CN119667163A

CN119667163A - Application of IL-1R2 in identifying the immune status of subjects

Info

Publication number: CN119667163A
Application number: CN202411462291.6A
Authority: CN
Inventors: 魏海明; 周永刚; 傅斌清; 陈剑; 徐秀秀
Original assignee: University of Science and Technology of China USTC
Current assignee: University of Science and Technology of China USTC
Priority date: 2024-10-18
Filing date: 2024-10-18
Publication date: 2025-03-21

Abstract

本申请提供了鉴定对象免疫状态的方法，和用于检测来自对象的样品中IL‑1R2水平的试剂在制备用于鉴定所述对象免疫状态的产品中的用途。本申请还提供了结合人IL‑1R2的抗体及其用途，以及产生结合人IL‑1R2的抗体的杂交瘤细胞及其用途。The present application provides a method for identifying the immune status of a subject, and the use of a reagent for detecting the level of IL-1R2 in a sample from a subject in the preparation of a product for identifying the immune status of the subject. The present application also provides antibodies that bind to human IL-1R2 and their uses, as well as hybridoma cells that produce antibodies that bind to human IL-1R2 and their uses.

Description

Use of IL-1R2 in identifying immune status of a subject

Technical Field

The present application relates to the field of biological detection and antibody preparation, in particular, the present application provides a method for identifying an immune status of a subject, the use of an agent for detecting the level of IL-1R2 in a sample from a subject in the manufacture of a product for identifying said immune status of a subject, antibodies binding to human IL-1R2 and uses thereof, and hybridoma cells producing antibodies binding to human IL-1R2 and uses thereof.

Background

Systemic inflammatory response syndrome caused by infection can lead to sepsis. Sepsis is a dysfunction of life-threatening organs caused by deregulation of the host's response to infection. Sepsis has high incidence and mortality rate, and is still the main cause of death in hospital and outside infection at present. A recent statistical report emphasizes nearly 5000 thousands of sepsis patients worldwide each year, with the age of onset covering all age groups. Sepsis deaths in 2017 account for nearly 20% of all deaths worldwide. In addition, patients who have developed sepsis in the past have a higher risk of readmission and death. Sepsis is a serious threat to human health and also creates a significant socioeconomic burden.

Immune dysfunction is closely related to the occurrence and progression of sepsis, and patients may manifest as both excessive inflammatory responses and immunosuppression. The former may cause early tissue damage and organ dysfunction, while the latter, when severe and persistent, may further induce a number of fatal complications, thereby significantly increasing mortality in late patients with sepsis. In recent years, with the increasing progress of organ support technology, the mortality rate in the pro-inflammatory reaction stage has decreased, but the mortality rate in the immunosuppressive stage has remained high, so the focus of research on sepsis immune dysfunction has been changed from excessive inflammation to immunosuppression. However, the exact mechanism of immune dysfunction of the organism caused by serious injury and the action of the mechanism in sepsis are not known for a long time, and clinically effective immune monitoring and conditioning measures are lacking. Dynamic and accurate assessment of immune status is a prerequisite for timely identification of immune dysfunction in sepsis patients and determination of the timing of immunomodulation therapy. However, due to the lack of knowledge 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.

The current lymphocyte count and monocyte human leukocyte antigen DR (mHLA-DR) are widely used to assess immune function changes in sepsis patients, as indicated by the specialists in the latest published "sepsis immunosuppression diagnosis expert consensus". Although lymphocyte counts are readily available, their specificity is relatively low due to a number of confounding factors. Meanwhile, mHLA-DR detection requires a flow cytometer, equipment is complex, detection cost is high, and definition of an early warning threshold value is not yet determined. In recent years, researchers have explored immunomonitoring using a multi-genetics approach aimed at finding new sepsis-related cell subsets and biomarkers, but their transformation significance and value need to be further verified by large-scale clinical trials.

Therefore, it is of great biological and medical significance to develop new methods and products for identifying immune status in patients with sepsis or even systemic inflammatory response syndrome.

Disclosure of Invention

In a first aspect, the application provides a method of identifying an immune status of a subject comprising:

determining the level of interleukin 1 receptor 2 (IL-1R 2) in a sample from the subject.

In one or more embodiments of the application, the determining the level of IL-1R2 in a sample from the subject comprises contacting the sample with an agent that detects the level of IL-1R 2.

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

Wherein the first antibody comprises a heavy chain variable region comprising HCDR1 having an amino acid sequence shown as SEQ ID NO. 2, HCDR2 having an amino acid sequence shown as SEQ ID NO. 3 and HCDR3 having an amino acid sequence shown as SEQ ID NO. 4, and a light chain variable region comprising LCDR1 having an amino acid sequence shown as SEQ ID NO. 5, LCDR2 having an amino acid sequence shown as SEQ ID NO. 6 and LCDR3 having an amino acid sequence shown as SEQ ID NO. 7, and/or

The second antibody comprises a heavy chain variable region containing HCDR1 with an amino acid sequence shown as SEQ ID NO. 8, HCDR2 with an amino acid sequence shown as SEQ ID NO. 9 and HCDR3 with an amino acid sequence shown as SEQ ID NO. 10, and a light chain variable region containing LCDR1 with an amino acid sequence shown as SEQ ID NO. 11, LCDR2 with an amino acid sequence shown as SEQ ID NO. 12 and LCDR3 with an amino acid sequence shown as SEQ ID NO. 13;

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

In one or more embodiments of the application, the contacting is simultaneous or sequential.

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

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

In a second aspect, the application provides the use of an agent for detecting the level of IL-1R2 in a sample from a subject in the manufacture of a product for identifying the immune status of said subject.

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

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

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

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

In one or more embodiments of the application, the product for identifying the immune status of the subject is selected from the group consisting of a kit, a test strip, a test card, and a microfluidic test device.

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

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

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

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

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

Wherein the first antibody comprises a heavy chain variable region comprising HCDR1 with an amino acid sequence shown as SEQ ID NO. 2, HCDR2 with an amino acid sequence shown as SEQ ID NO. 3, and HCDR3 with an amino acid sequence shown as SEQ ID NO. 4, and a light chain variable region comprising LCDR1 with an amino acid sequence shown as SEQ ID NO. 5, LCDR2 with an amino acid sequence shown as SEQ ID NO. 6, and LCDR3 with an amino acid sequence shown as SEQ ID NO. 7, and/or

In one or more embodiments of the application, the cut-off value of 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 the application, the immune status of the subject with the IL-1R2 level greater than the cut-off value is identified as an immunosuppressive status. In one or more embodiments of the application, the immune status of the subject in which the IL-1R2 level is less than the cut-off value is identified as a non-immunosuppressive status.

In one or more embodiments of the application, the immune status (e.g., immunosuppressive status) of the subject is also identified in combination with at least one clinical indicator selected from lymphocyte absolute numbers (Absolute Lymphocyte Count, ALC), monocyte HLA-DR positive rate, disease history, medication history, age, body mass index, and albumin level.

In a third aspect, the application provides an antibody that binds human IL-1R2 comprising a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 and a light chain variable region comprising LCDR1, LCDR2, and LCDR3, wherein

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

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

In one or more embodiments of the application, the antibody is a whole antibody, a Fab fragment, a Fab 'fragment, a 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 monoclonal antibody, and/or

The antibody comprises a heavy chain constant region selected from the group consisting of an IgG1 subtype, an IgG2 subtype, or an IgG4 subtype, and/or

The antibody comprises a light chain constant region selected from the kappa subtype or lambda subtype, and/or

The antibody is combined with human IL-1R2 with an amino acid sequence shown as SEQ ID NO. 1.

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

In a fourth aspect, the application provides hybridoma cells producing antibodies that bind human IL-1R2, which are deposited with the accession number CCTCC NO: C2024249 or CCTCC NO: C2024250.

In a fifth aspect, the application provides a product for detecting the level of human IL-1R2 in a sample comprising a first antibody and/or a second antibody;

In a sixth aspect, the application provides a method for detecting the level of human IL-1R2 in a sample, the method comprising contacting a first antibody and a second antibody with the sample, optionally, the contacting being simultaneous or sequential;

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

In a seventh aspect, the application provides the use of an antibody according to the third aspect, or a hybridoma cell according to the fourth aspect, for the preparation of a product for detecting the level of human IL-1R2 in a sample.

In one or more embodiments of the application, the product for detecting the level of human IL-1R2 in a sample is selected from the group consisting of a kit, a test strip, a test card, and a microfluidic detection device.

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

In one or more embodiments of the application, the first antibody or the second antibody is labeled with a detectable label selected from the group consisting of an enzyme, a fluorescent molecule, a radioisotope, a chemiluminescent molecule, latex particles, gold particles, a detectable ligand, and any combination thereof.

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

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

Drawings

FIG. 1 shows the results of correct expression and purity identification of human soluble IL-1R2 recombinant proteins. Wherein A shows the result of verifying correct expression of soluble IL-1R2 in cell culture supernatant using His tag antibody Western Blot, and B shows the result of verifying purity of human soluble IL-1R2 recombinant protein using SDS-PAGE.

FIG. 2 shows SPR detection of binding and dissociation curves of different concentrations of monoclonal antibody 2E10 to IL-1R2 protein.

FIG. 3 shows SPR detection of binding and dissociation curves of different concentrations of monoclonal antibody 3D10 to IL-1R2 protein.

FIG. 4 shows the results of a ROC curve analysis for the detection of human plasma IL-1R2 levels using monoclonal antibody 2E10 paired with 3D10 to identify immunosuppression.

FIG. 5 shows plasma IL-1R2 levels results for apparently healthy, non-immunosuppressive sepsis patients and immunosuppressive sepsis patients detected using monoclonal antibody 2E10 paired with 3D 10.

Preservation description

1. Cell name hybridoma cell line (Hybridoma cell line) ZIL R-2E10

China center for type culture Collection

CCTCC for preservation organization

Address of university of Wuhan in China (No. 299 of Wuhan City of Hubei province, eight paths of Wuhan)

The preservation date is 2024, 08 and 22 days

Registration number of preservation center CCTCC NO: C2024249

2. Cell name hybridoma cell line (Hybridoma cell line) ZIL R-3D10

China center for type culture Collection

CCTCC for preservation organization

The preservation date is 2024, 08 and 22 days

Registration number of preservation center CCTCC NO: C2024250

DESCRIPTION OF THE SEQUENCES

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

SEQ ID NOS.2-4 show the amino acid sequences of HCDR1, HCDR2 and HCDR3, respectively, of monoclonal antibody 2E10 directed against soluble human IL-1R 2.

SEQ ID NOS 5-7 show the amino acid sequences of LCDR1, LCDR2 and LCDR3, respectively, of monoclonal antibody 2E10 directed against soluble human IL-1R 2.

SEQ ID NOS 8-10 show the amino acid sequences of HCDR1, HCDR2 and HCDR3, respectively, of monoclonal antibody 3D10 directed against soluble human IL-1R 2.

SEQ ID NOS.11-13 show the amino acid sequences of LCDR1, LCDR2 and LCDR3, respectively, of monoclonal antibody 3D10 directed against soluble human IL-1R 2.

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

SEQ ID NO. 15 shows the amino acid sequence of the heavy chain variable region of monoclonal antibody 3D10 against soluble human IL-1R 2.

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

SEQ ID NO. 17 shows the amino acid sequence of the light chain variable region of monoclonal antibody 3D10 against soluble human IL-1R 2.

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

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

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

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

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

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

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

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

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

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

SEQ ID NO. 28 shows a nucleotide sequence encoding the heavy chain variable region of monoclonal antibody 3D10 against soluble human IL-1R 2.

SEQ ID NO. 29 shows the amino acid sequence of FR1 of the heavy chain variable region of monoclonal antibody 3D10 against soluble human IL-1R 2.

SEQ ID NO. 30 shows the amino acid sequence of FR2 of the heavy chain variable region of monoclonal antibody 3D10 against soluble human IL-1R 2.

SEQ ID NO. 31 shows the amino acid sequence of FR3 of the heavy chain variable region of monoclonal antibody 3D10 against soluble human IL-1R 2.

SEQ ID NO. 32 shows the amino acid sequence of FR4 of the heavy chain variable region of monoclonal antibody 3D10 against soluble human IL-1R 2.

SEQ ID NO. 33 shows a nucleotide sequence encoding the light chain variable region of monoclonal antibody 3D10 against soluble human IL-1R 2.

SEQ ID NO. 34 shows the amino acid sequence of FR1 encoding the light chain variable region of monoclonal antibody 3D10 against soluble human IL-1R 2.

SEQ ID NO. 35 shows the amino acid sequence of FR2 encoding the light chain variable region of monoclonal antibody 3D10 against soluble human IL-1R 2.

SEQ ID NO. 36 shows the amino acid sequence of FR3 encoding the light chain variable region of monoclonal antibody 3D10 against soluble human IL-1R 2.

SEQ ID NO. 37 shows the amino acid sequence of FR4 encoding the light chain variable region of monoclonal antibody 3D10 against soluble human IL-1R 2.

Detailed Description

Interleukin-1 (IL-1) is a cytokine with potent pro-inflammatory and amplified immune response, produced mainly by macrophages during the defensive response. IL-1 is one of several pro-inflammatory cytokines produced during infection, sepsis and Systemic Inflammatory Response Syndrome (SIRS) and is used to initiate the host inflammatory response and integrate nonspecific immunity. IL-1 is an important mediator involved in SIRS pathogenesis. The leading cytokines in the early stages of SIRS are IL-1 and TNF- α, which induce the release of other pro-inflammatory cytokines (IL-6, IL-8, IFN-gamma) and trigger the coagulation cascade. IL-1 may also induce TNF- α production.

Interlukin-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 in chromosome 2q12 band, and, like other IL-1 receptor family members, consists of an extracellular portion containing three glycosylated immunoglobulin (Ig) -like domains, and has 28% amino acid homology with the extracellular portion of IL-1R 1. However, compared to other members of this family, downstream signaling cannot be initiated due to their shorter cytoplasmic structure and lack of Toll/IL-1 receptor (TIR) domains. IL-1R2 proteins exist in both cell membrane-bound and soluble forms (sIL-1R 2).

IL-1 forms an IL-1/IL-1R1/IL-1RAP complex with IL-1R1 under the action of IL-1RAP, activates IL-1 downstream signal transduction pathways, and exerts pro-inflammatory biological functions. The membrane-bound protein IL-1R2 competitively captures IL-1 at the cell surface, whereas sIL-1R2 binds IL-1 in the extracellular microenvironment, preventing both IL-1 binding to IL-1R1 at the cell surface and activation of cell activation, is one of the main mechanisms of inhibiting IL-1 activity and is defined as a decoy receptor.

IL-1R2 has a high affinity for IL-1β and a low affinity for interleukin 1 receptor antagonist (IL-1 Ra), and is therefore able to exert its effect of inhibiting IL-1 activity and is only partially interfered with by IL-1 Ra. Compared to membrane-bound protein IL-1R2, sIL-1R2 has a different ligand binding capacity. The binding capacity of sIL-1R2 to IL-1 beta is equivalent to that of a membrane receptor, but the binding capacity to IL-1 alpha is superior to that of the membrane receptor. More importantly, sIL-1R2 did not bind to IL-1Ra, indicating that sIL-1R2 is a better IL-1 inhibitor than membrane-bound protein IL-1R2 because its effect is not interfered with by IL-1 Ra. In addition, sIL-1R2 can also be combined with IL-1 beta precursor Pro-IL-1 beta to prevent Pro-IL-1 beta from being processed and converted into mature IL-1 beta by protease in cells, thereby exerting negative regulation and control on inflammatory response. In summary, soluble IL-1R2 functions to suppress immune responses by inhibiting the pro-inflammatory effects of IL-1.

The inventors of the present application found that in critically ill patients suffering from systemic inflammatory response syndrome such as sepsis, the plasma levels of soluble IL-1R2 are significantly elevated and that plasma IL-1R2 levels are positively correlated with the severity of the disease. The IL-1R2 elevation reflects IL-1 imbalance, indicating a SIRS transition to the compensatory anti-inflammatory response syndrome (CARS). In addition, IL-1R2 can be up-regulated by stimulation with anti-inflammatory factors such as IL-4, IL-10, glucocorticoid, etc. IL-1R2 plays a major negative regulatory role in inflammatory responses in different cell types. In conclusion, the rise of the soluble IL-1R2 indicates that the patient is in a relative immunosuppression state, and can be used as one of auxiliary detection indexes of the immunosuppression state of patients with sepsis and even systemic inflammatory response syndrome.

Furthermore, in order to achieve the determination of the level of human IL-1R2 (in particular human soluble IL-1R 2), the inventors of the present application have also obtained novel antibodies binding to human IL-1R2 (in particular human soluble IL-1R 2) by antibody engineering techniques.

In various aspects of the application, methods are provided for identifying an immune status of a subject, the use of an agent for detecting the level of IL-1R2 in a sample from a subject in the manufacture of a product for identifying the immune status of the subject, antibodies that bind to human IL-1R2 and uses thereof, hybridoma cells that produce antibodies that bind to human IL-1R2 and uses thereof, products for detecting the level of human IL-1R2 in a sample, and methods for detecting the level of human IL-1R2 in a sample.

The practice of the present application will employ, unless otherwise indicated, molecular biology, microbiology, cell biology, biochemistry and immunology techniques which are conventional in the art.

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

Unless otherwise indicated, the experimental reagents were all commercially available products.

Definition of the definition

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

As used herein, the term "immunosuppressive state" refers to a state in which an individual's immune system function is impaired or inhibited, resulting in a reduced ability to respond to an antigen (e.g., pathogen, tumor cell, or vaccine). Such conditions may make an individual more susceptible to infection, develop certain diseases, or be difficult to effectively combat.

As used herein, the term "relative immunosuppressive state" refers to an individual whose immune system function is partially impaired or inhibited, but such inhibition is not absolute or complete, under certain specific conditions or relative to a healthy standard. Such conditions may make an individual more susceptible to infection or other immune related problems in certain situations (e.g., long term hospital patients), but may still maintain a degree of immune response in other situations.

As used herein, the term "antibody" refers to immunoglobulin molecules comprising four polypeptide chains, two heavy (H) and two light (L) chains interconnected by disulfide bonds, as well as multimers thereof (e.g., igM). Each heavy chain comprises a heavy chain variable region (abbreviated VH) and a heavy chain constant region (abbreviated CH). The heavy chain constant region comprises three domains, CH1, CH2 and CH3. Each light chain comprises a light chain variable region (abbreviated VL) and a light chain constant region (abbreviated CL). The light chain constant region comprises a domain (CL 1). VH and VL regions can be further subdivided into regions of hypervariability, termed Complementarity Determining Regions (CDRs), interspersed with regions that are conserved, termed Framework Regions (FR). In some embodiments, from N-terminal to C-terminal, the light and heavy chain variable regions each comprise FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

Antibodies specifically bind to an immunoglobulin molecule of a target via at least one antigen recognition site located in a variable region of the immunoglobulin molecule. Targets include, but are not limited to, carbohydrates, polynucleotides, lipids, polypeptides, and the like. As used herein, "antibody" includes not only intact (i.e., full length) antibodies, but also antigen binding fragments thereof, variants thereof, fusion proteins comprising antibody portions, humanized antibodies, chimeric antibodies, diabodies, linear antibodies, single chain antibodies, multispecific antibodies (e.g., bispecific antibodies), and any other modified configuration of immunoglobulin molecules comprising antigen-recognition sites of the desired specificity, including glycosylated variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies.

The full length antibody may be any kind of antibody, such as IgD, igE, igG, igA or IgM (or subclasses thereof as described above), but the antibody need not be of any particular class. Immunoglobulins can be assigned to different classes depending on the antibody amino acid sequence of the heavy chain constant region. Typically, immunoglobulins have five main classes IgA, igD, igE, igG and IgM, and several of these classes can be further divided into subclasses (isotypes), such as IgG1, igG2, igG3, igG4, igA1, and IgA2. Heavy chains corresponding to different immunoglobulin classes are referred to as α, δ, ε, γ, and μ, respectively. 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 an intact antibody molecule responsible for binding an antigen. The antigen binding fragment 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 intact antibody molecules using any suitable standard technique, including proteolytic digestion or recombinant genetic engineering techniques, and the like. 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 minimal recognition units (e.g., isolated CDRs) consisting of amino acid residues that mimic the hypervariable regions of an antibody. Antigen binding fragments may also include other engineered molecules such as diabodies, triabodies, tetrabodies, minibodies, and the like. For example, fd fragments as described herein refer to antibody fragments consisting of the VH and CH1 regions, fv fragments consisting of the VL and VH regions in a single arm of the antibody, dAb fragments (Ward et al, nature 1989; 341:544-546) consisting of the VH region.

It is well known to those skilled in the art that complementarity determining regions (CDRs, typically CDR1, CDR2 and CDR 3) are regions of the variable region that have the greatest influence on the affinity and specificity of an antibody. The CDR amino acid sequences of VH or VL are defined by common definition means, such as IMGT numbering scheme definition, C h o t h i a numbering scheme definition and K a b a t numbering scheme definition. See, for example, K a b a t , "S e q u e n c e s o f P r o t e i n s o f Immunological Interest",National Institutes of Health , Bethesda,Md . (1991) 7;A1-L a z i k a n i , et al., J .M o l .B i o l .2 7 3 : 9 2 7-9 4 8 ( 1 9 9 7 ) ; and MarTin, et al, proc. Natl. Acad. Sci. USA 86:9268-9272 (1989). For a given antibody variable region amino acid sequence, the CDR amino acid sequences in VH and VL amino acid sequences may be determined according to IMGT numbering scheme definition, chothia numbering scheme definition, or Kabat numbering scheme definition.

For a given antibody variable region amino acid sequence, the mid CDR amino acid sequence of the variable region amino acid sequence can be analyzed in a number of ways, for example, as determined using on-line 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 binding of an antibody to an epitope of an antigen, e.g., the ability of an antibody to bind to a specific antigen with an affinity that is at least twice greater than its affinity for a non-specific antigen. However, it will be appreciated that antibodies can specifically bind to two or more antigens associated with their sequences.

As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies that make up the population are identical except for the naturally occurring mutations that may be present in a small number of individuals.

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

As used herein, the term "reactivity of a monoclonal antibody" refers to the ability of the 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 by 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 introduction of gaps, if desired, to achieve a maximum percentage of homology. Methods and computer programs for alignment are well known in the art. "at least 90% homology" as used herein refers to any value of 90% to 100% homology, 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 the test result is above or below clinical or analytical. If the detection result is lower than the cut-off value, the detection result is negative, and if the detection result is higher than the cut-off value, the detection result is positive. In some embodiments of the application, when the human plasma IL-1R2 level is above the cut-off value, it is indicated that the patient is in an immunosuppressive state (e.g., a relatively immunosuppressive state), and when the human plasma IL-1R2 level is below the cut-off value, it is indicated that the patient is in a non-immunosuppressive state.

In one or more embodiments of the application, the method of identifying an immune state of a subject is a method of aiding in the identification of an immune state (e.g., an immunosuppressive state) of a subject.

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

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

In one or more embodiments of the application, the subject has systemic inflammatory response syndrome, cancer, dengue fever, or colitis. In one or more embodiments of the application, the systemic inflammatory response syndrome is sepsis or acute respiratory distress syndrome. In one or more embodiments of the 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 the application, the agent is a protein, such as an antibody. In one or more embodiments of the application, the antibody can be any antibody capable of binding to IL-1R2, such as a commercially available antibody that binds to IL-1R 2.

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

In one or more embodiments of the application, the product for identifying the immune status of the subject is selected from the group consisting of a kit, a test strip (e.g., a colloidal gold test strip), a test card, and a microfluidic test device.

In one or more embodiments of the application, the product for identifying the immune status of the subject may also include other reagents for detection, such as substrates, reference standards, dilutions, washing solutions, and the like.

In one or more embodiments of the application, the product for identifying the immune status of the subject may further comprise product instructions for use.

In one or more embodiments of the application, the product for identifying the immune status of the subject may further comprise a container for mixing the sample with the antibody. Such containers may be suitable for use in a detection instrument capable of detecting a signal generated by a detection monoclonal antibody.

In one or more embodiments of the application, the product for identifying an immune status of a subject is used to aid in identifying an immune status, such as an immunosuppressive status, of a subject.

In one or more embodiments of the application, the cut-off value of 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、26.3、26.4、26.5、26.6、26.7、26.8、26.9、27、27.1、27.2、27.3、27.4、27.5、27.6、27.7、27.8、27.9、28、28.1、28.2、28.3、28.4、28.5、28.6、28.7、28.8、28.9 or 29 ng/mL. In one or more embodiments of the application, the cut-off value of 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. In one or more specific embodiments of the application, the cut-off value of the IL-1R2 level used to identify the immune status of the subject is 24 ng/mL.

In one or more specific embodiments of the application, the immune status of the subject in which the IL-1R2 level is greater than the cut-off value is identified as an immunosuppressive status. In one or more embodiments of the application, the immune status of the subject in which the IL-1R2 level is less than the cut-off value is identified as a non-immunosuppressive status.

In one or more embodiments of the application, the immune status (e.g., immunosuppressive status) of the subject is also identified in combination with at least one clinical indicator selected from lymphocyte absolute number, monocyte HLA-DR positive rate, history of disease, history of medication, age, body mass index, and albumin level.

In one or more embodiments of the application, the immune status (e.g., immunosuppressive status) of the subject also needs to be identified by binding to lymphocyte absolute numbers and monocyte HLA-DR positive rates. In one or more embodiments of the application, the immune status of a patient in which the absolute number of lymphocytes in a three-day continuous blood routine report is less than 1.0X10 ⁹/L, and/or the HLA-DR positive rate of peripheral blood mononuclear cells is less than 60% is identified as an immunosuppressive status, such as a relative immunosuppressive status. In one or more embodiments of the application, whether the patient is in an immunosuppressive state requires a physician to combine clinical criteria (e.g., lymphocyte absolute number and/or factors such as HLA-DR positive rate of peripheral blood mononuclear cells) for identification.

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

In one or more embodiments of the application, the amino acid sequence of the light chain variable region of the antibody has at least 90% identity, e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity, to SEQ ID No. 16 or 17.

In one or more embodiments of the 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 more identity to 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 more identity to SEQ ID NO. 16 or 17.

In one or more embodiments of the application, the amino acid sequence of the heavy chain variable region of the antibody differs from the amino acid sequence set forth in SEQ ID NO. 14 or 15 by about 1,2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions, and/or additions.

In one or more embodiments of the application, the amino acid sequence of the light chain variable region of the antibody differs from the amino acid sequence set forth in SEQ ID NO. 16 or 17 by about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions, and/or additions.

In one or more embodiments of the application, the C-terminal or N-terminal region of the amino acid sequence shown in SEQ ID NO. 14 or 15 may also 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 maintaining similar functions of the heavy chain variable region of the antibody.

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

In one or more embodiments of the application, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids may also be added or deleted in a region other than the C-or N-terminus of the amino acid sequence shown in SEQ ID NO. 14 or 15, provided that the altered amino acid sequence substantially retains similar functions of the heavy chain variable region of the antibody.

In one or more embodiments of the application, the C-terminal or N-terminal region of the amino acid sequence shown in SEQ ID NO. 16 or 17 may also 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 maintaining the function of the light chain variable region of the similar antibody.

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

In one or more embodiments of the application, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids may also be added or deleted in a region other than the C-terminal or N-terminal region of the amino acid sequence shown in SEQ ID NO. 16 or 17, provided that the altered amino acid sequence substantially retains similar functions of the light chain variable region of the antibody.

In one or more embodiments of the 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 the 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 embodiments of the 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 as SEQ ID NO. 15, and the amino acid sequence of the light chain variable region of the antibody is shown as SEQ ID NO. 17.

In one or more embodiments of the application, 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.

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

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

In one or more embodiments of the application, the antibody comprises a heavy chain constant region selected from the group consisting of an IgG1 subtype, an IgG2 subtype, or an IgG4 subtype.

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

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

In some embodiments of the first aspect, the antibody is a monoclonal antibody 3D10 against soluble IL-1R2, comprising a heavy chain constant region of an IgG2 subtype (e.g., igG2 alpha subtype).

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

In one or more embodiments of the application, the antibodies bind to human IL-1R2 having the amino acid sequence shown in SEQ ID NO. 1.

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

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

In one or more embodiments of the application, the antibody or a product comprising the antibody may be used for Point of CARE TESTING (POCT).

In one or more embodiments of the application, the antibodies have the advantages of good specificity, high potency, and high sensitivity.

In one or more embodiments of the application, the antibody can effectively detect the IL-1R2 level in body fluid (such as blood plasma, blood or urine) of a patient so as to judge whether the patient is in an immunosuppression state, thereby guiding doctors to judge the critical degree of the illness state of the patient, timely taking effective treatment means and reducing the death rate, and having important clinical significance.

In one or more embodiments of the application, antibodies and/or antibody-producing cells can be obtained in vivo automatically after inoculation of an animal with a human IL-1R2 protein (e.g., human soluble IL-1R2 protein) antigen. Immortalized cell lines producing antibodies can be prepared from cells isolated from the immunized animal body. After immunization, animals are killed, lymph node and/or spleen B cells are subjected to immortalization treatment, and are fused with immortalized cells (such as myeloma cells, e.g., mouse myeloma cells SP 2/0) by treatment with oncogenic compounds and mutagenic compounds, thereby removing the activity of tumor suppressor genes. If a myeloma cell is used for fusion, the myeloma cell preferably does not secrete an immunoglobulin polypeptide (non-secretory cell line). Immortalized cells are selected 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 primary screening is performed using an enzyme-linked immunoassay (ELISA). Cells, such as hybridomas, which produce antibodies against human IL-1R2 protein (e.g., human soluble IL-1R2 protein) are selected for cloning, and further screened for desired characteristics, including good growth, high antibody production, and desired antibody characteristics. Methods of hybridoma screening, cloning and amplification are well known to those of ordinary skill in the art.

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

In one or more embodiments of the application, the hybridoma cell line is a mouse hybridoma cell line, e.g., a BALB/c mouse hybridoma cell line.

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

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

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

In one or more embodiments of the application, the product for detecting the level of human IL-1R2 in a sample is selected from the group consisting of a kit, a test strip (e.g., a colloidal gold test strip), a test card, and a microfluidic test device.

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

In one or more embodiments of the application, the product for detecting the level of human IL-1R2 in a sample may further comprise product instructions for use.

In one or more embodiments of the application, the product for detecting the level of human IL-1R2 in a sample may further comprise a container for mixing the sample with the antibody. Such containers may be suitable for use in a detection instrument capable of detecting a signal generated by a detection monoclonal antibody.

In one or more embodiments of the application, the level of IL-1R2, e.g., the level of human IL-1R2, in a sample is detected by an immunological method.

In one or more embodiments of the application, the immunological method is selected from the group consisting of an enzyme-linked immunosorbent assay, a fluorescent immunosorbent assay, a chemiluminescent immunoassay, an immunochromatographic assay, an immunonephelometric assay, an immunoprecipitation and any combination thereof.

According to the application, the level of IL-1R2 can be detected immunologically using antibodies that bind to IL-1R2. For example, IL-1R2 can be detected and quantified using a "sandwich" assay. In this method, typically, an antibody, which is thus referred to herein as a capture antibody, is immobilized on a solid surface in order to bind and capture IL-1R2. The other antibody is detectably labeled, e.g., with a fluorophore, an enzyme, or a colored particle, the binding of which to the IL-1R 2-capturing antibody complex indicates that IL-1R2 has been captured, and the intensity of the signal is proportional to the concentration of IL-1R2 in the sample. Thus, another antibody is also referred to herein as a detection antibody or a labeled antibody. Such an assay may be referred to as a two-site immunoassay, "sandwich" or (when the antibody is an adhesive) "sandwich immunoassay. The capture antibody and the detection antibody may be contacted with the test sample simultaneously or sequentially, as is known in the art. Sequential methods (Sequential method), sometimes referred to as the "forward" method, can be accomplished by incubating the capture antibody with the sample and thereafter adding the labeled detection antibody over a predetermined period of time. Alternatively, the labeled detection antibody may be first incubated with the sample, and then the sample may be contacted with the capture antibody (sometimes referred to as a "reverse" method). Such assays may be performed in many specific forms known to those of skill in the art, including through the use of different high-throughput clinical laboratory analyzers or with point of care or home test equipment.

The most commonly used Enzyme immunoassay is the "Enzyme-linked immunosorbent assay (ELISA)". ELISA is a technique that uses a labeled (e.g., enzyme-linked) form of an antibody to detect and measure antigen concentration. There are different forms of E L I S A known to those skilled in the art. Standard techniques for E L I S a known in the art are described in "M E t h o d S I n Immunodiagnosis", 2 nd 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 (e.g., anti-human IL-1R2 antibodies) are attached to a solid phase (i.e., a microtiter plate) and contacted with a biological sample containing an antigen (e.g., human IL-1R 2), the solid phase is then washed to remove unbound antigen. Labeled antibodies (e.g., enzyme-linked antibodies) are then bound to bound antigen, thereby forming an antibody-antigen-antibody sandwich. Examples of enzymes that can be attached to antibodies are horseradish peroxidase, alkaline phosphatase, luciferase, urease, and β -galactosidase. Enzyme-linked antibodies react with a substrate to produce a chromogenic reaction product that can be measured.

The fluorescent immunoquantitative analysis (Quantitative fluorescence immunoassay) is based on the principle of antigen-antibody reaction, in which known antigens or antibodies are labeled with fluorescein to prepare fluorescent markers, and the corresponding antigens (or antibodies) in cells or tissues are checked by using the fluorescent antibodies (or antigens) as molecular probes. The antigen-antibody complex formed in the cells or tissues contains fluorescein, the specimen is observed by a fluorescence microscope, the fluorescein emits bright fluorescence when irradiated by stimulated luminescence, and the cells or tissues where the fluorescence is located can be seen, so that the nature and the positioning of the antigen or the antibody are determined, and the content is determined by using a quantitative technology.

Chemiluminescent immunoassay (Chemiluminescence immunoassay, CLIA) is a combination of a chemiluminescent assay with high sensitivity and a highly specific immune reaction, and is used for detection and analysis of various antigens, haptens, antibodies, hormones, enzymes, fatty acids, vitamins, medicines and the like, and is a latest immunoassay developed after radioimmunoassay, enzyme immunoassay, fluorescent immunoassay and time-resolved fluorescent immunoassay. Chemiluminescent immunoassay the antigen or antibody is directly labeled with a chemiluminescent reagent for immunoassay. Among the chemiluminescent substances commonly used for labeling are acridine esters (Acridinium Ester, AE) which are effective luminescent labels that emit light by activating the action of a luminescent reagent, intense direct luminescence being completed within one second as rapid blinking. The acridinium ester is used as a marker for immunoassay, the chemical reaction is simple and rapid, a catalyst is not needed, a competition method is adopted for detecting small molecular antigens, a sandwich method is adopted for macromolecular antigen principle, the nonspecific binding is less, the background is low, and the generated light quantity is not reduced when the acridinium ester is combined with macromolecules, so that the sensitivity is increased.

The immunochromatography (Immunochromatography) is based on the principle that a specific antibody is fixed on a certain zone of a nitrocellulose membrane, after one end of the dried nitrocellulose membrane is immersed in a sample, the sample moves forward along the membrane due to capillary action, and when the sample moves to the zone fixed with the antibody, the corresponding antigen in the sample is specifically combined with the antibody, and if the zone is stained with immune colloidal gold or immune enzyme, the zone can display a certain color, so that specific immunodiagnosis is realized.

The immunological turbidimetry (Immunoturbidimetric assay) is an antigen-antibody binding dynamic measurement method, and the basic principle is that when antigen and antibody react in a special dilution system and the proportion is proper (the excessive antibody is generally regulated), the formed soluble immune complex is separated out from liquid phase under the action of a polymerization promoter in the dilution system to form particles, so that turbidity of the reaction solution occurs. When the concentration of the antibody is fixed, the amount of the immunocomplex formed increases with an increase in the amount of the antigen in the sample, and the turbidity of the reaction solution increases. The content of the antigen in the sample can be calculated by measuring the turbidity of the reaction liquid and comparing with a series of standard substances.

Immunoprecipitation (Immunoprecipitation) is a method of purifying and enriching a protein of interest using an antibody-specific reaction. After the antibody is combined with the corresponding protein in the sample, the antibody is incubated with protein A/G (ProteinA/G) or secondary antibody coupled agarose or agarose beads, the bead-protein A/G or secondary antibody-target protein complex is obtained through centrifugation, after precipitation is washed, the precipitate is resuspended in electrophoresis loading buffer solution, boiling is carried out, under the action of high temperature and reducing agent, the antigen and the antibody are dissociated, supernatant is collected through centrifugation, and the supernatant comprises the antibody, target protein and a small amount of hybrid protein.

In one or more embodiments of the application, the first or second antibody is labeled with a detectable label. In one or more embodiments of the application, the detectable label may be selected from any label generally known in the art. In one or more embodiments of the application, the detectable label is a label that allows for more accurate quantification. In one or more embodiments of the application, examples of the detectable label 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 the application, the detectable label is an enzyme or a fluorescent molecule. Methods for attaching the detectable label to an antibody are well known in the art and include covalent and non-covalent attachment.

In one or more embodiments of the application, the detection antibody is detectably labeled by linking the antibody to an enzyme such that the enzyme will react with its substrate upon contact, which reaction can be detected, for example, by spectrophotometry, fluorometry or visual inspection. Enzymes useful for detectably labeling antibodies of the application include, but are not limited to, horseradish peroxidase, malate dehydrogenase, staphylococcal nuclease, delta-V-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triose phosphate isomerase, alkaline phosphatase, luciferase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase, and acetylcholinesterase. In one or more embodiments of the application, the enzyme is horseradish peroxidase.

In one or more embodiments of the application, the antibodies may be labeled with fluorescent molecules. When a fluorescently labeled antibody is exposed to light of the appropriate wavelength, its presence can be detected by the emitted fluorescence. Among them, the most commonly used fluorescent molecules may be selected from Cy3 and Cy5 (a water-soluble fluorescent dye of the cyanine dye family-Cy dye), fluorescein isothiocyanate, rhodamine, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine.

In one or more embodiments of the application, detection may also be achieved using radiolabeled antibodies, which may then be detected by using radioimmunoassays. Radioisotopes, such as ³H、¹³¹I、³⁵S、¹⁴ C and ¹²⁵ I, may be detected using, for example, a gamma counter or scintillation counter, or using autoradiography.

In one or more embodiments of the application, the presence of chemiluminescent antibodies can also be determined by coupling the antibodies to a chemiluminescent molecule to detectably label the antibodies, and then detecting the presence of luminescence generated during the chemical reaction. The chemiluminescent molecule may be selected from the group consisting of luminol, luciferin, isophthaloyl dihydrazide, imidazole, acridinium salt and oxalate ester.

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

In one or more embodiments of the application, the solid support may be a plastic article (e.g., polystyrene plate), a microparticle (e.g., magnetized microparticle), or a membrane support (e.g., nitrocellulose membrane, glass cellulose membrane, or nylon membrane).

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

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

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

In other aspects, the application also provides nucleic acid molecules encoding the antibodies of the application that bind to human IL-1R2, vectors comprising the nucleic acid molecules, and host cells comprising the nucleic acid molecules or the vectors. In some embodiments, the nucleic acid molecule is operably linked to a regulatory nucleotide sequence that can be recognized by a host cell transformed with the vector.

It should be understood that the foregoing detailed description is only for the purpose of making apparent to those skilled in the art the contents of the application, and is not intended to be limiting in any way. Various modifications and changes to the described embodiments will occur to those skilled in the art.

Examples

EXAMPLE 1 eukaryotic expression and purification of human soluble IL-1R2 recombinant proteins

Artificially synthesizing a soluble IL-1R2 (Uniprot: P27930-1, the amino acid sequences of positions 1 to 343 are shown as SEQ ID NO: 1) codon-optimized gene, introducing 6 XHis-Tag at the C end, inserting a pTT5 expression vector through EcoRI/HindIII cleavage sites, transferring into 293 cells for eukaryotic expression, collecting cell culture supernatant, purifying protein, and finally obtaining the 5 mg soluble IL-1R2 protein. As shown in FIG. 1A, the result of the immunoblotting of His tag antibody in the 293 cell culture supernatant and the cell pellet showed that the 293 cell culture supernatant contained the target protein IL-1R2 (as indicated by the red arrow). As shown in FIG. 1B, SDS-PAGE electrophoresis of the purified protein confirmed that the purity and size of the IL-1R2 protein were expected (as indicated by the red arrow).

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

2.1 Immunized mice and cell fusion

5 Female BALB/c mice of 6 weeks old were subcutaneously and multi-point injected with Freund's complete adjuvant emulsified soluble IL-1R2 protein, respectively, and subjected to three immunizations. And after the three immunization is finished, blood is collected from tail vein, and serum is collected for ELISA detection of serum titer. Taking a mouse with the highest serum titer, performing intraperitoneal injection of IL-1R2 protein for impact immunization, taking the spleen of the mouse, collecting spleen cells, performing cell fusion with mouse myeloma cells SP2/0, and plating the mouse with the spleen cells in a 96-well plate for culture.

2.2 Hybridoma selection, stable cell line establishment and antibody subtype identification

ELISA detection is carried out on culture supernatant of the fused cells, positive holes are screened out, re-detection is carried out, and subcloning is carried out on the re-detection positive holes through a limiting dilution method. ELISA detection is carried out after subcloning, positive holes are selected and subcloning is carried out by limiting dilution method. And (3) subcloning for a plurality of times until the ELISA detection positive rate is 100%, selecting clones with high absorbance, selecting wells with vigorous growth for expansion culture, and freezing for seed preservation. ELISA detection is carried out on the culture supernatant of the stable cell strain, and the antibody subtype is identified.

2.3 Preparation of monoclonal antibodies

The hybridoma cells are injected into the abdominal cavity of the mouse, ascites is collected, the abdominal cavity is centrifuged, the supernatant is filtered through a 0.22 mu m filter membrane, then the antibody is purified through a protease A chromatographic column (HiScreen FibroTM PrismA, cytiva), the solution is exchanged and concentrated through an ultrafiltration tube, finally the solution is replaced into PBS, and the monoclonal antibody is obtained through sterile filtration through the 0.22 mu m filter membrane. The antibody is diluted by PBS buffer solution in a gradient way, ELISA is carried out to detect the antibody titer, and the titer of 2 strains of antibody obtained by screening is larger than 1:128000 as shown in the table 1.

TABLE 1 ELISA detection of the OD ₄₅₀ values of monoclonal antibody titers

2.4 Antibody pairing validation

Antibodies 2E10 and 3D10 obtained in section 2.3, and commercially available antibodies ALS11331 (rabbit anti-human IL-1R2 monoclonal antibody, purchased from Abgent, cat# A-ALS 11331) and R020 (rabbit anti-human IL-1R2 monoclonal antibody, purchased from Beijing Yiqiao Seisakusho technology Co., ltd., cat# 10111-R020) were used as the capture antibody and the labeled antibody, respectively, for pairing verification. Wherein the capture antibody was diluted to 1 μg/mL, added to the elisa plate, coated with antibody overnight at 4 ℃, followed by plate washing, blocking, and incubation of antigen IL-1R2. The diluted horseradish peroxidase (HRP) -coupled detection antibody is added, incubated, washed, developed and read, and the pairing result is shown in Table 2, wherein the sensitivity of the two paired antibodies of the antibody 2E10 (subtype IgG 1) and the antibody 3D10 (subtype IgG2 alpha) is the highest, and can reach 10 pg/mL.

TABLE 2 ELISA detection of OD ₄₅₀ values paired with anti-soluble IL-1R2 monoclonal antibody

Example 3 affinity identification of anti-human IL-1R2 monoclonal antibodies 2E10 and 3D10

SPR experiments were performed using Biacore8k+ surface plasmon resonance system (cytiva) to detect the affinity of both 2E10 and 3D10 antibodies to soluble human IL-1R2 protein. The eukaryotic expressed soluble human recombinant IL-1R2 protein was diluted to 10. Mu.g/mL with sodium acetate pH5.5 and coupled to a CM5 chip (cytiva). Two antibodies, 2E10 and 3D10, 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, and flowed as analytes over the chip surface in order from low to high concentration, with a binding time of 120 s, a dissociation time of 300 s, a flow rate of 30. Mu.L/min, glycine (pH 1.5) regeneration of 30 s, a flow rate of 30. Mu.L/min. Fitting model (1:1 binding), analytical method using multiple cycle dynamics (multi-CYCLE KINETICS), according to different concentration of antibody binding, dissociation response value curve fit, calculation of the binding rate constant K _a, dissociation rate constant K _d and affinity constant K _D value. The binding and dissociation curves of the 2E10 antibody and the IL-1R2 protein are shown in figure 2,3D10, and the binding and dissociation curves of the 2E10 antibody and the IL-1R2 protein are shown in figure 3. The K _a、k_d、K_D values obtained by fitting the binding and dissociation curves are shown in Table 3,2E10 with IL-1R2 affinity K _D of 1.19X10-10,3D10 and IL-1R2 affinity K _D of 1.35X10-11. The affinity of antibody 2E10 for IL-1R2 is comparable or better than the affinity of antibody ALS11331 or R020 for IL-1R 2. The affinity of antibody 3D10 for IL-1R2 is comparable or better than the affinity of antibody ALS11331 or R020 for IL-1R 2.

TABLE 3 affinity constants of monoclonal antibodies 2E10 and 3D10 to human IL-1R2

Example 4 elevation of plasma IL-R2 in sepsis patients reflects patient immunosuppression status

4.1 Identification of the immunosuppressive State of sepsis patients

According to the general consensus of sepsis immunosuppression diagnosis and treatment specialists (the committee of shock and sepsis profession of Chinese research hospital society, the committee of severe medical science of the liberation army of Chinese people, the collaborative group of severe immune studies, etc., [ J ], the medical science of sepsis immunosuppression diagnosis and treatment critical illness, 2020, 32 (11): 9. And Pei, Fei et al., "Expert consensus on the monitoring and treatment of sepsis-induced immunosuppression." Military Medical Research vol. 9,1 74. 26 Dec. 2022）,, the clinical examination result, the disease history and the medication information of patients with sepsis are collected, and the immune status (whether immunosuppression is not) of the patients is identified by taking indexes such as lymphocyte absolute numbers of less than 1.0X10 ⁹ per liter, HLA-DR positive rate of peripheral blood mononuclear cells of less than 60%, age of more than 65 years, BMI of less than 18.5, albumin decline, administration of steroid drugs, etc., in the regular report of three consecutive days.

4.2 HLA-DR positive rate detection for peripheral blood mononuclear cells of sepsis patients

EDTA anticoagulation was collected from patients with sepsis, mixed well, 100. Mu.L of blood was taken, 5. Mu.L of PE-Anti-HLA-DR antibody (Biolegend) was added to each, 5. Mu.L of PerCP-CY5.5 Anti-CD14 antibody (Biolegend) was mixed well, and incubated at four degrees of light-shielding for 30 min. 900. Mu.L of 1 XBD FACS lysate (10 Xsolution diluted with deionized water) was added, mixed well and incubated in the dark at 15 min. The flow meter was turned on, the voltage and compensation of each channel were adjusted, CD14 ⁺ monocytes were circled in the CD14-SSC plot, 2000 to 4000 monocytes were harvested, and PE-HLA-DR positive population ratios were circled.

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

Patient EDTA anticoagulation was collected, centrifuged at 4 ℃,4000 rpm at 15: 15min, and the supernatant was taken and plasma IL-1R2 levels were detected by ELISA using a kit for detecting IL-1R2 levels prepared from anti-human IL-1R2 monoclonal antibodies 2E10 and 3D 10.

4.4 Immune status contrast analysis

The immune status of the collected sepsis patients was judged according to a plurality of clinical criteria as shown in table 4 (lymphocyte absolute numbers ALC, monocyte HLA-DR positive rate, history of disease, history of medication, age, BMI, albumin level), a total of 47 sepsis patients were considered immunosuppressed, 20 sepsis patients were considered non-immunosuppressed, and 44 apparent healthy persons (non-immunosuppressed) were added as controls (as shown in table 5). ROC curves were plotted for the population considered immunosuppressed and non-immunosuppressed, as shown in FIG. 4, with areas under the ROC curves AUC=0.9302, P <0.0001, AUC above 0.9, with higher accuracy. The cut-off value of IL-1R2 was calculated to be 24 ng/mL from the ROC curve. As shown in FIG. 5, the distribution of plasma IL-1R2 levels in three groups of patients, in which the clinical index was determined to be immunosuppressed, was found to be that 39 groups of patients had IL-1R2 levels elevated, exceeding 24 ng/mL, and the sensitivity was found to be 82.98%, and in which the clinical index was determined to be non-immunosuppressed, 58 groups of patients had IL-1R2 levels below 24 ng/mL, and the specificity was found to be 90.63%. Pairing of anti-human IL-1R2 monoclonal antibodies 2E10 and 3D10, therefore, in the case of the measured plasma IL-1R2>24 ng/mL, identified a sensitivity of 82.98% and a specificity of 90.63% for the patient's immunosuppression status, can reflect the patient's immunosuppression status.

TABLE 4 determination of immunosuppression status in sepsis patients

TABLE 5 plasma IL-1R2 levels in apparently healthy (non-immunosuppressive) patients

It should be understood that while the application has been described in connection with the above specific forms, it is not intended to be limited to the specific form set forth herein. It will be obvious to those skilled in the art that various equivalent changes can be made to the technical features contained in the application as described without departing from the spirit of the application, and these changes shall fall within the scope of the application.

Sequence information

The sequencing result of the variable region sequence of the antibody is provided by the global gene.

SEQ ID NO:1

MLRLYVLVMGVSAFTLQPAAHTGAARSCRFRGRHYKREFRLEGEPVALRCPQVPYWLWASVSPRINLTWHKNDSARTVPGEEETRMWAQDGALWLLPALQEDSGTYVCTTRNASYCDKMSIELRVFENTDAFLPFISYPQILTLSTSGVLVCPDLSEFTRDKTDVKIQWYKDSLLLDKDNEKFLSVRGTTHLLVHDVALEDAGYYRCVLTFAHEGQQYNITRSIELRIKKKKEETIPVIISPLKTISASLGSRLTIPCKVFLGTGTPLTTMLWWTANDTHIESAYPGGRVTEGPRQEYSENNENYIEVPLIFDPVTREDLHMDFKCVVHNTLSFQTLRTTVKE

SEQ ID NO:2

GYTFTSYA

SEQ ID NO:3

IYPSRDYT

SEQ ID NO:4

ARDGGFFGGHAMDY

SEQ ID NO:5

QNINVW

SEQ ID NO:6

RAS

SEQ ID NO:7

QQGQSYPLT

SEQ ID NO:8

GYTFSSYW

SEQ ID NO:9

ILPGSGRN

SEQ ID NO:10

GRSGLY

SEQ ID NO:11

QSLLYSDGKTY

SEQ ID NO:12

LVS

SEQ ID NO:13

WQGTHSPFT

SEQ ID NO:14

QVQLQQSGAALARPGASVKMSCKASGYTFTSYAMHWVKQRPGRGLEWIGYIYPSRDYTNYNQRFKDKATLTADKSSSTAYMQLSSLTSEDSAVYYCARDGGFFGGHAMDYWGQGTSVTVSS

SEQ ID NO:15

QVQLQQSGAELMKPGASVKISCKATGYTFSSYWIEWVKQRPGHGLEWIGEILPGSGRNAYNEKFKGKATFTADSSSNTVYMQLSSLTSEDSAVYYCGRSGLYWGQGTLVTVSA

SEQ ID NO:16

DIQMNQSPSSLSASLGDTITITCHASQNINVWLSWYQQKPGNIPKLLIYRASNLHTGVPSRFSGSGSGTGFTFTISSLQPEDIATYYCQQGQSYPLTFGAGTKLELK

SEQ ID NO:17

DVVMTQTPLTLSVTIGQPASISCKSSQSLLYSDGKTYLNWLLQRPGQSPKRLIYLVSNLDSGVPDRFTGSGSGTDFTLKIRRVEAEDLGVYYCWQGTHSPFTFGSGTKLEIK

SEQ ID NO:18

CAGGTCCAGCTGCAGCAGTCTGGGGCTGCACTGGCAAGACCTGGGGCCTCAGTGAAGATGTCCTGCAAGGCTTCTGGCTACACCTTTACTAGCTACGCGATGCACTGGGTGAAACAGAGGCCTGGACGGGGTCTGGAATGGATTGGATACATTTATCCTAGTCGTGATTATACTAATTACAATCAGAGGTTCAAAGACAAGGCCACATTGACTGCAGACAAATCCTCCAGCACAGCCTACATGCAACTGAGCAGCCTGACATCTGAGGACTCTGCAGTCTATTACTGTGCAAGAGACGGAGGTTTCTTCGGGGGACATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA

SEQ ID NO:19

QVQLQQSGAALARPGASVKMSCKAS

SEQ ID NO:20

MHWVKQRPGRGLEWIGY

SEQ ID NO:21

NYNQRFKDKATLTADKSSSTAYMQLSSLTSEDSAVYYC

SEQ ID NO:22

WGQGTSVTVSS

SEQ ID NO:23

GACATCCAGATGAACCAGTCTCCATCCAGTCTGTCTGCATCCCTTGGAGACACAATTACCATCACTTGCCATGCCAGTCAGAACATTAATGTGTGGCTAAGCTGGTACCAGCAGAAACCAGGAAATATTCCTAAACTATTGATCTATAGGGCTTCCAACTTGCACACAGGCGTCCCATCAAGGTTTAGTGGCAGTGGATCTGGAACAGGTTTCACATTCACCATCAGCAGCCTGCAGCCTGAAGACATTGCCACTTACTACTGTCAACAGGGTCAAAGTTATCCTCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAA

SEQ ID NO:24

DIQMNQSPSSLSASLGDTITITCHAS

SEQ ID NO:25

LSWYQQKPGNIPKLLIY

SEQ ID NO:26

NLHTGVPSRFSGSGSGTGFTFTISSLQPEDIATYYC

SEQ ID NO:27

FGAGTKLELK

SEQ ID NO:28

CAGGTTCAGCTGCAGCAGTCTGGAGCTGAGCTGATGAAGCCTGGGGCCTCAGTGAAGATATCCTGCAAGGCTACTGGCTACACATTCAGTAGCTACTGGATAGAGTGGGTAAAGCAGAGGCCTGGACATGGCCTTGAGTGGATTGGAGAGATTTTACCTGGAAGTGGTCGTAATGCCTACAATGAGAAGTTCAAGGGCAAGGCCACGTTCACTGCAGATTCATCCTCCAACACAGTCTACATGCAACTCAGCAGCCTGACATCTGAGGACTCTGCCGTCTATTACTGTGGAAGATCTGGCCTCTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA

SEQ ID NO:29

QVQLQQSGAELMKPGASVKISCKAT

SEQ ID NO:30

IEWVKQRPGHGLEWIGE

SEQ ID NO:31

AYNEKFKGKATFTADSSSNTVYMQLSSLTSEDSAVYYC

SEQ ID NO:32

WGQGTLVTVSA

SEQ ID NO:33

GATGTTGTGATGACCCAGACTCCACTCACTTTGTCGGTTACCATTGGACAACCAGCCTCCATCTCTTGCAAGTCAAGTCAGAGCCTCTTATATAGTGATGGAAAGACATATTTGAATTGGTTGTTACAGAGGCCAGGCCAGTCTCCAAAGCGCCTAATCTATCTGGTGTCTAATCTGGACTCTGGAGTCCCTGACAGGTTCACTGGCAGTGGATCAGGGACAGATTTCACACTGAAAATCAGGAGAGTGGAGGCTGAGGATTTGGGAGTTTATTATTGCTGGCAAGGTACACATTCTCCATTCACGTTCGGCTCGGGGACAAAGTTGGAAATAAAA

SEQ ID NO:34

DVVMTQTPLTLSVTIGQPASISCKSS

SEQ ID NO:35

LNWLLQRPGQSPKRLIY

SEQ ID NO:36

NLDSGVPDRFTGSGSGTDFTLKIRRVEAEDLGVYYC

SEQ ID NO:37

FGSGTKLEIK

Claims

1. A method of identifying an immune status of a subject, comprising:

determining the level of interleukin 1 receptor 2 (IL-1R 2) in a sample from the subject;

preferably the immune state is an immunosuppressive state, more preferably the immune state is a relatively immunosuppressive state, and/or

Preferably, the subject is a mammal, more preferably, the subject 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 the group consisting of plasma, blood, urine, serum, lymph, gastric fluid, bile, saliva, sweat, spinal fluid, and any combination thereof, more preferably, the sample is plasma, blood, or urine;

optionally, the IL-1R2 is a soluble IL-1R2 or a membrane-bound protein IL-1R2.

2. The method of claim 1, wherein the determining the level of IL-1R2 in a sample from the subject comprises contacting the sample with an agent that detects the level of IL-1R 2;

Preferably, the agent is a protein, more preferably, the agent is an antibody.

3. The method of claim 1 or 2, wherein the determining the level of IL-1R2 in the sample from the subject comprises contacting the sample with a first antibody and a second antibody, optionally, the contacting is simultaneous or sequential;

Wherein the amino acid sequences of HCDR and LCDR are defined according to IMGT numbering scheme;

Preferably, the first antibody or the second antibody is labeled with a detectable label selected from the group consisting of an enzyme, a fluorescent molecule, a radioisotope, a chemiluminescent molecule, a latex particle, a gold particle, a detectable ligand, and any combination thereof, and/or

Preferably, the first antibody or the second antibody is attached to a solid support, and/or

Detecting the level of IL-1R2 in the sample by an immunological method, preferably selected from the group consisting of an enzyme-linked immunosorbent assay, a fluorescent immunosorbent assay, a chemiluminescent immunoassay, an immunochromatography, an immunonephelometry, an immunoprecipitation assay, and any combination thereof, more preferably an enzyme-linked immunosorbent assay, and/or

Preferably comparing the IL-1R2 level with a cut-off value for identifying the IL-1R2 level of the subject immune status, preferably the cut-off value for identifying the IL-1R2 level of the subject immune status is any value in the range of 19-29 ng/mL, more preferably the cut-off value for identifying the IL-1R2 level of the subject immune status is 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or 29 ng/mL, most preferably the cut-off value for identifying the IL-1R2 level of the subject immune status is 24 ng/mL, and/or

Preferably, the immune status of the subject with the IL-1R2 level greater than the cut-off value is identified as an immunosuppressive status, and/or the immune status of the subject with the IL-1R2 level less than the cut-off value is identified as a non-immunosuppressive status;

optionally, the immune status of the subject is also identified in combination with at least one clinical indicator selected from lymphocyte absolute number, monocyte HLA-DR positive rate, history of disease, history of medication, age, body mass index, and albumin level.

4. Use of an agent for detecting the level of IL-1R2 in a sample from a subject in the manufacture of a product for identifying the immune status of said subject;

Preferably, the sample is selected from the group consisting of plasma, blood, urine, serum, lymph, gastric fluid, bile, saliva, sweat, spinal fluid, and any combination thereof, more preferably, the sample is plasma, blood or urine, and/or

Preferably, the agent is a protein, more preferably, the agent is an antibody, and/or

Preferably, the product for identifying the immune status of the subject is selected from the group consisting of a kit, a test strip, a detection card, and a microfluidic detection device;

optionally, the IL-1R2 is a soluble IL-1R2 or a membrane-bound protein IL-1R2.

5. The use of claim 4, wherein the antibody comprises a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 and a light chain variable region comprising LCDR1, LCDR2 and LCDR3, wherein

6. The use of claim 4 or 5, wherein the product for identifying the immune status of the subject comprises a first antibody and/or a second antibody;

Preferably the cut-off value of the IL-1R2 level used to identify the immune status of the subject is any number in the range of 19-29 ng/mL, more preferably the cut-off value of 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 of the IL-1R2 level used to identify the immune status of the subject is 24ng/mL, and/or

Optionally, the subject's immune status (e.g., immunosuppressed status) also needs to be identified in combination with at least one clinical indicator of lymphocyte absolute number, monocyte HLA-DR positive rate, history of disease, history of medication, age, body mass index, and albumin level.

7. An antibody that binds human IL-1R2 comprising a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 and a light chain variable region comprising LCDR1, LCDR2, and LCDR3, wherein

preferably, the amino acid sequence of the heavy chain variable region of the antibody has at least 90% identity to SEQ ID NO 14 or 15, and/or

The amino acid sequence of the light chain variable region of said antibody has at least 90% identity to SEQ ID No. 16 or 17;

more preferably, the heavy chain variable region of the antibody has an amino acid sequence as shown in SEQ ID NO. 14 or 15, and/or

The amino acid sequence of the light chain variable region of the antibody is shown as SEQ ID NO. 16 or 17;

Most preferably, the amino acid sequence of the heavy chain variable region of the antibody is shown as SEQ ID NO. 14 and the amino acid sequence of the light chain variable region of the antibody is shown as SEQ ID NO. 16, or

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

Optionally, the human IL-1R2 is human soluble IL-1R2 or human membrane bound protein IL-1R2.

8. The antibody of claim 7, wherein

The antibody is a whole antibody, a Fab fragment, a Fab 'fragment, a 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, preferably a murine monoclonal antibody, and/or

The antibody comprises a heavy chain constant region selected from the group consisting of an IgG1 subtype, an IgG2 subtype, or an IgG4 subtype, preferably the antibody comprises a heavy chain constant region of the IgG1 subtype or the IgG2 subtype, and/or

The antibody binds to human IL-1R2 with the amino acid sequence shown as SEQ ID NO. 1 and/or

The antibody is produced by hybridoma cell strain with preservation number of CCTCC NO: C2024249 or CCTCC NO: C2024250.

9. Hybridoma cells which produce antibodies that bind human IL-1R2 and which are preserved at a preservation number of CCTCC NO: C2024249 or CCTCC NO: C2024250;

10. Use of the antibody of claim 7 or 8, or the hybridoma cell of claim 9, in the preparation of a product for detecting the level of human IL-1R2 in a sample;

Preferably, the product for detecting the level of human IL-1R2 in a sample is selected from the group consisting of a kit, a test strip, a detection card and a microfluidic detection device, and/or