Use of GSDMD in the diagnosis and efficacy evaluation of chest pain-related diseases

Abstract

This application relates to the use of GSDMD in the diagnosis and treatment evaluation of chest pain-related diseases. Quantitative analysis of serum / plasma GSDMD levels can provide an objective basis for early diagnosis of chest pain-related diseases and help develop reasonable treatment plans for chest pain-related diseases.

Description

Technical Field

[0001] The present application relates to the use of GSDMD in the diagnosis and efficacy evaluation of chest pain-related diseases, and belongs to the field of molecular biology technology. Background Art

[0002] Among chest pain-related diseases, acute myocardial infarction (AMI) is the most serious cardiovascular disease with a high mortality rate, requiring prompt treatment. Currently, the main method of diagnosis relies on a combination of "symptoms + electrocardiogram + high-sensitivity troponin (HS-cTn)." However, cTnI concentrations typically reach their highest level 12-18 hours after the onset of AMI and remain at a high level for 5-10 days. Therefore, diagnosis based on high-sensitivity troponin levels is delayed. If a patient suffers another MI, the high-sensitivity troponin level will not change significantly due to the 5-10 days of high concentration, making it difficult to diagnose a recurrent MI.

[0003] Among the many chest pain-related conditions besides heart and cardiovascular disease, pulmonary embolism (PE) is the third leading cause of death in humans, second only to malignant tumors and myocardial infarction, and reports of its occurrence are increasing. However, the clinical manifestations of PE lack specificity, leading to high rates of missed and misdiagnosis. Furthermore, the morbidity and mortality of lung cancer remain high, particularly among middle-aged and elderly individuals. Early detection of lung cancer significantly improves cure rates.

[0004] Based on the above situation, there is an urgent clinical need for a biomarker that can respond quickly in the early stages of the disease, has a short half-life, higher specificity and sensitivity, and is more cost-effective, so as to serve as an early differential diagnosis and efficacy evaluation tool for chest pain-related diseases. Summary of the Invention

[0005] In view of the above technical problems, the present application provides a hybridoma cell line with a deposit number of CGMCC No.45331.

[0006] The present application also provides a monoclonal antibody or an antigen-binding fragment thereof that specifically binds to GSDMD-CT, which contains antigen-complementarity determining regions CDR1, CDR2 and CDR3 of a heavy chain variable region, and antigen-complementarity determining regions CDR1, CDR2 and CDR3 of a light chain variable region; wherein the amino acid sequences of the antigen-complementarity determining regions CDR1, CDR2 and CDR3 of the heavy chain variable region are SEQ ID NO.1: GYTFTDYN, SEQ ID NO.2: VRPYNGRA, and SEQ ID NO.3: ARFYRFDGWCFDV, respectively; and the amino acid sequences of the antigen-complementarity determining regions CDR1, CDR2 and CDR3 of the light chain variable region are SEQ ID NO.4: QDITNF, HTS, and SEQ ID NO.5: QQYNNLPLT, respectively; or wherein the sequence of the heavy chain variable region is SEQ ID NO.11, and the sequence of the light chain variable region is SEQ ID NO.12.

[0007] The present application also provides a hybridoma cell line, with the deposit number being CGMCC No.45332.

[0008] The present application also provides a monoclonal antibody or an antigen-binding fragment thereof that specifically binds to GSDMD-CT, which contains antigen-complementarity determining regions CDR1, CDR2 and CDR3 of a heavy chain variable region, and antigen-complementarity determining regions CDR1, CDR2 and CDR3 of a light chain variable region; wherein the amino acid sequences of the antigen-complementarity determining regions CDR1, CDR2 and CDR3 of the heavy chain variable region are SEQ ID NO.6: GFSLSTSGM, SEQ ID NO.7: IYWDDDK, and SEQ ID NO.8: ARTYSTIITDY, respectively; and the amino acid sequences of the antigen-complementarity determining regions CDR1, CDR2 and CDR3 of the light chain variable region are SEQ ID NO.9: QNVGHF, LAS, and SEQ ID NO.10: QQYSRSPYT, respectively; or wherein the sequence of the heavy chain variable region is SEQ ID NO.13, and the sequence of the light chain variable region is SEQ ID NO.14.

[0009] In some embodiments, the antigen-binding fragment comprises a Fab fragment, a Fab', a F(ab')2 fragment; an Fv fragment, a single-chain scFv, a single-chain scFv Fc fragment, or a single-chain antibody ScAb.

[0010] The present application also provides a cell line that produces the aforementioned monoclonal antibody or antigen-binding fragment thereof. The present application also provides a nucleic acid molecule that encodes the aforementioned monoclonal antibody or antigen-binding fragment thereof. The present application also provides an expression cassette, recombinant vector, or recombinant microorganism that comprises the aforementioned nucleic acid molecule.

[0011] The present application also provides a kit, or a composition, or a system, or a device, comprising the aforementioned cell line, or the aforementioned monoclonal antibody or its antigen-binding fragment, or the aforementioned cell line, or the aforementioned nucleic acid molecule, or the aforementioned expression cassette, recombinant vector, or recombinant microorganism.

[0012] The present application also provides a use of a substance or system, which includes: the aforementioned cell line, or the aforementioned monoclonal antibody or its antigen-binding fragment, or the aforementioned cell line, or the aforementioned nucleic acid molecule, or the aforementioned expression cassette, recombinant vector, or recombinant microorganism and / or the aforementioned kit, composition, system, or device; the use is one of the following: (a) use in preparing a preparation for determining the degree of cell pyroptosis; (b) use in preparing a preparation for diagnosing inflammation in a patient; (c) use in preparing a preparation for assessing the degree of inflammation of a patient's inflammatory disease or the prognosis of an inflammatory disease; (d) use in preparing a preparation for monitoring the progression of a patient's inflammatory disease; (e) use in preparing a preparation for diagnosing a patient's chest pain disease or monitoring the progression of a patient's chest pain disease; (f) use in preparing a preparation for judging the severity of a patient's chest pain disease (such as myocardial infarction area, lesion size, etc.); (g) use in preparing a preparation for predicting the prognosis of a patient's chest pain disease; (h) use in screening drugs, wherein the drugs are used to treat chest pain-related diseases.

[0013] In some embodiments, methods for using the formulation include the following steps: (i) detecting one or more proteins or nucleic acids in a patient sample and quantifying their levels; (ii) comparing the quantified levels to normal thresholds or to the levels of the corresponding proteins or nucleic acids in a control population; the proteins include markers for GSDMD or the nucleic acids include nucleic acids encoding GSDMD. Optionally, the method further includes (iii) determining the risk or severity of a chest pain-related disease in the patient.

[0014] In some embodiments, the method of using the preparation comprises measuring the level of GSDMD or a fragment thereof from a patient sample; and performing early diagnosis, severity assessment, recurrence diagnosis, efficacy monitoring, and prognosis assessment of chest pain diseases based on the level of GSDMD or a fragment thereof.

[0015] In some embodiments, the method of using the preparation further includes combining other diagnostic or assessment means, such as the level of one or more other markers or one or more other diagnostic and treatment results. In some embodiments, the one or more other markers include D-dimer, CRP, PCT, SAA, IL-6, Pro-GRP, CEA, SCC, NSE, CYFRA21-1, troponin, high-sensitivity troponin, NT pro-BNP or BNP, myeloperoxidase, neopterin, GDF-15, ST2, CK-MB, myoglobin, lactate dehydrogenase, cystatin C, C-reactive protein, and various mature peptides, precursors and prohormone forms of peptides, the peptides being natriuretic peptides, adrenomedullin, endothelin or vasopressin. In some embodiments, the one or more other diagnostic and treatment results include electrocardiogram, chest CT, or angiography.

[0016] In some embodiments, "comparing" includes comparing the level of GSDMD or a fragment thereof with a threshold level, comparing the level of GSDMD or a fragment thereof with values before and after myocardial ischemia-reperfusion, comparing the level of GSDMD or a fragment thereof with values before and after medication or surgical treatment, comparing the level of GSDMD or a fragment thereof with the area of vascular stenosis or occlusion (cardiac damage area) as determined by imaging, or comparing the level of GSDMD or a fragment thereof with the patient's clinical condition. In some embodiments, the threshold value refers to the 99% normal percentile, for example, 15-20 ng / L.

[0017] In some embodiments, the inflammatory disease includes a condition of cell inflammatory necrosis caused by or associated with cell pyroptosis; or the inflammatory disease includes an inflammatory disease caused by infection; preferably, the inflammatory disease caused by infection includes inflammation caused by bacterial infection or inflammation caused by viral infection, or the inflammatory disease caused by infection includes pneumonia, peritonitis, cholangitis, urinary tract infection, cellulitis, meningitis, abscess, trauma, multiple trauma, burns, sepsis, septicemia and septic shock.

[0018] In some embodiments, the chest pain disease is a heart disease or a lung disease; preferably, the heart disease is heart failure or myocardial infarction (such as acute coronary syndrome); or the lung disease is a lung infection, pulmonary embolism, lung nodule or lung tumor.

[0019] In some embodiments, the patient is a human or an animal; or the sample is serum, plasma, urine, blood, cerebrospinal fluid, cell culture fluid, or cell lysate.

[0020] The present application provides a partner that can specifically recognize a marker, namely, the GSDMD full protein or a fragment thereof. In one embodiment, the partner comprises an aptamer, a peptide aptamer, a peptibody, a mimetic, a phage, an inhibitor, a compound, and / or an antibody. In one embodiment, the antibody comprises a nanobody, a monospecific antibody, and / or a polyclonal antibody. In one embodiment, the partner carries a recognizable label; preferably, the label can be a dye, an epitope tag, a fluorescent moiety, a luminescent moiety, a chemiluminescent moiety, an enzyme label, a magnetic label, a paramagnetic label, a contrast agent, a nanoparticle, a radioisotope, biotin, streptavidin, and a quencher.

[0021] The present application also provides a kit or composition comprising the aforementioned marker, the aforementioned biomaterial, and / or the aforementioned partner. In one embodiment, the kit or composition further comprises a partner combined with other markers, and / or a reagent for qualitative or quantitative detection and analysis. In one embodiment, one or more of qualitative or quantitative immunoassays, chemical assays, and / or counting assays. In one embodiment, the kit or composition further comprises a preparation or drug used as a standard and for calibration. In one embodiment, the preparation or drug used as a standard and for calibration includes GSDMD-CN, GSDMD-NT, and GSDMD-CT. The present application also provides the use of the aforementioned marker, the aforementioned biomaterial, the aforementioned partner, the aforementioned kit or composition, the aforementioned system or device in the differential diagnosis and efficacy evaluation of chest pain-related diseases. In some embodiments, chest pain-related diseases are diseases such as infection, lesions, and infarction necrosis in the heart and cardiovascular parts, especially acute coronary syndrome, lung infection, pulmonary embolism, or lung cancer.

[0022] The present application provides a method, characterized in that it includes utilizing the cell line of the present application, or the monoclonal antibody or its antigen-binding fragment, or the cell line of the present application, or the nucleic acid molecule of the present application, or the expression cassette, recombinant vector, or recombinant microorganism of the present application, and / or the kit, composition, system, or device of the present application; the method is one of the following: (a) a method for preparing a preparation for determining the degree of cell pyroptosis; (b) a method for preparing a preparation for diagnosing inflammation; (c) a method for preparing a preparation for assessing the degree of inflammation of an inflammatory disease or the prognosis of a patient with an inflammatory disease; (d) a method for preparing a preparation for monitoring the progression of an inflammatory disease; (e) a method for preparing a preparation for diagnosing chest pain disease or monitoring the progression of chest pain disease; (f) a method for preparing a preparation for judging the severity of chest pain disease (such as myocardial infarction area, lesion size, etc.); (g) a method for preparing a preparation for predicting the prognosis of chest pain disease; (h) a method for screening drugs, wherein the drugs are used to treat chest pain-related diseases. Specifically, the method comprises qualitatively or quantitatively detecting a marker in a sample, wherein the marker is GSDMD or a fragment thereof, and / or a modified variant of GSDMD or a fragment thereof, and / or a derivative of GSDMD or a fragment thereof. In one embodiment, the detection is performed using the aforementioned partner and / or the aforementioned kit.

[0023] The present application also provides a use of a GSDMD partner or a kit containing the partner, the use being one of the following: (a) preparing a preparation for determining the degree of cell pyroptosis; (b) preparing a preparation for diagnosing inflammation; (c) preparing a preparation for assessing the degree of inflammation of an inflammatory disease or the prognosis of a patient with an inflammatory disease; (d) preparing a preparation for monitoring the progression of an inflammatory disease; (e) use in preparing a preparation for diagnosing chest pain or monitoring the progression of a chest pain disease; (f) use in preparing a preparation for determining the severity of a chest pain disease (such as myocardial infarction area, lesion size, etc.); (g) use in preparing a preparation for predicting the prognosis of a chest pain disease; (h) use for screening drugs for treating chest pain-related diseases. In some embodiments, the method includes using a kit to measure clinical samples to monitor the treatment process, determine the efficacy, etc.

[0024] In some embodiments, GSDMD includes the full-length GSDMD protein (GSDMD-CN), partial GSDMD protein fragments (such as the C-terminal fragment GSDMD-CT or the N-terminal fragment GSDMD-NT), and modified variants and / or derivatives of the full-length GSDMD protein and fragments.

[0025] In some embodiments, the application also provides systems, methods and devices for testing multiple or single small-volume clinical samples or aliquots thereof for the presence of one or more of a variety of markers. In embodiments, the system, method or device is a point of service (POS) system, method or device.

[0026] In some embodiments, the methods or uses of the present application can be used for early diagnosis of acute coronary syndrome (ACS), myocardial ischemia-reperfusion injury, therapeutic efficacy monitoring, cardiac damage area, and prognosis in patients with ACS. In some embodiments, the methods or uses include the following steps: measuring the level of GSDMD or a fragment thereof in a sample obtained from the patient; and correlating the level of GSDMD or a fragment thereof with the early diagnosis of ACS, myocardial ischemia-reperfusion injury, therapeutic efficacy monitoring, cardiac damage area, and prognosis, wherein the correlation includes comparing the level of GSDMD or a fragment thereof with a threshold level, comparing the level of GSDMD fragment with that before and after myocardial ischemia-reperfusion, comparing the level of GSDMD or a fragment thereof with that before and after medication or surgical treatment, comparing the level of GSDMD or a fragment thereof with the area of vascular stenosis or occlusion on imaging, i.e., cardiac damage area, comparing the level of GSDMD or a fragment thereof with the patient's clinical condition, or combining other markers to assess the patient's prognosis. Alternatively, when the level of GSDMD or a fragment thereof exceeds a threshold level, the patient is prone to early diagnosis, myocardial ischemia-reperfusion injury, therapeutic efficacy monitoring, and assessment of cardiac damage area and prognosis, wherein the threshold level is 15-20 ng / L, which can also refer to the 99% percentile of a normal individual. The analytical method comprises a sandwich assay using antibodies targeting different portions of GSDMD or a fragment thereof. Similar diagnostic methods are also used for acute aortic dissection and acute pulmonary embolism.

[0027] In other embodiments, the use of lung diseases further includes correlating the level of the GSDMD or a fragment thereof with the level of one or more additional markers, whereby the combination of the level of the GSDMD or a fragment thereof and the level of the marker increases the value of the level of the GSDMD or a fragment thereof or the level of the related marker for the early detection, efficacy assessment and prognosis of the pulmonary infection, pulmonary embolism and lung cancer, wherein the additional marker is selected from D-dimer, CRP, PCT, SAA, IL-6, Pro-GRP, CEA, SCC, NSE, CYFRA21-1, etc.

[0028] In some embodiments, the use of the present application for cardiac diseases further comprises correlating the level of GSDMD or a fragment thereof with the level of one or more additional markers, whereby the combination of the level of GSDMD or a fragment thereof and the level of the marker increases the value of the level of GSDMD or a fragment thereof or the level of the associated marker for early diagnosis, myocardial ischemia-reperfusion injury, therapeutic efficacy monitoring, cardiac damaged area, and prognosis, wherein the additional marker is selected from troponin, high-sensitivity troponin, NT-pro-BNP or BNP, myeloperoxidase, neopterin, GDF-15, ST2, CK-MB, myoglobin, lactate dehydrogenase, cystatin C, C-reactive protein, and the mature peptide, precursor, and prohormone forms of the following peptides: natriuretic peptide, adrenomedullin, endothelin, and vasopressin. Furthermore, the method or use of the present application further comprises analyzing electrocardiogram, chest CT, and angiography data measured from the patient.

[0029] In some embodiments, the methods or uses of the present application include determining the level of one or more additional markers in a sample obtained from the patient, and correlating the level of GSDMD or a fragment thereof and the level of the one or more additional markers with the early diagnosis, myocardial ischemia-reperfusion injury, therapeutic efficacy monitoring, cardiac damaged area, and prognosis. Thus, the combination of the level of GSDMD or a fragment thereof and the level of the one or more additional markers increases the predictive value of total GSDMD or its fragments for the early diagnosis, myocardial ischemia-reperfusion injury, therapeutic efficacy monitoring, cardiac damaged area, and prognosis of cardiac diseases, as well as the early diagnosis, therapeutic efficacy monitoring, and prognosis of lung diseases. In some embodiments, the correlation between the level of GSDMD or a fragment thereof and the level of the one or more additional markers and electrocardiogram, and / or chest CT, and / or angiography is performed using a mathematical algorithm. The methods or uses of the present application also include companion diagnostic applications for drug therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Standard curve of GSDMD chemiluminescence detection system.

[0031] Figure 2 Schematic diagram of the amino acid sequence of the full-length and fragments of GSDMD protein.

[0032] Figure 3 The expression levels of GSDMD in normal controls and lung infection, myocardial infarction, heart failure, and lung cancer.

[0033] Figure 4 Different markers were used to continuously monitor the condition of a patient with mild myocardial infarction from onset to treatment to discharge from treatment.

[0034] Figure 5 Different markers were used to continuously monitor the situation of a patient with severe myocardial infarction from onset - treatment - reinfarction - treatment and discharge.

[0035] Figure 6 Different markers were used to continuously monitor the condition of a patient with severe myocardial infarction from onset - treatment - secondary cardiogenic heart failure - treatment and discharge.

[0036] Figure 7 Use different markers to continuously monitor a patient with severe myocardial infarction from onset to treatment to discharge from treatment.

[0037] Figure 8 The location (area) of infarction on cardiac angiography was positively correlated with the expression of GSDMD.

[0038] Figure 9 One of the sensitivity of antibody pairing.

[0039] Figure 10 Antibody pairing sensitivity 2.

[0040] Figure 11 Antibody pairing sensitivity three.

[0041] Figure 12 Antibody pairing sensitivity 4.

[0042] Figure 13 Application of GSDMD in the detection of lung cancer and pulmonary nodules. DETAILED DESCRIPTION

[0043] The following describes the embodiments of the present application through specific examples. Those skilled in the art can easily understand other advantages and effects of the present application from the content disclosed in this specification. The present application can also be implemented or applied through other different specific embodiments. The details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present application. The following examples are only used as examples to more clearly illustrate the technical solutions of the present application and should not be used to limit the scope of protection of the present application.

[0044] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this application belongs; the terms used herein are only for the purpose of describing specific embodiments and are not intended to limit this application; the terms "including" and "having" and any variations thereof in the specification and claims of this application and the above-mentioned figure descriptions are intended to cover non-exclusive inclusions.

[0045] In the description of the embodiments of this application, the term "and / or" is simply a description of the association relationship between associated objects, indicating that three relationships can exist. For example, A and / or B can represent the following three situations: A exists alone, A and B exist simultaneously, and B exists alone. In addition, the character " / " in this document generally indicates that the associated objects are in an "or" relationship.

[0046] Reference herein to "one or more" or "at least one" means that at least one of the elements is present; a plurality of such elements may be present unless expressly specified otherwise.

[0047] As used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0048] In the medical context, "diagnosis" is the act or process of identifying one or more health conditions (including diseases and / or injuries) to identify or determine the nature and / or cause of the disease by evaluating one or more factors, which may include patient history, physical examination, review of symptoms, and review of data from one or more laboratory tests. In this application, if not otherwise defined, they include not only diagnosis in the sense of identifying a specific disease, but also screening asymptomatic or high-risk populations who are at risk for or suspected of having a disease, or monitoring untreated or treated patients, as well as monitoring the course of treatment and making early prognostics and / or survival predictions.

[0049] As used herein, "monitoring" involves following a disease, condition, complication, or risk that has been diagnosed, such as analyzing the progression of a disease, or analyzing the effect of a particular treatment on the progression of a disease or condition.

[0050] As used herein, "assessment" refers to the prognosis of a disease or complication before symptoms or markers of the disease become apparent or have changed significantly.

[0051] As used herein, the term "chest pain-related diseases" includes diseases such as infection, lesions, infarction or necrosis of the heart and cardiovascular system, and also includes lung diseases. Specifically, diseases such as infection, lesions, infarction or necrosis of the heart and cardiovascular system include acute coronary syndrome, coronary heart disease, myocardial infarction or acute myocardial infarction (AMI), acute inferior myocardial infarction, acute anterior myocardial infarction, heart failure (acute or chronic), aortic dissection, or myocarditis; and lung diseases include lung infection, pulmonary embolism, pulmonary nodules, or lung cancer.

[0052] "Inflammation-related diseases" refer to inflammatory diseases or conditions with cellular inflammatory necrosis caused by cell pyroptosis, including but not limited to: inflammatory diseases caused by infection, especially inflammation caused by bacterial infection, inflammation caused by viral infection, such as common pneumonia, peritonitis, cholangitis, urinary tract infection, cellulitis, meningitis, abscesses, etc. and their complications of infection, surgery, multiple injuries, trauma, burns, sepsis, sepsis and septic shock, etc.; autoimmune diseases, such as arthritis, gout, scleroderma, etc.; chronic diseases such as diabetes, chronic obstructive bronchitis, leukemia, aplastic anemia and urinary stones, etc.; other diseases such as one or more of cancer, diabetes, kidney disease, Alzheimer's disease, etc.

[0053] "Infection," as used herein, refers to a pathological process caused by the invasion of normally sterile tissues or body fluids by potentially pathogenic agents / pathogens, organisms, and / or microorganisms, such as fungal, bacterial, viral, and / or parasitic infections. An infection can be localized or systemic. Furthermore, a patient experiencing an infection can be simultaneously exposed to more than one source of infection. For example, a patient can experience both a viral infection and a fungal infection; a bacterial infection and a fungal infection; and a bacterial, fungal, and viral infection.

[0054] As used herein, "patient" or "individual" or "subject" refers to a human or an animal. "Drug", "compound", "composition" or "formulation" may have the same meaning.

[0055] As used herein, the term "control population" or "control sample" can be a negative control population or a positive control population. In some embodiments, the control population is a population of individuals suffering from the same disease as the individual being tested. In some embodiments, the control population is a population of individuals suffering from the same disease as the individual being tested and who do not have a crisis reaction and / or a life-threatening reaction to the disease. In some embodiments, the control population is a population of normal individuals. In some embodiments, the control population is a population of individuals in which the majority of the members of the control population do not suffer from the same disease as the individual being tested. In some embodiments, the above-mentioned population is an unbiased population.

[0056] The "marker" is not limited to a certain form and can be a biomarker or a body fluid marker, or a protein, polypeptide or antibody.

[0057] As used herein, "sample" or "biological sample" or "clinical sample" refers to a sample of fluid, tissue, secretion or excretion obtained from a subject, including but not limited to blood, serum, plasma, saliva, sputum, urine, gastric fluid, digestive fluid, tears, sweat, feces, semen, vaginal fluid, interstitial fluid, fluid from tumor tissue, intraocular fluid, mucus, earwax, oil, glandular secretions, spinal fluid, skin, cerebrospinal fluid from within the skull, tissue, fluid or material from a nasal swab, throat swab, oral swab (e.g., cheek swab), vaginal swab or nasopharyngeal wash, biopsy fluid or material, fetal fluid, amniotic fluid, umbilical cord blood, lymph fluid, cavity fluid, pus, microbial flora obtained from a subject, meconium, milk or other sample of secretion or excretion.

[0058] The "threshold" refers to the 99th percentile concentration of normal individuals. Based on reagents from different sources, the 99th percentile concentration is calculated to be the upper limit of the reference interval, for example, 20 ng / L.

[0059] The term "antibody" encompasses monoclonal antibodies and polyclonal antibodies and also encompasses antigen-binding fragments of antibodies. The "antigen-binding fragment of an antibody" (or simply "antibody portion" or "antibody fragment") refers to one or more fragments of a full-length antibody that retain the ability to specifically bind to the marker of the present application. These antibody fragments are obtained using conventional techniques known to those skilled in the art and screened in the same manner as intact antibodies. In a preferred embodiment, the antibody is labeled with a detectable marker, such as a fluorescent marker, a chemiluminescent marker, or the like. In some embodiments, the antibodies of the present application are directed against non-N-terminal amino acid sequences of GSDMD-CT, and these antibodies can also form antibody pairs (such as antibody sandwich assays) to detect partial and full-length GSDMD.

[0060] Example 1 Establishment of Detection Method (Optimization Screening of Magnetic Microparticle Chemiluminescence Method)

[0061] According to methods known to those skilled in the art, several paired antibodies were screened using the ELISA double antibody sandwich method. Magnetic microparticles were then coated with the antibodies, and the detection antibodies were coupled to acridinium esters. GSDMD and / or fragments were used as calibrators and quality controls. Through different pairings, antibody pairs consistent with actual clinical cases were screened, and the most sensitive pair of monoclonal antibodies (produced by cell lines MD202533# and MD210647#, see Figure 2 , 9-12 and Table 1). On this basis, by adjusting the sample loading amount, coupled antibody concentration, reaction time, temperature, reaction sequence, different reaction buffers, stability and other methods known to those skilled in the art, a kit with a functional sensitivity of 0.5 pg / ml and a linear range of 6000 pg / ml was developed (see Figure 1The following examples all used monoclonal antibodies (for simplicity, they can be directly referred to as monoclonal antibody MD202533# and monoclonal antibody MD210647#) for detection.

[0062] Table 1 Antibody information

[0063]

[0064]

[0065] Example 2 Detecting the concentration of GSDMD or its fragments in samples from healthy individuals and patients with chest pain diseases

[0066] Hundreds of samples were collected from clinical patients and health centers during hospitalization at Tangshan Workers' Hospital and Hebei Provincial People's Hospital, including myocardial infarction samples, heart failure samples, lung cancer samples, lung nodule samples, and healthy controls. This study was conducted in accordance with the principles of the Declaration of Helsinki and approved by the Medical Ethics Committee of Tangshan Workers' Hospital and Hebei Provincial People's Hospital. Serum and / or plasma were collected from healthy individuals and patients suffering from various chest pain diseases (including myocardial infarction, heart failure, and lung cancer), and the immunoreactivity of GSDMD or its fragments was measured. Compared with healthy individuals, the immunoreactivity of GSDMD or its fragments was surprisingly increased in disease states (see Figure 3 The following examples are only some specific examples among many typical cases.

[0067] Example 3 Detection of 4 cases of chest pain with complete onset cycle

[0068] Patient samples from the hospital's clinical patients were collected throughout the entire disease cycle (2024), including complete samples collected from the time the patient was admitted for chest pain to the final diagnosis, hospitalization, post-treatment testing, and discharge. The inventors of this work (Li Jun, Liu Ying, and Lü Yuexian from the Laboratory Department of Tangshan Workers' Hospital) performed serum and / or plasma assays for GSDMD or its fragments. The results showed that GSDMD or its fragments were present at high concentrations when the patient was admitted for chest pain. After treatment, transient ischemia-reperfusion injury caused an increase in GSDMD or its fragments. If the patient did not develop heart disease during treatment, GSDMD or its fragments levels decreased rapidly, while other indicators such as high-sensitivity troponin remained at a low level. If heart disease recurred during treatment, GSDMD or its fragments levels increased accordingly, while other indicators such as high-sensitivity troponin did not change accordingly. At the same time, combined with cardiac angiography and other imaging techniques, it was found that the level of GSDMD or its fragments was positively correlated with the area of myocardial infarction, with a cutoff value of approximately 20 pg / ml (see Table 2). Among them, the 99th percentile concentration of high-sensitivity troponin (Hs-cTnI) was 0.1 ng / ml, and the critical value was 0.5 ng / ml.

[0069] Table 2 Continuous monitoring of myocardial infarction patients from onset to treatment to reinfarction to discharge using different markers

[0070]

[0071]

[0072] Table 2 lists samples from four patients with myocardial infarction at different stages of their lifespan, and the GSDMD and high-sensitivity troponin levels were analyzed. (1) The GSDMD and high-sensitivity troponin levels were significantly elevated in all four patients at the time of their myocardial infarction, and therefore, they could both predict the occurrence of myocardial infarction. High-sensitivity troponin is a myocardial-specific protein, so simultaneous testing of GSDMD and high-sensitivity troponin is more accurate in predicting myocardial infarction. (2) The data from the four patients in Table 2 show that both GSDMD and high-sensitivity troponin levels are elevated in the early stages of myocardial infarction, and therefore, they can both predict early myocardial infarction. However, GSDMD can return to normal values more quickly after the patient recovers. High-sensitivity troponin has a more severe tailing and a longer half-life, making GSDMD more meaningful for monitoring patient treatment. (3) In Case 2 and Case 3, the patients experienced heart discomfort again during treatment. Since high-sensitivity troponin has a long half-life and cannot reflect the patient's latest condition, high-sensitivity troponin has poor predictive ability for recurrent myocardial infarction or heart failure. However, GSDMD was more sensitive in Case 2 and Case 3, and the patients' recurrent discomfort could be well monitored.

[0073] Example 4 Testing of Lung Cancer Patients and Lung Nodule Patients

[0074] Samples from lung cancer patients were collected from the hospital and compared with cancer markers such as Pro-GRP, CEA, SCC, NSE, and CYFRA21-1. The ROC curve was used to analyze the diagnostic efficacy of plasma GSDMD or its fragments for lung cancer. The results showed that the area under the ROC curve for plasma GSDMD or its fragments for lung cancer diagnosis was 0.972, which was superior to the diagnostic efficacy of carcinoembryonic antigen (CEA) (0.809), squamous cell carcinoma antigen (SCC) (0.717), and neuronal enolase (NSE) (0.774). This indicates that GSDMD or its fragments are potential biomarkers for lung cancer (see Table 3, Figure 8 ).

[0075] Table 3 ROC curves of GGSDMD, Pro-GRP, CEA, SCC, NSE, and CYFRA21-1 for the diagnosis of lung cancer

[0076]

[0077] Samples from clinical lung cancer patients, hospitalized patients with lung nodules, and healthy checkups were collected. Statistical analysis of both lung cancer samples and lung nodule samples compared with healthy controls showed significant differences (P<0.0001), which can be used as potential monitoring markers for the development of lung nodules into lung cancer (see Figure 13 ). The inventors of the work of this embodiment are Huo Lijing, Liu Xuexin, Wei Changmei, Ding Nan, and Yu Fang from the Laboratory Department of Hebei Provincial People's Hospital. The present application further clarifies that serum / plasma GSDMD can be used as a new marker to achieve early diagnosis and efficacy evaluation of chest pain diseases. Prior to the present application, the relationship between serum / plasma GSDMD levels and chest pain diseases was still unclear. The present application discovered for the first time that in patients with heart failure and myocardial infarction, GSDMD levels were significantly increased and positively correlated with the severity of the disease, making it a reliable biomarker for differential diagnosis of cardiovascular diseases; at the same time, GSDMD levels were significantly correlated with the therapeutic efficacy of the above-mentioned cardiovascular diseases. By quantitatively analyzing serum and plasma GSDMD levels, an objective basis can be provided for the early judgment of common cardiovascular diseases, and an objective basis can be provided for the formulation of reasonable treatment plans for the treatment of cardiovascular diseases.

[0078] This application has endeavored to describe the principles of the invention and evidence of its effectiveness. The scope of this application is defined by the appended claims. Those skilled in the art will clearly understand the scope defined by the claims in conjunction with this specification and common knowledge in the art. Without departing from the spirit and scope of this application, those skilled in the art may make any modifications or changes to the technical solutions of this application, and such modifications and changes are also within the scope of this application.

Claims

1. A hybridoma cell line, characterized in that: The deposit number is CGMCC No. 45331.

2. A monoclonal antibody or antigen-binding fragment thereof that specifically binds to GSDMD-CT, characterized in that: It contains the antigen complementary determining regions CDR1, CDR2 and CDR3 of the heavy chain variable region, and the antigen complementary determining regions CDR1, CDR2 and CDR3 of the light chain variable region; wherein the amino acid sequences of the antigen complementary determining regions CDR1, CDR2 and CDR3 of the heavy chain variable region are SEQ ID NO.1: GYTFTDYN, SEQ ID NO.2: VRPYNGRA, SEQ ID NO.3: ARFYRFDGWCFDV, respectively; the amino acid sequences of the antigen complementary determining regions CDR1, CDR2 and CDR3 of the light chain variable region are SEQ ID NO.4: QDITNF, HTS, SEQ ID NO.5: QQYNNLPLT, respectively; or wherein the sequence of the heavy chain variable region is SEQ ID NO.11, and the sequence of the light chain variable region is SEQ ID NO.

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3. A hybridoma cell line, characterized in that: The deposit number is CGMCC No. 45332.

4. A monoclonal antibody or antigen-binding fragment thereof that specifically binds to GSDMD-CT, characterized in that: It contains the antigen complementary determining regions CDR1, CDR2 and CDR3 of the heavy chain variable region, and the antigen complementary determining regions CDR1, CDR2 and CDR3 of the light chain variable region; wherein the amino acid sequences of the antigen complementary determining regions CDR1, CDR2 and CDR3 of the heavy chain variable region are SEQ ID NO.6: GFSLSTSGM, SEQ ID NO.7: IYWDDDK, SEQ ID NO.8: ARTYSTIITDY, respectively; the amino acid sequences of the antigen complementary determining regions CDR1, CDR2 and CDR3 of the light chain variable region are SEQ ID NO.9: QNVGHF, LAS, SEQ ID NO.10: QQYSRSPYT, respectively; or wherein the sequence of the heavy chain variable region is SEQ ID NO.13, and the sequence of the light chain variable region is SEQ ID NO.

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5. A pair of monoclonal antibodies or antigen-binding fragments thereof that specifically bind to GSDMD-CT, characterized in that: The antibody is an antibody pair consisting of the monoclonal antibody described in claim 2 and claim 4.

6. The monoclonal antibody or antigen-binding fragment thereof according to any one of claims 2, 4, and 5, characterized in that: The antigen-binding fragment is selected from Fab fragment, Fab', F(ab')2 fragment, Fv fragment, single-chain scFv or single-chain antibody ScAb.

7. A cell line that produces the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 2, 4-6.

8. A nucleic acid molecule, characterized in that It encodes the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 2, 4-6.

9. An expression cassette, recombinant vector, or recombinant microorganism, characterized in that: It comprises the nucleic acid molecule according to claim 8.

10. A kit, or a composition, or a system, or a device, characterized in that: Comprising the cell line of claim 1 or 3, or the monoclonal antibody or antigen-binding fragment thereof of any one of claims 2, 4-6, or the cell line of claim 7, or the nucleic acid molecule of claim 8, or the expression cassette, recombinant vector, or recombinant microorganism of claim 9.

11. A use of a product, characterized in that: The product is: the cell line according to claim 1 or 3, or the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 2, 4-6, or the cell line according to claim 7, or the nucleic acid molecule according to claim 8, or the expression cassette, recombinant vector, or recombinant microorganism according to claim 9 and / or the kit or composition according to claim 10; the use is one of the following: (a) use in preparing a preparation for determining the degree of cell pyroptosis; (b) use in preparing a preparation for diagnosing inflammation in a patient; (c) use in preparing a preparation for assessing the prognosis of an inflammatory disease in a patient; (d) use in preparing a preparation for monitoring the progression of an inflammatory disease in a patient; (e) use in preparing a preparation for diagnosing chest pain in a patient; (f) use in preparing a preparation for monitoring the progression of chest pain in a patient; (g) use in preparing a preparation for predicting the prognosis of a chest pain in a patient; (h) use in screening a drug for treating a chest pain disease; The inflammation or inflammatory disease is sepsis or septic shock; The chest pain disease is heart disease or lung disease, the heart disease is heart failure or myocardial infarction; the lung disease is lung infection or lung tumor.

12. The use according to claim 11, characterized in that The patient is a human.

13. The use according to claim 11, characterized in that The patient is an animal.

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