from position 1 to position 343 of soluble IL-1R2 with Uniprot number P27930-1.

SEQ ID NO:2-4 show the amino acid sequences of HCDR1, HCDR2 and HCDR3 of the monoclonal antibody 2E10 against soluble human IL-1R2, respectively.

SEQ ID NO:5-7 show the amino acid sequences of LCDR1, LCDR2 and LCDR3 of the monoclonal antibody 2E10 against soluble human IL-1R2, respectively.

SEQ ID NO:8-10 show the amino acid sequences of HCDR1, HCDR2 and HCDR3 of the monoclonal antibody 3D10 against soluble human IL-1R2, respectively.

SEQ ID NO:11-13 show the amino acid sequences of LCDR1, LCDR2 and LCDR3 of the monoclonal antibody 3D10 against soluble human IL-1R2, respectively.

SEQ ID NO:14 shows the amino acid sequence of the heavy chain variable region of the monoclonal antibody 2E10 against soluble human IL-1R2.

SEQ ID NO:15 shows the amino acid sequence of the heavy chain variable region of the monoclonal antibody 3D10 against soluble human IL-1R2.

SEQ ID NO:16 shows the amino acid sequence of the light chain variable region of monoclonal antibody 2E10 against soluble human IL-1R2.

SEQ ID NO:17 shows the amino acid sequence of the light chain variable region of the monoclonal antibody 3D10 against soluble human IL-1R2.

SEQ ID NO:18 shows the nucleotide sequence of the heavy chain variable region of monoclonal antibody 2E10 encoding soluble human IL-1R2.

SEQ ID NO:19 shows the amino acid sequence of FR1 in the heavy chain variable region of monoclonal antibody 2E10 against soluble human IL-1R2.

SEQ ID NO:20 shows the amino acid sequence of FR2 in the heavy chain variable region of monoclonal antibody 2E10 against soluble human IL-1R2.

SEQ ID NO:21 shows the amino acid sequence of FR3 in the heavy chain variable region of monoclonal antibody 2E10 against soluble human IL-1R2.

SEQ ID NO:22 shows the amino acid sequence of FR4 in the heavy chain variable region of the monoclonal antibody 2E10 against soluble human IL-1R2.

SEQ ID NO:23 shows the nucleotide sequence of the light chain variable region of monoclonal antibody 2E10 encoding soluble human IL-1R2.

SEQ ID NO:24 shows the amino acid sequence of FR1 in the light chain variable region of monoclonal antibody 2E10 against soluble human IL-1R2.

SEQ ID NO:25 shows the amino acid sequence of FR2 in the light chain variable region of monoclonal antibody 2E10 against soluble human IL-1R2.

SEQ ID NO:26 shows the amino acid sequence of FR3 in the light chain variable region of monoclonal antibody 2E10 against soluble human IL-1R2.

SEQ ID NO:27 shows the amino acid sequence of FR4 of the light chain variable region of the monoclonal antibody 2E10 against soluble human IL-1R2.

SEQ ID NO:28 shows the nucleotide sequence of the heavy chain variable region of monoclonal antibody 3D10 encoding anti-soluble human IL-1R2.

SEQ ID NO:29 shows the amino acid sequence of FR1 in the heavy chain variable region of the monoclonal antibody 3D10 against soluble human IL-1R2.

SEQ ID NO:30 shows the amino acid sequence of FR2 in the heavy chain variable region of the monoclonal antibody 3D10 against soluble human IL-1R2.

SEQ ID NO:31 shows the amino acid sequence of FR3 in the heavy chain variable region of the monoclonal antibody 3D10 against soluble human IL-1R2.

SEQ ID NO:32 shows the amino acid sequence of FR4 in the heavy chain variable region of the monoclonal antibody 3D10 against soluble human IL-1R2.

SEQ ID NO:33 shows the nucleotide sequence of the light chain variable region of monoclonal antibody 3D10 encoding anti-soluble human IL-1R2.

SEQ ID NO:34 shows the amino acid sequence of FR1 in the light chain variable region of monoclonal antibody 3D10 against soluble human IL-1R2.

SEQ ID NO:35 shows the amino acid sequence of FR2 in the light chain variable region of the monoclonal antibody 3D10 against soluble human IL-1R2.

SEQ ID NO:36 shows the amino acid sequence of FR3 in the light chain variable region of monoclonal antibody 3D10 against soluble human IL-1R2.

SEQ ID NO:37 shows the amino acid sequence of FR4 in the light chain variable region of the monoclonal antibody 3D10 against soluble human IL-1R2.

Detailed Implementation

Interleukin-1 (IL-1) is a potent pro-inflammatory cytokine that amplifies the immune response, primarily produced by macrophages during the defense process. IL-1 is one of several pro-inflammatory cytokines produced during infection, sepsis, and systemic inflammatory response syndrome (SIRS), used to initiate the host inflammatory response and integrate non-specific immunity. IL-1 is an important mediator involved in the pathogenesis of SIRS. In the early stages of SIRS, IL-1 and TNF-α are the dominant cytokines, inducing the release of other pro-inflammatory cytokines (IL-6, IL-8, IFN-γ) and triggering the coagulation cascade. IL-1 can also induce the production of TNF-α.

Interleukin-1 receptor type 2 (IL-1R2) belongs to the IL-1 receptor family. IL-1R1, IL-1R2, and IL-1RAP are important components of the IL-1 receptor family. The IL-1R2 gene is located on chromosome 2q12 and, like other members of the IL-1 receptor family, consists of an extracellular portion containing three glycosylated immunoglobulin (Ig)-like domains, sharing 28% amino acid homology with the extracellular portion of IL-1R1. However, compared to other members of this family, it has a shorter cytoplasmic structure and lacks the Toll/IL-1 receptor (TIR) domain, thus failing to initiate downstream signal transduction. The IL-1R2 protein exists in both a membrane-bound form and a soluble form (sIL-1R2).

IL-1 forms an IL-1/IL-1R1/IL-1RAP complex with IL-1R1 under the action of IL-1RAP, activating downstream IL-1 signaling pathways and exerting pro-inflammatory biological functions. The membrane-bound protein IL-1R2 competitively captures IL-1 on the cell surface, while sIL-1R2 binds to IL-1 in the extracellular microenvironment. This prevents both IL-1 binding to IL-1R1 on the cell surface and the initiation of cell activation, and is one of the main mechanisms inhibiting IL-1 activity, thus being defined as a decoy receptor.

IL-1R2 has a high affinity for IL-1β but a low affinity for the interleukin-1 receptor antagonist (IL-1Ra), thus enabling it to inhibit IL-1 activity with only partial interference from IL-1Ra. Compared to the membrane-bound protein IL-1R2, sIL-1R2 exhibits different ligand-binding abilities. sIL-1R2's binding affinity for IL-1β is comparable to that of the membrane receptor, but its binding affinity for IL-1α is superior. More importantly, sIL-1R2 cannot bind to IL-1Ra, indicating that sIL-1R2 is a better IL-1 inhibitor than the membrane-bound protein IL-1R2 because its action is not interfered with by IL-1Ra. Furthermore, sIL-1R2 can also bind to the IL-1β precursor Pro-IL-1β, preventing Pro-IL-1β from being cleaved and converted into mature IL-1β by proteases within the cell, thereby exerting a negative regulatory effect on the inflammatory response. In summary, soluble IL-1R2 exerts its function of suppressing the immune response by inhibiting the pro-inflammatory effect of IL-1.

The inventors of this application discovered that plasma levels of soluble IL-1R2 are significantly elevated in critically ill patients with sepsis and other systemic inflammatory response syndromes (SIRS), and that plasma IL-1R2 levels are positively correlated with disease severity. Elevated IL-1R2 reflects IL-1 dysregulation, indicating a shift from SIRS to compensatory anti-inflammatory response syndrome (CARS). Furthermore, stimulation by anti-inflammatory factors such as IL-4, IL-10, and glucocorticoids can upregulate IL-1R2. IL-1R2 primarily plays a negative regulatory role in inflammatory responses across different cell types. In summary, elevated soluble IL-1R2 indicates a state of relative immunosuppression in patients and can serve as an auxiliary indicator of immunosuppression in patients with sepsis and even systemic inflammatory response syndromes.

In addition, in order to achieve the determination of human IL-1R2 (especially human soluble IL-1R2) levels, the inventors of this application have also obtained new antibodies that bind to human IL-1R2 (especially human soluble IL-1R2) through antibody engineering technology.

In several aspects of this application, methods for identifying the immune status of a subject are provided, the use of reagents for detecting IL-1R2 levels in samples from a subject in the preparation of products for identifying the immune status of said subject is provided, antibodies that bind to human IL-1R2 and their use therewith are provided, hybridoma cells that generate antibodies that bind to human IL-1R2 and their use therewith are provided, products for detecting human IL-1R2 levels in samples are provided, and methods for detecting human IL-1R2 levels in samples are provided.

Unless otherwise specified, the implementation of this application will employ conventional molecular biology, microbiology, cell biology, biochemistry, and immunology techniques in the art.

Unless otherwise specified, the terms used in this application have the meanings commonly understood by those skilled in the art.

Unless otherwise specified, all reagents used in the experiments are commercially available products.

definition

As used in this article, the term "immune status" refers to the functional state and responsiveness of an individual's immune system at a specific point in time. It reflects the immune system's ability to respond to antigens (such as pathogens, vaccines, or other foreign substances) and its overall health.

As used in this article, the term "immunosuppressive state" refers to a weakened or suppressed immune system that reduces an individual's ability to respond to antigens such as pathogens, tumor cells, or vaccines. Such a state may make an individual more susceptible to infection, develop certain diseases, or have difficulty fighting off diseases effectively.

As used in this article, the term "relative immunosuppression" refers to a condition in which an individual's immune system function is partially weakened or suppressed under certain specific conditions or relative to healthy standards, but this suppression is not absolute or complete. This state may make an individual more susceptible to infections or other immune-related problems in certain situations (such as long-term hospitalized patients), but in other situations, they may still maintain a certain level of immune responsiveness.

As used herein, the term "antibody" refers to an immunoglobulin molecule comprising four polypeptide chains: two heavy chains (H) and two light chains (L) linked by disulfide bonds, and its multimers (e.g., IgM). Each heavy chain contains a heavy chain variable region (abbreviated as VH) and a heavy chain constant region (abbreviated as CH). The heavy chain constant region contains three domains: CH1, CH2, and CH3. Each light chain contains a light chain variable region (abbreviated as VL) and a light chain constant region (abbreviated as CL). The light chain constant region contains one domain (CL1). The VH and VL regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), interspersed with conserved regions called framework regions (FRs). In some embodiments, from the N-terminus to the C-terminus, both the light and heavy chain variable regions contain FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

An antibody is an immunoglobulin molecule that specifically binds to a target immunoglobulin molecule via at least one antigen recognition site located in the variable region of the immunoglobulin molecule. Targets include, but are not limited to, carbohydrates, polynucleotides, lipids, peptides, etc. As used herein, "antibody" includes not only complete (i.e., full-length) antibodies, but also their antigen-binding fragments, their variants, fusion proteins containing antibody portions, humanized antibodies, chimeric antibodies, biantibodies, linear antibodies, single-chain antibodies, multispecific antibodies (e.g., bispecific antibodies), and any other modified configurations of immunoglobulin molecules containing antigen recognition sites of desired specificity, including glycosylated variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies.

Full-length antibodies can be any type of antibody, such as IgD, IgE, IgG, IgA, or IgM (or subclasses mentioned above), but they do not need to belong to any specific class. Immunoglobulins can be classified into different classes based on the antibody's amino acid sequence in the heavy chain constant region. Generally, there are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these classes can be further subdivided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain regions corresponding to different immunoglobulin classes are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional structures of different classes of immunoglobulins are well known.

As used herein, the term "antigen-binding fragment" of an antibody refers to a portion or segment of the complete antibody molecule responsible for binding an antigen. Antigen-binding fragments may comprise a heavy chain variable region (VH), a light chain variable region (VL), or both. Antigen-binding fragments of antibodies can be prepared from complete antibody molecules using any suitable standard technique, including proteolytic digestion or recombinant genetic engineering. Non-limiting examples of antigen-binding fragments include: Fab fragments, Fab' fragments, F(ab') ₂ fragments, Fv fragments, single-chain Fv fragments (scFv), Fd fragments, single-domain antibodies, dAb fragments, and the smallest recognition unit (e.g., isolated CDR) consisting of amino acid residues mimicking the hypervariable region of an antibody. Antigen-binding fragments may also include other engineered molecules, such as biantibodies, triantibodies, tetraantibodies, and microantibodies. For example, the Fd fragment mentioned in this article refers to an antibody fragment composed of the VH and CH1 regions; the Fv fragment is composed of the VL and VH regions in the single arm of the antibody; and the dAb fragment (Ward et al., Nature 1989; 341: 544-546) is composed of the VH region.

It is well known to those skilled in the art that complementarity-determining regions (CDRs, typically CDR1, CDR2, and CDR3) are the regions in the variable region that have the greatest impact on antibody affinity and specificity. The CDR amino acid sequences of VH or VL are defined using common methods, such as the IMGT numbering scheme, the Chothia numbering scheme, and the Kabat numbering scheme. See, for example, Kabat, “Sequences of Proteins of Immunological Interest”, National Institutes of Health, Bethesda, Md. (1991) 7; Al-Lazikani, et al., J. Mol. Biol. 273: 927-948 (1997); and Martin, et al., Proc. Natl. Acad. Sci. USA 86: 9268-9272 (1989). For a given antibody's variable region amino acid sequence, the CDR amino acid sequence in the VH and VL amino acid sequences can be determined according to the IMGT numbering scheme, the Chothia numbering scheme, or the Kabat numbering scheme.

For a given antibody's variable region amino acid sequence, the CDR amino acid sequence can be analyzed in various ways, such as using the online software Abysis (http://www.abysis.org/).

As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as the binding of an antibody to an antigenic epitope, or the ability of an antibody to bind to a specific antigen with an affinity at least twice that of a nonspecific antigen. However, it should be understood that antibodies can specifically bind to two or more antigens associated with their sequence.

As used in this article, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, that is, the individual antibodies that make up the population are identical except for the possibility of naturally occurring mutations in a small number of individuals.

As used herein, the term "mouse-derived antibody" refers to any antibody in which all constant region sequences are mouse sequences. Such antibodies can be produced via hybridomas.

As used herein, the term "monoclonal antibody reactivity" refers to the ability of a monoclonal antibody to bind to an antigen under suitable reaction conditions.

As used herein, the term "cell line" refers to a single-cell culture obtained from a primary culture or cell line through screening or limiting dilution methods.

As used herein, the term "homology" is defined as the percentage of identical residues in an amino acid or nucleotide sequence variant after sequence alignment and vacancy introduction, reaching the maximum percentage of homology if desired. Methods and computer programs used for alignment are well known in the art. "At least 90% homology" as used herein means any value between 90% and 100%, such as 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

As used herein, the term "cut-off value" is a threshold value used to determine whether a test result is abnormal. In medical testing, the cut-off value is a decision point used to determine whether a test result is higher or lower than a clinical or analytical threshold. If the test result is lower than the cut-off value, the test result is negative; if it is higher than the cut-off value, the test result is positive. In some embodiments of this application, when the human plasma IL-1R2 level is higher than the cut-off value, it indicates that the patient is in an immunosuppressed state (e.g., a relative immunosuppressed state); when the human plasma IL-1R2 level is lower than the cut-off value, it indicates that the patient is in a non-immunosuppressed state.

Firstly, this application provides a method for identifying the immune status of a subject, comprising:

Determine the level of interleukin-1 receptor 2 (IL-1R2) in samples from the subject.

In one or more embodiments of this application, determining the level of IL-1R2 in a sample from the object includes contacting the sample with a reagent for detecting the level of IL-1R2.

In one or more embodiments of this application, determining the level of IL-1R2 in a sample from the object includes contacting the sample with a first antibody and a second antibody.

The first antibody comprises a heavy chain variable region containing HCDR1 as shown in SEQ ID NO:2, HCDR2 as shown in SEQ ID NO:3, and HCDR3 as shown in SEQ ID NO:4, and a light chain variable region containing LCDR1 as shown in SEQ ID NO:5, LCDR2 as shown in SEQ ID NO:6, and LCDR3 as shown in SEQ ID NO:7; and/or

The second antibody comprises a heavy chain variable region containing HCDR1 as shown in SEQ ID NO:8, HCDR2 as shown in SEQ ID NO:9, and HCDR3 as shown in SEQ ID NO:10, and a light chain variable region containing LCDR1 as shown in SEQ ID NO:11, LCDR2 as shown in SEQ ID NO:12, and LCDR3 as shown in SEQ ID NO:13;

The amino acid sequences of HCDR and LCDR are defined according to the IMGT numbering scheme.

In one or more embodiments of this application, the method for identifying the immune status of a subject is a method for assisting in the identification of the immune status of a subject (e.g., immunosuppressive status).

In one or more embodiments of this application, the level of IL-1R2 is compared with a cut-off value of the IL-1R2 level used to identify the immune status of the subject.

Secondly, this application provides the use of reagents for detecting IL-1R2 levels in samples from an object in the preparation of products for identifying the immune status of the object.

In one or more embodiments of this application, the immune state is an immunosuppressive state. In one or more embodiments of this application, the immune state is a relative immunosuppressive state.

In one or more embodiments of this application, the object is a mammal, such as a human.

In one or more embodiments of this application, the subject suffers from systemic inflammatory response syndrome, cancer, dengue fever, or colitis. In one or more embodiments of this application, the systemic inflammatory response syndrome is sepsis or acute respiratory distress syndrome. In one or more embodiments of this application, the cancer is gastric cancer, breast cancer, colon cancer, pancreatic cancer, ovarian cancer, prostate cancer, lung cancer, or leukemia.

In one or more embodiments of this application, the reagent is a protein, such as an antibody. In one or more embodiments of this application, the antibody can be any antibody capable of binding to IL-1R2, such as commercially available antibodies that bind to IL-1R2.

In one or more embodiments of this application, the IL-1R2 is soluble IL-1R2 or membrane-bound protein IL-1R2. In one or more embodiments of this application, the IL-1R2 is soluble IL-1R2, such as human soluble IL-1R2.

In one or more embodiments of this application, the antibody comprises a heavy chain variable region containing HCDR1, HCDR2, and HCDR3 and a light chain variable region containing LCDR1, LCDR2, and LCDR3, wherein

The amino acid sequence of HCDR1 is shown in SEQ ID NO:2, the amino acid sequence of HCDR2 is shown in SEQ ID NO:3, the amino acid sequence of HCDR3 is shown in SEQ ID NO:4, the amino acid sequence of LCDR1 is shown in SEQ ID NO:5, the amino acid sequence of LCDR2 is shown in SEQ ID NO:6, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:7; or

The amino acid sequence of HCDR1 is shown in SEQ ID NO:8, the amino acid sequence of HCDR2 is shown in SEQ ID NO:9, the amino acid sequence of HCDR3 is shown in SEQ ID NO:10, the amino acid sequence of LCDR1 is shown in SEQ ID NO:11, the amino acid sequence of LCDR2 is shown in SEQ ID NO:12, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:13.

The amino acid sequences of HCDR and LCDR are defined according to the IMGT numbering scheme.

In one or more embodiments of this application, the product for identifying the immune status of the subject is selected from: reagent kits, test strips (e.g., colloidal gold test strips), test cards, and microfluidic detection devices.

In one or more embodiments of this application, the product for identifying the immune status of the subject comprises a first antibody and/or a second antibody;

The first antibody comprises a heavy chain variable region containing HCDR1 (as shown in SEQ ID NO:2), HCDR2 (as shown in SEQ ID NO:3), and HCDR3 (as shown in SEQ ID NO:4), and a light chain variable region containing LCDR1 (as shown in SEQ ID NO:5), LCDR2 (as shown in SEQ ID NO:6), and LCDR3 (as shown in SEQ ID NO:7); and/or

The second antibody comprises a heavy chain variable region containing HCDR1 as shown in SEQ ID NO:8, HCDR2 as shown in SEQ ID NO:9, and HCDR3 as shown in SEQ ID NO:10, and a light chain variable region containing LCDR1 as shown in SEQ ID NO:11, LCDR2 as shown in SEQ ID NO:12, and LCDR3 as shown in SEQ ID NO:13;

The amino acid sequences of HCDR and LCDR are defined according to the IMGT numbering scheme.

In one or more embodiments of this application, the product for identifying the immune status of the subject may further include other reagents for detection, such as substrates, reference standards, diluents, washing solutions, etc.

In one or more embodiments of this application, the product for identifying the immune status of the subject may further include a product instruction manual.

In one or more embodiments of this application, the product for identifying the immune status of the subject may further include a container for mixing the sample with the antibody. Such a container may be suitable for use with detection instruments capable of detecting signals generated by detecting monoclonal antibodies.

In one or more embodiments of this application, the product for identifying the immune status of the subject is used to assist in identifying the subject's immune status, such as an immunosuppressive state.

In one or more embodiments of this application, the cut-off value for the IL-1R2 level used to identify the immune status of the subject is any value in the range of 19-29 ng/mL, such as 19, 19.1, 19.2, 19.3, 19.4, 19.5, 19.6, 19.7, 19.8, 19.9, 20, 20.1, 20.2, 20.3, 20. 4, 20.5, 20.6, 20.7, 20.8, 20.9, 21, 21.1, 21.2, 21.3, 21.4, 21.5, 21.6, 21.7, 21.8, 21.9, 22, 22.1, 22.2, 22.3, 22.4, 22.5, 22.6, 22.7, 22.8, 22.9, 23, 23.1, 23.2, 23.3 23.4, 23.5, 23.6, 23.7, 23.8, 23.9, 24, 24.1, 24.2, 24.3, 24.4, 24.5, 24.6, 24.7, 24.8, 24.9, 25, 25.1, 25.2, 25.3, 25.4, 25.5, 25.6, 25.7, 25.8, 25.9, 26, 26.1, 26.2 The cut-off values for the IL-1R2 level used to identify the immune status of the subject are 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29 ng/mL. In one or more embodiments of this application, the cut-off value for the IL-1R2 level used to identify the immune status of the subject is 24 ng/mL. In one or more specific embodiments of this application, the cut-off value for the IL-1R2 level used to identify the immune status of the subject is 24 ng/mL.

In one or more embodiments of this application, the immune status of an object whose IL-1R2 level is greater than the cut-off value is identified as an immunosuppressed state. In one or more embodiments of this application, the immune status of an object whose IL-1R2 level is less than the cut-off value is identified as a non-immunosuppressed state.

In one or more embodiments of this application, the immune status of the subject (e.g., immunosuppressed status) also needs to be identified in conjunction with at least one of the following clinical indicators: absolute lymphocyte count, HLA-DR positivity rate of monocytes, medical history, medication history, age, body mass index, and albumin level.

In one or more embodiments of this application, the immune status of the subject (e.g., immunosuppressive status) also needs to be identified in conjunction with the absolute lymphocyte count and the HLA-DR positivity rate of monocytes. In one or more embodiments of this application, the immune status of patients with an absolute lymphocyte count of less than 1.0 × ^10⁹ cells/L and/or an HLA-DR positivity rate of peripheral blood monocytes of less than 60% in three consecutive days of routine blood tests is identified as immunosuppressive status, such as relative immunosuppression. In one or more embodiments of this application, whether the patient is in an immunosuppressive state requires a physician to comprehensively determine it by combining clinical indicators (e.g., absolute lymphocyte count and/or HLA-DR positivity rate of peripheral blood monocytes).

Thirdly, this application provides an antibody that binds to human IL-1R2, comprising a heavy chain variable region containing HCDR1, HCDR2, and HCDR3 and a light chain variable region containing LCDR1, LCDR2, and LCDR3, wherein...

The amino acid sequence of HCDR1 is shown in SEQ ID NO:2, the amino acid sequence of HCDR2 is shown in SEQ ID NO:3, the amino acid sequence of HCDR3 is shown in SEQ ID NO:4, the amino acid sequence of LCDR1 is shown in SEQ ID NO:5, the amino acid sequence of LCDR2 is shown in SEQ ID NO:6, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:7; or

The amino acid sequence of HCDR1 is shown in SEQ ID NO:8, the amino acid sequence of HCDR2 is shown in SEQ ID NO:9, the amino acid sequence of HCDR3 is shown in SEQ ID NO:10, the amino acid sequence of LCDR1 is shown in SEQ ID NO:11, the amino acid sequence of LCDR2 is shown in SEQ ID NO:12, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:13.

The amino acid sequences of HCDR and LCDR are defined according to the IMGT numbering scheme.

In one or more embodiments of this application, the amino acid sequence of the heavy chain variable region of the antibody has at least 90% identity with SEQ ID NO:14 or 15, for example, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity.

In one or more embodiments of this application, the amino acid sequence of the light chain variable region of the antibody has at least 90% identity with SEQ ID NO:16 or 17, for example, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity.

In one or more embodiments of this application, the amino acid sequence of the heavy chain variable region of the antibody has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with SEQ ID NO:14 or 15; and the amino acid sequence of the light chain variable region of the antibody has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with SEQ ID NO:16 or 17.

In one or more embodiments of this application, the amino acid sequence of the heavy chain variable region of the antibody differs from the amino acid sequence shown in SEQ ID NO: 14 or 15 by substitution, deletion, and/or addition of approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids.

In one or more embodiments of this application, the amino acid sequence of the variable region of the light chain of the antibody differs from the amino acid sequence shown in SEQ ID NO: 16 or 17 by substitution, deletion, and/or addition of approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids.

In one or more embodiments of this application, the C-terminal or N-terminal region of the amino acid sequence shown in SEQ ID NO: 14 or 15 may be truncated by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids while still retaining the function of a similar heavy chain variable region of the antibody.

In one or more embodiments of this application, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids may be added to the C-terminal or N-terminal region of the amino acid sequence shown in SEQ ID NO: 14 or 15, and the resulting amino acid sequence still retains the function of the heavy chain variable region of the antibody.

In one or more embodiments of this application, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids may be added or deleted in regions other than the C-terminus or N-terminus of the amino acid sequence shown in SEQ ID NO: 14 or 15, as long as the altered amino acid sequence substantially maintains the function of the heavy chain variable region of the antibody.

In one or more embodiments of this application, the C-terminal or N-terminal region of the amino acid sequence shown in SEQ ID NO: 16 or 17 may be truncated by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids while still retaining the function of a similar light chain variable region of the antibody.

In one or more embodiments of this application, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids may be added to the C-terminal or N-terminal region of the amino acid sequence shown in SEQ ID NO: 16 or 17, and the resulting amino acid sequence still retains the function of the light chain variable region similar to that of the antibody.

In one or more embodiments of this application, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids may be added or deleted in regions other than the C-terminus or N-terminus of the amino acid sequence shown in SEQ ID NO: 16 or 17, as long as the altered amino acid sequence substantially maintains the function of the light chain variable region of the antibody.

In one or more embodiments of this application, the amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO: 14 or 15.

In one or more embodiments of this application, the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO: 16 or 17.

In one or more specific embodiments of this application, the amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO:14, and the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO:16; or

The amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO:15, and the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO:17.

In one or more embodiments of this application, the antibody is a whole antibody, a Fab fragment, a Fab' fragment, an F(ab') ₂ fragment, an Fv fragment, a single-chain Fv fragment (scFv), an Fd fragment, a single-domain antibody, or a dAb fragment.

In one or more embodiments of this application, the antibody is a monoclonal antibody.

In one or more specific embodiments of this application, the antibody is a murine monoclonal antibody.

In one or more embodiments of this application, the antibody comprises a heavy chain constant region selected from IgG1, IgG2 or IgG4 subtypes.

In one or more embodiments of this application, the antibody comprises a heavy chain constant region of IgG1 or IgG2 subtypes.

In one or more specific embodiments of this application, when the antibody is a monoclonal antibody 2E10 against soluble IL-1R2, it contains the heavy chain constant region of the IgG1 subtype.

In some specific embodiments of the first aspect, when the antibody is a monoclonal antibody 3D10 against soluble IL-1R2, it contains a heavy chain constant region of an IgG2 subtype (e.g., an IgG2α subtype).

In one or more embodiments of this application, the antibody comprises a light chain constant region selected from the κ or λ subtype.

In one or more embodiments of this application, the antibody binds to human IL-1R2 with an amino acid sequence as shown in SEQ ID NO:1.

In one or more embodiments of this application, the antibody is produced by a hybridoma cell line with accession number CCTCC NO:C2024249 or CCTCC NO:C2024250.

In one or more embodiments of this application, the monoclonal antibody 2E10 against human soluble IL-1R2 is produced by a hybridoma cell line with accession number CCTCC NO:C2024249.

In one or more embodiments of this application, the monoclonal antibody 3D10 against human soluble IL-1R2 is produced by a hybridoma cell line with accession number CCTCC NO:C2024250.

In one or more embodiments of this application, the antibody or a product containing the antibody can be used for point-of-care testing (POCT).

In one or more embodiments of this application, the antibody has the advantages of high specificity, high titer, and high sensitivity.

In one or more embodiments of this application, the antibody can effectively detect the level of IL-1R2 in a patient's body fluids (e.g., plasma, blood, or urine) to determine whether the patient is in an immunosuppressed state. This can guide doctors to assess the severity of the patient's condition, take timely and effective treatment measures, reduce mortality, and has important clinical significance.

Fourthly, this application provides hybridoma cells that produce antibodies that bind to human IL-1R2, which are deposited under accession numbers CCTCC NO:C2024249 or CCTCC NO:C2024250.

In one or more embodiments of this application, after an animal is inoculated with a human IL-1R2 protein (e.g., human soluble IL-1R2 protein) antigen, antibodies and/or antibody-producing cells can be automatically obtained within the animal. The antibody-producing immortalized cell line can be prepared from cells isolated from an immunized animal. After immunization, the animal is killed, and lymph node and/or spleen B cells are immortalized, treated with carcinogenic and mutagenic compounds, and fused with immortalized cells (e.g., myeloma cells, such as mouse myeloma cells SP2/0) to remove the activity of tumor suppressor genes. If myeloma cells are used for fusion, these myeloma cells preferably do not secrete immunoglobulin peptides (non-secreting cell line). Immortalized cells are screened using human IL-1R2 protein (e.g., human soluble IL-1R2 protein) or cells expressing human IL-1R2 protein (e.g., human soluble IL-1R2 protein). In some embodiments, the initial screening is performed using an enzyme-linked immunosorbent assay (ELISA). Cells that produce antibodies against human IL-1R2 protein (e.g., soluble human IL-1R2 protein), such as hybridomas, are selected for cloning. Further screening is then conducted to determine desired characteristics, including good growth, high antibody yield, and possession of the desired antibody properties. Methods for screening, cloning, and amplifying hybridomas are well-known to those skilled in the art.

In one or more embodiments of this application, the immunized animal is a non-human animal, wherein spleen B cells are fused with a myeloma cell line from the same species as the non-human animal.

In one or more embodiments of this application, the immunized animal is a BALB/c mouse.

In one or more embodiments of this application, the hybridoma cell line is a mouse hybridoma cell line, such as the BALB/c mouse hybridoma cell line.

Fifthly, this application provides a product for detecting human IL-1R2 levels in a sample, which comprises a first antibody and/or a second antibody;

The first antibody comprises a heavy chain variable region containing HCDR1 (as shown in SEQ ID NO:2), HCDR2 (as shown in SEQ ID NO:3), and HCDR3 (as shown in SEQ ID NO:4), and a light chain variable region containing LCDR1 (as shown in SEQ ID NO:5), LCDR2 (as shown in SEQ ID NO:6), and LCDR3 (as shown in SEQ ID NO:7); and/or

The second antibody comprises a heavy chain variable region containing HCDR1 as shown in SEQ ID NO:8, HCDR2 as shown in SEQ ID NO:9, and HCDR3 as shown in SEQ ID NO:10, and a light chain variable region containing LCDR1 as shown in SEQ ID NO:11, LCDR2 as shown in SEQ ID NO:12, and LCDR3 as shown in SEQ ID NO:13;

The amino acid sequences of HCDR and LCDR are defined according to the IMGT numbering scheme.

In a sixth aspect, this application provides a method for detecting human IL-1R2 levels in a sample, the method comprising contacting the sample with a first antibody and a second antibody;

The first antibody comprises a heavy chain variable region containing HCDR1 (as shown in SEQ ID NO:2), HCDR2 (as shown in SEQ ID NO:3), and HCDR3 (as shown in SEQ ID NO:4), and a light chain variable region containing LCDR1 (as shown in SEQ ID NO:5), LCDR2 (as shown in SEQ ID NO:6), and LCDR3 (as shown in SEQ ID NO:7); and/or

The second antibody comprises a heavy chain variable region containing HCDR1 as shown in SEQ ID NO:8, HCDR2 as shown in SEQ ID NO:9, and HCDR3 as shown in SEQ ID NO:10, and a light chain variable region containing LCDR1 as shown in SEQ ID NO:11, LCDR2 as shown in SEQ ID NO:12, and LCDR3 as shown in SEQ ID NO:13;

The amino acid sequences of HCDR and LCDR are defined according to the IMGT numbering scheme.

In a seventh aspect, this application provides the use of the antibody described in the first aspect or the hybridoma cell described in the second aspect in the preparation of a product for detecting human IL-1R2 levels in a sample.

In one or more embodiments of this application, the human IL-1R2 level is an important indicator for monitoring the immune status (e.g., immunosuppression) of the subject.

In one or more embodiments of this application, the product for detecting human IL-1R2 levels in a sample is selected from: kits, test strips (e.g., colloidal gold test strips), test cards, and microfluidic detection devices.

In one or more embodiments of this application, the product for detecting human IL-1R2 levels in a sample may further include other reagents for detection, such as substrates, reference standards, diluents, washing solutions, etc.

In one or more embodiments of this application, the product for detecting human IL-1R2 levels in a sample may further include a product instruction manual.

In one or more embodiments of this application, the product for detecting human IL-1R2 levels in a sample may further include a container for mixing the sample with the antibody. Such a container may be suitable for use in detection instruments capable of detecting signals generated by the detection monoclonal antibody.

In one or more embodiments of this application, the contact is simultaneous or successive.

In one or more embodiments of this application, the level of IL-1R2 in a sample, such as the level of human IL-1R2, is detected by immunological methods.

In one or more embodiments of this application, the immunological method is selected from: enzyme-linked immunosorbent assay (ELISA), fluorescence immunoassay, chemiluminescence immunoassay, immunochromatography, immunoturbidimetric assay, immunoprecipitation, and any combination thereof.

According to this application, the level of IL-1R2 can be detected by immunological methods using antibodies that bind to IL-1R2. For example, IL-1R2 can be detected and quantified using a "sandwich" assay. In this method, typically, one antibody is immobilized on a solid surface to bind and capture IL-1R2; this antibody is therefore referred to herein as a capture antibody. Another antibody is detectably labeled, for example, with a fluorescent group, enzyme, or colored particles, and its binding to the IL-1R2-capture antibody complex indicates that IL-1R2 has been captured, with the signal intensity proportional to the concentration of IL-1R2 in the sample. Therefore, this other antibody is also referred to herein as a detection antibody or labeled antibody. Such assays may be referred to as two-site immunoassays, "sandwich" assays, or (when the antibody is a binder) "sandwich immunoassays." As is known in the art, the capture antibody and the detection antibody can be contacted with the test sample simultaneously or sequentially. A sequential method, sometimes called a "forward" method, can be accomplished by incubating the capture antibody with the sample and subsequently adding the labeled detection antibody at predetermined times. Alternatively, the labeled detection antibody can be incubated with the sample first, and then the sample can be contacted with the capture antibody (sometimes referred to as the "reverse" method). Such assays can be performed in many specific forms known to those skilled in the art, including by using various high-throughput clinical laboratory analyzers or by utilizing point-of-care testing or home testing devices.

The most commonly used enzyme immunoassay is the enzyme-linked immunosorbent assay (ELISA). ELISA is a technique that uses labeled antibodies (e.g., enzyme-linked) to detect and measure antigen concentrations. Different forms of ELISA exist, known to those skilled in the art. Standard techniques known in the art for ELISA are described in "Methods in Immunodiagnosis," 2nd edition, Rose and Bigazzi, eds. John Wiley & Sons, 1980; Campbell, et al., "Methods and Immunology," W.A. Benjamin, Inc., 1964; and Oelleric, M. (1984, J. Clin. Chem. Clin. Biochem. 22: 895-904). In a sandwich ELISA, antibodies are... An enzyme-linked antibody (e.g., an anti-human IL-1R2 antibody) is attached to a solid phase (i.e., a microtiter plate) and contacted with a biological sample containing an antigen (e.g., human IL-1R2). The solid phase is then washed to remove unbound antigen. A labeled antibody (e.g., an enzyme-linked antibody) is then bound to the antigen, forming an antibody-antigen-antibody sandwich. Examples of enzymes that can be linked to antibodies include horseradish peroxidase, alkaline phosphatase, luciferase, urease, and β-galactosidase. The enzyme-linked antibody reacts with the substrate to produce a measurable colorimetric product. This measurement can be used, for example, to deduce the concentration of human IL-1R2 present in the sample by comparing the measured value to a human IL-1R2 standard curve.

Quantitative fluorescence immunoassay is based on the principle of antigen-antibody reactions. First, known antigens or antibodies are labeled with fluorescein to create fluorescent markers. Then, these fluorescent antibodies (or antigens) are used as molecular probes to examine the corresponding antigens (or antibodies) within cells or tissues. The antigen-antibody complexes formed in cells or tissues contain fluorescein. When the specimen is observed using a fluorescence microscope, the fluorescein emits bright fluorescence when exposed to excitation light, making the cells or tissues containing the fluorescence visible. This allows for the determination of the nature and location of the antigen or antibody, as well as the quantitative determination of its concentration.

Chemiluminescence immunoassay (CLIA) combines highly sensitive chemiluminescence assays with highly specific immunoreactions for the detection and analysis of various antigens, haptens, antibodies, hormones, enzymes, fatty acids, vitamins, and drugs. It is a cutting-edge immunoassay technique developed after radioimmunoassay, enzyme immunoassay, fluorescence immunoassay, and time-resolved fluorescence immunoassay. CLI uses chemiluminescent agents to directly label antigens or antibodies for immunoassay. Commonly used chemiluminescent substances include acridinium esters (AE), which are effective luminescent labels. They emit light by initiating the luminescent reagent, producing intense, direct luminescence within one second, exhibiting rapid flash luminescence. Acridinium esters, used as labels in immunoassays, offer a simple, rapid, and catalyst-free chemical reaction. They can be used for detecting small molecule antigens using a competitive method and for large molecule antigens using a sandwich method. They exhibit low non-specific binding and low background; binding to large molecules does not reduce the amount of light produced, thus increasing sensitivity.

The principle of immunochromatography is to first immobilize specific antibodies on a certain zone of a nitrocellulose membrane. When one end of the dried nitrocellulose membrane is immersed in the sample, the sample will move forward along the membrane due to capillary action. When it moves to the area where the antibody is immobilized, the corresponding antigen in the sample will specifically bind to the antibody. If immunochromatography is used to stain the area with colloidal gold or immunoenzymes, a certain color can be displayed, thereby achieving specific immunodiagnosis.

Immunoturbidimetric assay is a dynamic method for determining antigen-antibody binding. Its basic principle is as follows: when antigen and antibody react in a specific dilution system with an appropriate ratio (generally, antibody is in excess), the resulting soluble immune complexes precipitate from the liquid phase under the action of an aggregation promoter in the dilution system, forming microparticles and causing turbidity in the reaction solution. When the antibody concentration is constant, the amount of immune complexes formed increases with the amount of antigen in the sample, and the turbidity of the reaction solution also increases accordingly. By measuring the turbidity of the reaction solution and comparing it with a series of standards, the antigen content in the sample can be calculated.

Immunoprecipitation is a method for purifying and enriching target proteins using antibody-specific reactions. After the antibody binds to the corresponding protein in the sample, it is incubated with agarose or agarose beads conjugated with Protein A/G or a secondary antibody. Centrifugation yields bead-protein A/G or secondary antibody-antibody-target protein complexes. The precipitate is washed, resuspended in electrophoresis loading buffer, and boiled. Under high temperature and the action of a reducing agent, the antigen and antibody dissociate. Centrifugation collects the supernatant, which contains the antibody, the target protein, and a small amount of other proteins.

In one or more embodiments of this application, the first antibody or the second antibody is labeled with a detectable marker. In one or more embodiments of this application, the detectable marker may be selected from any marker commonly known in the art. In one or more embodiments of this application, the detectable marker is a marker that allows for more precise quantification. Examples of the detectable marker in one or more embodiments of this application include, but are not limited to: enzymes, fluorescent molecules, radioisotopes, chemiluminescent molecules, latex particles, gold particles, detectable ligands, and any combination thereof.

In one or more embodiments of this application, the detectable marker is an enzyme or a fluorescent molecule. Methods for attaching the detectable marker to an antibody are well known in the art and include covalent and non-covalent linkages.

In one or more embodiments of this application, a detection antibody is detectably labeled by linking it to an enzyme, such that the enzyme reacts with its substrate upon contact, a reaction which can be detected, for example, by spectrophotometry, fluorescence assay, or visual inspection. Enzymes that can be used to detectably label the antibodies of this application include, but are not limited to: horseradish peroxidase, malate dehydrogenase, staphylococcal nuclease, δ-V-steroid isomerase, yeast alcohol dehydrogenase, α-glycerophosphate dehydrogenase, triose phosphate isomerase, alkaline phosphatase, luciferase, asparaginase, glucose oxidase, β-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucosylamylase, and acetylcholinesterase. In one or more embodiments of this application, the enzyme is horseradish peroxidase.

In one or more embodiments of this application, antibodies can be labeled with fluorescent molecules. When the fluorescently labeled antibody is exposed to light of an appropriate wavelength, its presence can be detected by the emitted fluorescence. Commonly used fluorescent molecules may be selected from: Cy3 and Cy5 (water-soluble fluorescent dyes of the cyanine dye family – “Cy” dyes), fluorescein isothiocyanate, rhodamine, phycocyanin, allophycocyanin, phthalaldehyde, and fluorescein.

In one or more embodiments of this application, detection may also be achieved using radiolabeled antibodies, which can then be detected by radioimmunoassay. Radioisotopes, such as ^3H , ^131I , ^35S , ^14C , and ^125I , can be detected using methods such as gamma counters or scintillation counters or by using autoradiography.

In one or more embodiments of this application, the antibody can also be detectably labeled by conjugating it to a chemiluminescent molecule, and the presence of the chemiluminescent antibody can be determined by detecting the presence of light emitted during the chemical reaction. The chemiluminescent molecule can be selected from: luminol, luciferin, isoaminobenzoyl hydrazide, imidazole, acridine salts, and oxalates.

In one or more embodiments of this application, the first antibody or the second antibody is attached to a solid support.

In one or more embodiments of this application, the solid support may be a plastic product (e.g., polystyrene board), microparticles (e.g., magnetized microparticles) or a membrane support (e.g., nitrocellulose membrane, glass cellulose membrane, or nylon membrane).

In one or more embodiments of this application, one of a first antibody and a second antibody is used as a capture antibody and immobilized on a solid-phase support for capturing IL-1R2. The other of the first antibody and the second antibody is used as a detection antibody and conjugated to a detectable marker.

In one or more embodiments of this application, the sample is selected from: plasma, blood, urine, serum, lymph, gastric juice, bile, saliva, sweat, cerebrospinal fluid, and any combination thereof. In one or more embodiments of this application, the sample may be plasma, blood, or urine.

In one or more embodiments of this application, the human IL-1R2 is either human soluble IL-1R2 or human membrane-bound protein IL-1R2.

In one or more specific embodiments of this application, the human IL-1R2 is human soluble IL-1R2.

In other respects, this application also provides a nucleic acid molecule encoding an antibody that binds to human IL-1R2 as described in this application, a vector comprising the nucleic acid molecule, and a host cell comprising the nucleic acid molecule or the vector. In some embodiments, the nucleic acid molecule is operatively linked to a regulatory nucleotide sequence that can be recognized by a host cell transformed with the vector.

It should be understood that the above detailed description is only intended to provide a clearer understanding of the contents of this application to those skilled in the art, and is not intended to limit in any way. Those skilled in the art can make various modifications and variations to the described embodiments.

Example

Example 1: Eukaryotic expression and purification of recombinant human soluble IL-1R2 protein

A codon-optimized gene of soluble IL-1R2 (Uniprot: P27930-1, amino acid sequence from 1 to 343 as shown in SEQ ID NO: 1) was synthesized. A 6×His-Tag was introduced at the C-terminus, and the gene was inserted into the pTT5 expression vector via EcoRI/HindIII restriction sites. The vector was then transfected into 293 cells for eukaryotic expression. Cell culture supernatant was collected, and the protein was purified to obtain 5 mg of soluble IL-1R2 protein. As shown in Figure 1A, Western blot analysis of the His-tag antibody in the 293 cell culture supernatant and cell pellet showed that the 293 cell culture supernatant contained the target protein IL-1R2 (as indicated by the red arrow). As shown in Figure 1B, SDS-PAGE electrophoresis of the purified protein confirmed that the purity and size of the IL-1R2 protein met expectations (as indicated by the red arrow).

Example 2: Preparation of anti-human IL-1R2 monoclonal antibody and screening of antibody pairs

2.1 Immunization of mice and cell fusion

Five 6-week-old female BALB/c mice were subcutaneously injected at multiple sites with soluble IL-1R2 protein emulsified with Freund's complete adjuvant for three immunizations. After the three immunizations, blood was collected from the tail vein, and serum was used to determine serum titers by ELISA. The mouse with the highest serum titer was intraperitoneally injected with IL-1R2 protein for a pulse immunization. The spleen of this mouse was harvested, and spleen cells were collected and fused with mouse myeloma cells SP2/0. The cells were then cultured in 96-well plates.

2.2 Hybridoma screening, establishment of stable cell lines and identification of antibody subtypes

ELISA was performed on the culture supernatant of fused cells to screen out positive wells. These wells were then retested, and subcloning was performed on the retested positive wells using limiting dilution. After subcloning, ELISA was performed again, and positive wells were selected for further subcloning using limiting dilution. Subcloning was repeated until the ELISA positive rate reached 100%. Clones with high absorbance were selected, and wells with vigorous growth were chosen for expansion culture and cryopreservation. ELISA was then performed on the culture supernatant of stable cell lines to identify antibody subtypes.

2.3 Preparation of Monoclonal Antibodies

Hybridoma cells were injected intraperitoneally into mice, and ascites fluid was collected, centrifuged, and the supernatant was filtered through a 0.22 μm filter membrane. The resulting supernatant was then purified using a protein A chromatography column (HiScreen Fibro ^™ PrismA, Cytiva). The purified antibody was concentrated using an ultrafiltration tube, and finally transferred to PBS and aseptically filtered through a 0.22 μm filter membrane to obtain the monoclonal antibody. The antibody was serially diluted with PBS buffer, and the antibody titer was detected by ELISA. As shown in Table 1, the titers of the two selected antibody strains were both greater than 1:128000.

Table 1. OD ₄₅₀ values for ELISA detection of monoclonal antibody titers

2.4 Antibody pairing verification

The antibodies 2E10 and 3D10 obtained in Section 2.3, as well as the commercially available antibodies ALS11331 (rabbit anti-human IL-1R2 monoclonal antibody, purchased from Abcepta, catalog number A-ALS11331) and R020 (rabbit anti-human IL-1R2 monoclonal antibody, purchased from Beijing Yiqiao Shenzhou Technology Co., Ltd., catalog number Cat:10111-R020), were used as capture antibodies and labeled antibodies for pairing verification, respectively. The capture antibody was diluted to 1 μg/mL, added to an ELISA plate, and coated overnight at 4°C. The plate was then washed, blocked, and incubated with the IL-1R2 antigen. A diluted detection antibody conjugated with horseradish peroxidase (HRP) was added, followed by incubation, washing, color development, and reading. The pairing results are shown in Table 2. Antibodies 2E10 (subtype IgG1) and 3D10 (subtype IgG2α) showed the highest sensitivity, reaching 10 pg/mL.

Table 2. OD ₄₅₀ values of anti-soluble IL-1R2 monoclonal antibody pairings detected by ELISA

Example 3: Affinity determination of anti-human IL-1R2 monoclonal antibodies 2E10 and 3D10

SPR experiments were performed using the Biacore 8k+ surface plasmon resonance system (Cytiva) to detect the affinity of antibodies 2E10 and 3D10 for soluble human IL-1R2 protein. Control antibodies were commercially available antibody R020 (rabbit anti-human IL-1R2 monoclonal antibody, purchased from Beijing Sinocare Medical Technology Co., Ltd., catalog number Cat:10111-R020) and AP73653 (purchased from Abcepta, catalog number AP73653). The eukaryotically expressed soluble recombinant human IL-1R2 protein was diluted to 10 μg/mL with sodium acetate at pH 5.5 and conjugated onto a CM5 chip (Cytiva). Four antibodies, 2E10, 3D10, R020, and AP73653, were diluted with PBS + 0.025% P20 to concentrations of 1.5625 nM, 3.125 nM, 6.25 nM, 12.5 nM, 25 nM, and 50 nM, respectively. These concentrations, from lowest to highest, were used as analytical streams and flowed through the chip surface. The binding time was 120 s, the dissociation time was 300 s, and the flow rate was 30 μL/min. Regeneration with glycine (pH 1.5) was performed for 30 s at a flow rate of 30 μL/min. A 1:1 binding model was fitted, and multi-cycle kinetics was used for analysis. The binding and dissociation response curves of different antibody concentrations were fitted to calculate the binding rate constant _ka , the dissociation rate constant _kd , and the affinity constant _KD . The binding and dissociation curves of 2E10 antibody with IL-1R2 protein are shown in Figure 2, 3D10 antibody with IL-1R2 protein in Figure 3, R020 antibody with IL-1R2 protein in Figure 4, and AP73653 antibody with IL-1R2 protein in Figure 5. The k_{_a}, k_{_d} , and K_{_D} values obtained from fitting the binding and dissociation curves are shown in Table 3. The K_{_D} value of 2E10 with IL-1R2 is 1.19 × 10^-10, the K_{_D} value of 3D10 with IL-1R2 is 1.35 × 10^-11, the K _{_D} value of R020 with IL-1R2 is 5.25 × 10^-10, and AP73653 antibody does not bind to active secreted IL-1R2 protein.

The affinity of antibody 2E10 for IL-1R2 is comparable to or better than that of antibody ALS11331 or R020 for IL-1R2. The affinity of antibody 3D10 for IL-1R2 is comparable to or better than that of antibody ALS11331 or R020 for IL-1R2.

Table 3. Affinity constants of monoclonal antibodies 2E10 and 3D10 with human IL-1R2

Example 4: Elevated plasma IL-R2 in sepsis patients reflects their immunosuppressive state.

4.1 Identification of Immunosuppressive Status in Patients with Sepsis

Based on the expert consensus on the diagnosis and treatment of sepsis immunosuppression (Chinese Research Hospital Association Shock and Sepsis Professional Committee, Chinese People's Liberation Army Critical Care Medicine Professional Committee, Critical Care Immunology Research Collaboration Group, etc., Expert consensus on the diagnosis and treatment of sepsis immunosuppression [J], Chinese Journal of Critical Care Medicine, 2020, 32(11):9. and Pei, Fei et al., “Expert consensus on the monitoring and treatment of sepsis-induced immunosuppression.”, Military Medical Research vol.9, 174.26 Dec.2022), the following indicators were used to identify immunosuppression in patients: absolute lymphocyte count less than 1.0× ^10⁹ /L in three consecutive days of routine blood tests, HLA-DR positivity rate of peripheral blood mononuclear cells less than 60%, age greater than 65 years, BMI less than 18.5, decreased albumin, and use of steroid drugs. Clinical test results, medical history, and medication information of sepsis patients were collected to identify their immune status (whether they were immunosuppressed).

4.2 HLA-DR positivity rate of peripheral blood mononuclear cells in sepsis patients

Collect EDTA-anticoagulated blood from sepsis patients, mix well, and take 100 μL of blood. Add 5 μL of PE-Anti-HLA-DR antibody (Biolegend) and 5 μL of PerCP-CY5.5 Anti-CD14 antibody (Biolegend), mix well, and incubate at room temperature in the dark for 30 min. Add 900 μL of 1×BD FACS lysis buffer (10× solution diluted with deionized water), mix well, and incubate in the dark for 15 min. Turn on the flow cytometer, adjust the voltage and compensation of each channel, circle CD14 ⁺ monocytes in the CD14-SSC image, collect 2000 to 4000 monocytes, and circle the proportion of PE-HLA-DR positive cells.

4.3 Detection of plasma IL-1R2 levels in sepsis patients

EDTA-anticoagulated blood was collected from patients, centrifuged at 4000 rpm for 15 min at 4℃, and the supernatant was collected. Plasma IL-1R2 levels were detected by ELISA using a kit prepared with anti-human IL-1R2 monoclonal antibodies 2E10 and 3D10.

4.4 Comparative Analysis of Immune Status

The immune status of sepsis patients was determined based on multiple clinical indicators, as shown in Table 4 (absolute lymphocyte count (ALC), HLA-DR positivity rate of monocytes, medical history, medication history, age, BMI, and albumin level). A total of 47 sepsis patients were considered immunosuppressed, and 20 were considered non-immunosuppressed. Forty-four apparent healthy individuals (non-immunosuppressed) were included as controls (as shown in Table 5). ROC curves were plotted for the immunosuppressed and non-immunosuppressed groups, as shown in Figure 6. The area under the ROC curve (AUC) was 0.9302, P < 0.0001. An AUC above 0.9 indicates high accuracy. The cut-off value of IL-1R2 was calculated to be 24 ng/mL based on the ROC curve. Figure 7 shows the distribution of plasma IL-1R2 levels in three groups. Among the 47 patients clinically diagnosed with immunosuppression, 39 had elevated IL-1R2 levels exceeding 24 ng/mL, with a sensitivity of 82.98%. Among the 64 patients clinically diagnosed as non-immunosuppressed, 58 had IL-1R2 levels below 24 ng/mL, with a specificity of 90.63%. Therefore, using the paired anti-human IL-1R2 monoclonal antibodies 2E10 and 3D10 to identify the immunosuppressive status of patients with measured plasma IL-1R2 > 24 ng/mL has a sensitivity of 82.98% and a specificity of 90.63%, which can reflect the immunosuppressive status of patients.

Table 4. Determination of Immunosuppressive Status in Patients with Sepsis

Table 5. Plasma IL-1R2 levels in epigenetic healthy individuals (non-immunosuppressed)

It is understood that although the inventions described in this application are in the specific forms described above, these inventions are not limited to the specific content described in these specific forms. It will be apparent to those skilled in the art that various equivalent changes can be made to the technical features contained in the inventions described herein without departing from the spirit of the inventions described herein, and all such changes should fall within the scope of the inventions.

Sequence information

The sequencing results of the antibody variable region in this application were provided by Universal Gene.

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Claims (10)

Methods for identifying the immune status of a subject include: Determine the level of interleukin-1 receptor 2 (IL-1R2) in samples from said subjects; Preferably, the immune state is an immunosuppressive state; more preferably, the immune state is a relative immunosuppressive state; and/or Preferably, the object is a mammal; more preferably, the object is a human; and/or Preferably, the subject suffers from systemic inflammatory response syndrome, cancer, dengue fever, or colitis; more preferably, the systemic inflammatory response syndrome is sepsis or acute respiratory distress syndrome; and/or, the cancer is gastric cancer, breast cancer, colon cancer, pancreatic cancer, ovarian cancer, prostate cancer, lung cancer, or leukemia; and/or Preferably, the sample is selected from: plasma, blood, urine, serum, lymph, gastric juice, bile, saliva, sweat, cerebrospinal fluid, and any combination thereof; more preferably, the sample is plasma, blood, or urine. Optionally, the IL-1R2 is soluble IL-1R2 or membrane-bound protein IL-1R2. The method of claim 1, wherein determining the level of IL-1R2 in a sample from the object comprises contacting the sample with a reagent for detecting the level of IL-1R2; Preferably, the reagent is a protein; more preferably, the reagent is an antibody. The method of claim 1 or 2, wherein determining the level of IL-1R2 in a sample from the object comprises contacting the sample with a first antibody and a second antibody, optionally, the contact being simultaneous or sequential; The first antibody comprises a heavy chain variable region containing HCDR1 (as shown in SEQ ID NO:2), HCDR2 (as shown in SEQ ID NO:3), and HCDR3 (as shown in SEQ ID NO:4), and a light chain variable region containing LCDR1 (as shown in SEQ ID NO:5), LCDR2 (as shown in SEQ ID NO:6), and LCDR3 (as shown in SEQ ID NO:7); and/or The second antibody comprises a heavy chain variable region containing HCDR1 as shown in SEQ ID NO:8, HCDR2 as shown in SEQ ID NO:9, and HCDR3 as shown in SEQ ID NO:10, and a light chain variable region containing LCDR1 as shown in SEQ ID NO:11, LCDR2 as shown in SEQ ID NO:12, and LCDR3 as shown in SEQ ID NO:13; The amino acid sequences of HCDR and LCDR are defined according to the IMGT numbering scheme. Preferably, the first antibody or the second antibody is labeled with a detectable marker selected from: enzymes, fluorescent molecules, radioactive isotopes, chemiluminescent molecules, latex particles, gold particles, detectable ligands, and any combination thereof; and/or Preferably, the first antibody or the second antibody is attached to a solid support; and/or The level of IL-1R2 in a sample is detected by an immunological method; preferably, the immunological method is selected from: enzyme-linked immunosorbent assay (ELISA), quantitative fluorescence immunoassay, chemiluminescent immunoassay, immunochromatography, immunoturbidimetry, immunoprecipitation, and any combination thereof; more preferably, ELISA; and/or Preferably, the IL-1R2 level is compared with a cut-off value for the IL-1R2 level used to identify the subject's immune status; preferably, the cut-off value for the IL-1R2 level used to identify the subject's immune status is any value within the range of 19-29 ng/mL; more preferably, the cut-off value for the IL-1R2 level used to identify the subject's immune status is 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29 ng/mL; most preferably, the cut-off value for the IL-1R2 level used to identify the subject's immune status is 24 ng/mL; and/or Preferably, the immune status of the subject whose IL-1R2 level is greater than the cut-off value is identified as an immunosuppressed state; and/or, the immune status of the subject whose IL-1R2 level is less than the cut-off value is identified as a non-immunosuppressed state; Optionally, the immune status of the subject also needs to be identified in conjunction with at least one of the following clinical indicators: absolute lymphocyte count, HLA-DR positivity rate of monocytes, medical history, medication history, age, body mass index, and albumin level. Use of reagents for detecting IL-1R2 levels in samples from subjects in the preparation of products for identifying the immune status of said subjects; Preferably, the immune state is an immunosuppressive state; more preferably, the immune state is a relative immunosuppressive state; and/or Preferably, the object is a mammal; more preferably, the object is a human; and/or Preferably, the subject suffers from systemic inflammatory response syndrome, cancer, dengue fever, or colitis; more preferably, the systemic inflammatory response syndrome is sepsis or acute respiratory distress syndrome; and/or, the cancer is gastric cancer, breast cancer, colon cancer, pancreatic cancer, ovarian cancer, prostate cancer, lung cancer, or leukemia; and/or Preferably, the sample is selected from: plasma, blood, urine, serum, lymph, gastric juice, bile, saliva, sweat, cerebrospinal fluid, and any combination thereof; more preferably, the sample is plasma, blood, or urine; and/or Preferably, the reagent is a protein; more preferably, the reagent is an antibody; and/or Preferably, the product for identifying the immune status of the subject is selected from: reagent kits, test strips, test cards, and microfluidic detection devices; Optionally, the IL-1R2 is soluble IL-1R2 or membrane-bound protein IL-1R2. The use as described in claim 4, wherein the antibody comprises a heavy chain variable region containing HCDR1, HCDR2, and HCDR3 and a light chain variable region containing LCDR1, LCDR2, and LCDR3, wherein The amino acid sequence of HCDR1 is shown in SEQ ID NO:2, the amino acid sequence of HCDR2 is shown in SEQ ID NO:3, the amino acid sequence of HCDR3 is shown in SEQ ID NO:4, the amino acid sequence of LCDR1 is shown in SEQ ID NO:5, the amino acid sequence of LCDR2 is shown in SEQ ID NO:6, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:7; or The amino acid sequence of HCDR1 is shown in SEQ ID NO:8, the amino acid sequence of HCDR2 is shown in SEQ ID NO:9, the amino acid sequence of HCDR3 is shown in SEQ ID NO:10, the amino acid sequence of LCDR1 is shown in SEQ ID NO:11, the amino acid sequence of LCDR2 is shown in SEQ ID NO:12, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:13. The amino acid sequences of HCDR and LCDR are defined according to the IMGT numbering scheme. The use as described in claim 4 or 5, wherein the product for identifying the immune status of the subject comprises a first antibody and/or a second antibody; The first antibody comprises a heavy chain variable region containing HCDR1 (as shown in SEQ ID NO:2), HCDR2 (as shown in SEQ ID NO:3), and HCDR3 (as shown in SEQ ID NO:4), and a light chain variable region containing LCDR1 (as shown in SEQ ID NO:5), LCDR2 (as shown in SEQ ID NO:6), and LCDR3 (as shown in SEQ ID NO:7); and/or The second antibody comprises a heavy chain variable region containing HCDR1 as shown in SEQ ID NO:8, HCDR2 as shown in SEQ ID NO:9, and HCDR3 as shown in SEQ ID NO:10, and a light chain variable region containing LCDR1 as shown in SEQ ID NO:11, LCDR2 as shown in SEQ ID NO:12, and LCDR3 as shown in SEQ ID NO:13; The amino acid sequences of HCDR and LCDR are defined according to the IMGT numbering scheme. Preferably, the first antibody or the second antibody is labeled with a detectable marker selected from: enzymes, fluorescent molecules, radioactive isotopes, chemiluminescent molecules, latex particles, gold particles, detectable ligands, and any combination thereof; and/or Preferably, the first antibody or the second antibody is attached to a solid support; and/or Preferably, the cut-off value for the IL-1R2 level used to identify the immune status of the subject is any value within the range of 19-29 ng/mL; more preferably, the cut-off value for the IL-1R2 level used to identify the immune status of the subject is 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29 ng/mL; most preferably, the cut-off value for the IL-1R2 level used to identify the immune status of the subject is 24 ng/mL; and/or Preferably, the immune status of the subject whose IL-1R2 level is greater than the cut-off value is identified as an immunosuppressed state; and/or, the immune status of the subject whose IL-1R2 level is less than the cut-off value is identified as a non-immunosuppressed state; Optionally, the immune status of the subject (e.g., immunosuppression) also needs to be identified in conjunction with at least one of the following clinical indicators: absolute lymphocyte count, HLA-DR positivity rate of monocytes, medical history, medication history, age, body mass index, and albumin level. The antibody that binds to human IL-1R2 contains heavy chain variable regions containing HCDR1, HCDR2, and HCDR3, and light chain variable regions containing LCDR1, LCDR2, and LCDR3. The amino acid sequence of HCDR1 is shown in SEQ ID NO:2, the amino acid sequence of HCDR2 is shown in SEQ ID NO:3, the amino acid sequence of HCDR3 is shown in SEQ ID NO:4, the amino acid sequence of LCDR1 is shown in SEQ ID NO:5, the amino acid sequence of LCDR2 is shown in SEQ ID NO:6, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:7; or The amino acid sequence of HCDR1 is shown in SEQ ID NO:8, the amino acid sequence of HCDR2 is shown in SEQ ID NO:9, the amino acid sequence of HCDR3 is shown in SEQ ID NO:10, the amino acid sequence of LCDR1 is shown in SEQ ID NO:11, the amino acid sequence of LCDR2 is shown in SEQ ID NO:12, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:13. The amino acid sequences of HCDR and LCDR are defined according to the IMGT numbering scheme. Preferably, the amino acid sequence of the heavy chain variable region of the antibody has at least 90% identity with SEQ ID NO: 14 or 15; and/or The amino acid sequence of the variable region of the light chain of the antibody has at least 90% identity with SEQ ID NO: 16 or 17; More preferably, the amino acid sequence of the heavy chain variable region of the antibody is as shown in SEQ ID NO: 14 or 15; and/or The amino acid sequence of the variable region of the light chain of the antibody is shown in SEQ ID NO: 16 or 17; Most preferably, the amino acid sequence of the heavy chain variable region of the antibody is as shown in SEQ ID NO:14, and the amino acid sequence of the light chain variable region of the antibody is as shown in SEQ ID NO:16; or The amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO:15, and the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO:17. Optionally, the human IL-1R2 is either human soluble IL-1R2 or human membrane-bound protein IL-1R2. The antibody as claimed in claim 7, wherein The antibody is a whole antibody, Fab fragment, Fab' fragment, F(ab') ₂ fragment, Fv fragment, single-chain Fv fragment (scFv), Fd fragment, single-domain antibody, or dAb fragment; and/or The antibody is a monoclonal antibody, preferably a murine monoclonal antibody; and/or The antibody comprises a heavy chain constant region selected from IgG1, IgG2, or IgG4 isotypes; preferably, the antibody comprises a heavy chain constant region of IgG1 or IgG2 isotypes; and/or The antibody contains a light chain constant region selected from the κ or λ subtype; and/or The antibody binds to human IL-1R2 with the amino acid sequence shown in SEQ ID NO:1; and/or The antibody was produced by a hybridoma cell line with accession number CCTCC NO:C2024249 or CCTCC NO:C2024250. Hybridoma cells that produce antibodies that bind to human IL-1R2 are deposited with accession numbers CCTCC NO: C2024249 or CCTCC NO: C2024250. Optionally, the human IL-1R2 is either human soluble IL-1R2 or human membrane-bound protein IL-1R2. Use of the antibody of claim 7 or 8, or the hybridoma cell of claim 9, in the preparation of a product for detecting human IL-1R2 levels in a sample; Preferably, the product for detecting human IL-1R2 levels in a sample is selected from: kits, test strips, test cards, and microfluidic detection devices; and/or Preferably, the sample is selected from: plasma, blood, urine, serum, lymph, gastric juice, bile, saliva, sweat, cerebrospinal fluid, and any combination thereof; more preferably, the sample is plasma, blood, or urine. Optionally, the human IL-1R2 is either human soluble IL-1R2 or human membrane-bound protein IL-1R2.