Use of gsdmd in diagnosis and therapeutic effect evaluation of diseases related to chest pain

Abstract

The present application relates to the use of GSDMD in the diagnosis and therapeutic effect evaluation of diseases related to chest pain. Quantitative analysis of the GSDMD levels in serum / plasma can provide an objective basis for the early diagnosis of diseases related to chest pain, and help formulate a rational regimen for the treatment of diseases related to chest pain.

Description

Use of GSDMD in diagnosis and efficacy evaluation of chest pain related diseases TECHNICAL FIELD

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

[0002] Among chest pain related diseases, acute myocardial infarction (AMI) is the most serious cardiovascular disease with high mortality, which needs to be treated in time. At present, the diagnosis mainly relies on "symptoms + electrocardiogram + high-sensitivity troponin (HS-cTn)". However, usually 12-18 hours after the onset of AMI, the concentration of cTnI can reach the highest, and it will maintain a high concentration for 5-10 days. Therefore, the diagnosis relying on the level of high-sensitivity troponin is delayed, and if the patient has another heart attack, the level of high-sensitivity troponin will not change significantly because it maintains a high concentration for 5-10 days, so it cannot diagnose the recurrence of heart attack well.

[0003] Among chest pain related diseases other than heart and cardiovascular diseases, pulmonary embolism (PE) is the third leading cause of death next to malignant tumors and myocardial infarction, and its related reports are increasing. However, the clinical manifestations of pulmonary embolism lack specificity, and its misdiagnosis rate is high. In addition, the incidence and mortality of lung cancer are high, especially in the middle-aged and elderly population. If lung cancer can be detected early, the cure rate can be greatly improved.

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

[0005] In view of the above technical problems, the present application provides a hybridoma cell strain, the preservation number of which is CGMCC No. 45331.

[0006] The application also provides a monoclonal antibody or an antigen-binding fragment thereof specifically binding to GSDMD-CT, which contains antigenic complementarity determining regions CDR1, CDR2 and CDR3 of a heavy chain variable region, and antigenic complementarity determining regions CDR1, CDR2 and CDR3 of a light chain variable region; wherein the amino acid sequences of the antigenic complementarity 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; and the amino acid sequences of the antigenic complementarity 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. 12.

[0007] The application also provides a hybridoma cell strain, with a preservation number of CGMCC No. 45332.

[0008] The application also provides a monoclonal antibody or an antigen-binding fragment thereof specifically binding to GSDMD-CT, which contains antigenic complementarity determining regions CDR1, CDR2 and CDR3 of a heavy chain variable region, and antigenic complementarity determining regions CDR1, CDR2 and CDR3 of a light chain variable region; wherein the amino acid sequences of the antigenic complementarity 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; and the amino acid sequences of the antigenic complementarity 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. 14.

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

[0010] The application also provides a cell line producing the aforementioned monoclonal antibody or antigen-binding fragment thereof. The application also provides a nucleic acid molecule encoding the aforementioned monoclonal antibody or antigen-binding fragment thereof. The application also provides an expression cassette, a recombinant vector, or a recombinant microorganism comprising 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 strain, or the aforementioned monoclonal antibody or antigen-binding fragment thereof, 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 a system, which comprises the aforementioned cell strain, or the aforementioned monoclonal antibody or antigen-binding fragment thereof, 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) a use in the preparation of a preparation for determining the degree of pyroptosis of cells; (b) a use in the preparation of a preparation for diagnosing inflammation of a patient; (c) a use in the preparation of a preparation for evaluating the degree of inflammation of an inflammatory disease or the prognosis of an inflammatory disease of a patient; (d) a use in the preparation of a preparation for monitoring the progression of an inflammatory disease of a patient; (e) a use in the preparation of a preparation for diagnosing or monitoring the progression of a chest pain disease of a patient; (f) a use in the preparation of a preparation for determining the severity (such as the area of myocardial infarction, the size of the lesion, etc.) of a chest pain disease of a patient; (g) a use in the preparation of a preparation for predicting the prognosis of a chest pain disease of a patient; (h) a use for screening drugs for treating chest pain related diseases.

[0013] In some embodiments, the method of using the preparation comprises the following steps: (i) detecting one or more proteins or nucleic acids in a sample of a patient and quantifying the level thereof, (ii) comparing the quantified level with the normal threshold or the level of the corresponding protein or nucleic acid in a control population; the protein includes a marker of GSDMD or the nucleic acid includes a nucleic acid encoding GSDMD, and optionally further comprising (iii) determining the risk or degree of a chest pain related disease of the patient.

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

[0015] In some embodiments, the method of using the formulation further comprises combining with other diagnostic or assessment means, such as the level of one or more other markers or one or more other diagnostic or therapeutic outcomes. In some embodiments, the one or more other markers comprises 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 peptide, precursor and prohormone forms of the peptides, urodilatin, adrenomedullin, endothelin or vasopressin. In some embodiments, the one or more other diagnostic or therapeutic outcomes comprises electrocardiogram, chest CT, or angiography.

[0016] In some embodiments, "comparing" comprises comparing the level of GSDMD or the fragment thereof to a threshold level, comparing the level of GSDMD or the fragment thereof to a pre- and post-myocardial ischemia reperfusion value, comparing the level of GSDMD or the fragment thereof to a pre- and post- treatment with medication or surgery value, comparing the level of GSDMD or the fragment thereof to a patient's angiographic stenosis or occlusion area (area of heart damage), or comparing the level of GSDMD or the fragment thereof to a patient's clinical condition. In some embodiments, the threshold refers to the 99th percentile of normal, for example, 15-20 ng / L.

[0017] In some embodiments, the inflammatory disease comprises a condition caused by, or associated with, pyroptosis; or the inflammatory disease comprises an inflammatory disease caused by an infection; preferably, the inflammatory disease caused by an infection comprises inflammation caused by a bacterial infection or inflammation caused by a viral infection or the inflammatory disease caused by an infection comprises pneumonia, peritonitis, cholangitis, urinary tract infection, cellulitis, meningitis, abscess, trauma, polytrauma, burn, 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, or cell lysate.

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

[0021] The present application also provides a kit or composition comprising the aforementioned marker, the aforementioned biological material, and / or the aforementioned partner. In one embodiment, the kit or composition further comprises a partner that binds to other markers, and / or reagents for qualitative or quantitative detection analysis. In one embodiment, one or more of qualitative or quantitative immunoassay, chemical assay, and / or counting assay. 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, GSDMD-CT. The present application also provides the use of the aforementioned marker, the aforementioned biological material, 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 ways, the chest pain related diseases are diseases such as infection, lesion, and infarction necrosis in the heart and cardiovascular sites, especially acute coronary syndrome, pulmonary infection, pulmonary embolism, or lung cancer.

[0022] The present application provides a method, which is characterized in that, comprising using the cell strain of the present application, or the monoclonal antibody or antigen-binding fragment thereof of the present application, 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: (a) a method for preparing a preparation for determining the degree of pyroptosis of cells; (b) a method for preparing a preparation for diagnosing inflammation; (c) a method for preparing a preparation for evaluating the degree of inflammation of an inflammatory disease or the prognosis condition 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 a chest pain disease or monitoring the progression of a chest pain disease; (f) a method for preparing a preparation for judging the severity of a chest pain disease (such as the area of myocardial infarction, the size of injury, etc.); (g) a method for preparing a preparation for predicting the prognosis of a chest pain disease; (h) a method for screening a drug for treating a chest pain related disease. Specifically, the method comprises qualitatively or quantitatively detecting a marker in a sample, the marker being 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 an embodiment, the detection is performed using the aforementioned partner and / or the aforementioned kit.

[0023] The present application also provides a use of a partner of GSDMD or a use of a kit containing the partner, the use being one of: (a) preparing a preparation for determining the degree of pyroptosis of cells; (b) preparing a preparation for diagnosing inflammation; (c) preparing a preparation for evaluating the degree of inflammation of an inflammatory disease or the prognosis condition of a patient with an inflammatory disease; (d) preparing a preparation for monitoring the progression of an inflammatory disease; (e) a use in preparing a preparation for diagnosing a chest pain disease or monitoring the progression of a chest pain disease; (f) a use in preparing a preparation for judging the severity of a chest pain disease (such as the area of myocardial infarction, the size of injury, etc.); (g) a use in preparing a preparation for predicting the prognosis of a chest pain disease; (h) a use in screening a drug for treating a chest pain related disease. In some embodiments, the method comprises using a kit to determine a clinical sample to monitor the treatment process, judge the efficacy, etc.

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

[0025] In some embodiments, the present application also provides systems, methods, and devices for testing a plurality of or a single small volume clinical sample or aliquot thereof for the presence of one or more of a plurality 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, myocardial ischemia reperfusion injury, efficacy monitoring, area of heart damage, and prognosis in patients. In some embodiments, the methods comprise the steps of determining the level of GSDMD or fragments thereof in a sample obtained from the patient; and correlating the level of GSDMD or fragments thereof with the early diagnosis of acute coronary syndrome, myocardial ischemia reperfusion injury, efficacy monitoring, area of heart damage, and prognosis, wherein the correlation comprises comparing the level of GSDMD or fragments thereof to a threshold level, comparing the level of GSDMD fragments before and after myocardial ischemia reperfusion, comparing the level of GSDMD or fragments thereof before and after treatment with a drug or surgery, comparing the level of GSDMD or fragments thereof to the area of vascular stenosis or occlusion in the patient, i.e., the area of heart damage, comparing the level of GSDMD or fragments thereof to the clinical condition of the patient, or combining the level of GSDMD or fragments thereof with other markers to assess the prognosis of the patient. Alternatively, when the level of GSDMD or fragments thereof exceeds the threshold level, which is 15-20 ng / L, or 99th percentile of normal, the patient is likely to be diagnosed early, to have myocardial ischemia reperfusion injury, to have efficacy monitoring, to have area of heart damage, and to have prognosis. The assay methods include antibody sandwich assays for different parts of GSDMD or fragments thereof. Similar methods can be used for acute aortic dissection and acute pulmonary embolism.

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

[0028] In some embodiments, the use of the application in the use of a cardiac site disease further comprises correlating the level of said GSDMD or fragment thereof with the level of one or more additional markers, whereby the combination of the level of said GSDMD or fragment thereof and the level of said markers increases the predictive value of said GSDMD or fragment thereof or the level of said additional markers for said early diagnosis, myocardial ischemia reperfusion injury, efficacy monitoring, area of heart damage and prognostic, wherein said additional markers are selected from the group consisting of troponin, high sensitive 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: urodilatin, adrenomedullin, endothelin, vasopressin. Further, the method or use of the application further comprises the determination of electrocardiogram, chest CT, contrast analysis data from said patient.

[0029] In some embodiments, the method or use of the application comprises the determination of the level of one or more additional markers in a sample obtained from said patient and correlating the level of said GSDMD or fragment thereof and the level of said one or more additional markers, whereby the combination of the level of said GSDMD or fragment thereof and the level of said one or more additional markers increases the predictive value of said total GSDMD or fragment thereof for said early diagnosis, myocardial ischemia reperfusion injury, efficacy monitoring, area of heart damage and prognostic of a cardiac site disease and for said early diagnosis, efficacy monitoring, prognostic of a pulmonary disease. In some embodiments, the correlation between the level of GSDMD or fragment thereof and the level of said one or more additional markers and the electrocardiogram, and or chest CT, and or contrast is performed using a mathematical algorithm. The method or use of the application further comprises the companion diagnostic use of a drug treatment. BRIEF DESCRIPTION OF DRAWINGS

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

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

[0032] Figure 3 Expression level of GSDMD in normal controls and in pulmonary infection, myocardial infarction, heart failure, lung cancer.

[0033] Figure 4 Continuous monitoring of a patient with mild symptoms of myocardial infarction using different markers from the onset of the disease - treatment - hospital discharge after treatment.

[0034] Figure 5 Continuous monitoring of a patient with severe symptoms of myocardial infarction using different markers from the onset of the disease - treatment - reinfarction - hospital discharge after treatment.

[0035] Figure 6 Serial monitoring of a severely symptomatic MI patient with different markers from onset-treatment- secondary cardiogenic heart failure - treatment discharge.

[0036] Figure 7 Serial monitoring of a severely symptomatic MI patient with different markers from onset-treatment- treatment discharge.

[0037] Figure 8 Cardiac angiography infarct size (area) positively correlates with GSDMD expression.

[0038] Figure 9 Sensitivity of antibody pair 1.

[0039] Figure 10 Sensitivity of antibody pair 2.

[0040] Figure 11 Sensitivity of antibody pair 3.

[0041] Figure 12 Sensitivity of antibody pair 4.

[0042] Figure 13 GSDMD in detecting lung cancer and lung nodules. DETAILED DESCRIPTION

[0043] The present application is described in detail below by specific embodiments, and other advantages and effects of the present application can be easily understood by those skilled in the art from the description disclosed herein. The present application can also be implemented or applied by other different specific embodiments, and various modifications or changes can be made to the details herein based on different views and applications without departing from the spirit of the present application. The following examples are only used to more clearly illustrate the technical solutions of the present application, and should not limit the protection scope of the present application.

[0044] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs; the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application; the terms "comprising" and "having," and any variations thereof, as used herein are intended to cover a non-exclusive inclusion.

[0045] In the description of the embodiments of the present application, the term "and / or" is only a description of the association relationship of the associated objects, which means that there can be three relationships, for example, A and / or B, which can represent the three cases of A alone, A and B together, and B alone. In addition, the character " / " herein generally represents an "or" relationship between the front and rear associated objects.

[0046] Reference to "one or more" or "at least one" in the present document indicates that there is at least one of the elements; there can be more than one of such element.

[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 or determining the nature and / or cause of a disease by evaluating one or more factors, which can include patient history, physical examination, review of symptoms, and review of data from one or more laboratory tests. In the present application, if not otherwise defined, they include not only diagnosis in the sense of identifying a particular disease, but also screening of asymptomatic or high-risk persons who are at risk for or suspected of having a certain disease, or monitoring of those who are untreated or treated patients, and monitoring the course of treatment and early prognosis and / or survival prediction.

[0049] As used herein, "monitoring" relates to tracking of a disease, disorder, complication or risk that has already been diagnosed, for example analyzing the progression of the disease, or analyzing the influence of a particular treatment on the progression of the disease or disorder.

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

[0051] As used herein, the term "chest pain related disease" includes diseases such as infection, lesion, infarction or necrosis in the heart and cardiovascular sites, and also includes lung diseases. Specifically, the diseases such as infection, lesion, infarction or necrosis in the heart and cardiovascular sites include, for example, 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 the lung diseases include, for example, lung infection, pulmonary embolism, lung nodule or lung cancer.

[0052] "Inflammation-related disease" refers to an inflammatory disease, or a condition with cell inflammatory necrosis caused by pyroptosis, including but not limited to one or more of: inflammatory diseases caused by infection, particularly inflammation caused by bacterial infection, inflammation caused by viral infection, such as common pneumonia, peritonitis, cholangitis, urinary system infection, cellulitis, meningitis, abscess, and complications of infection thereof, surgical operation, polytrauma, trauma, burn, sepsis, septicemia, 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 stone, etc.; and other diseases such as cancer, diabetes, nephropathy, senile dementia, etc.

[0053] "Infection" within the scope of the present application refers to a pathological process caused by invasion of a potential pathogenic agent / pathogen, organism, and / or microorganism into normal sterile tissue or body fluid, such as infection of fungi, bacteria, viruses, and / or parasites. The infection can be a local infection or a systemic infection. In addition, a patient suffering from an infection can suffer from more than one source of infection at the same time. For example, a patient can suffer from viral infection and fungal infection; bacterial infection and fungal infection, and bacterial infection, fungal infection, and viral infection.

[0054] As used herein, "patient" or "individual" or "subject" refers to a human or an animal. "Drug", "compound", "composition", or "preparation" can 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 having the same disease as the subject individual. In some embodiments, the control population is a population of individuals having the same disease as the subject individual and not exhibiting a crisis reaction and / or 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 a majority of members of the control population do not have the same disease as the subject individual. In some embodiments, the aforementioned population of humans is an unbiased population of humans.

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

[0057] As used herein, a "sample" or "biological sample" or "clinical sample" refers to a sample of fluid, tissue, excretion, or exudate obtained from a subject, including but not limited to blood, serum, plasma, saliva, sputum, urine, gastric fluid, digestive fluid, tears, sweat, fecal matter, semen, vaginal fluid, interstitial fluid, fluid derived from tumor tissue, intraocular fluid, mucus, earwax, oil, glandular secretion, spinal fluid, cerebrospinal fluid from within the skull, tissue, fluid, or matter from a nasal swab, throat swab, mouth swab (e.g., buccal swab), vaginal swab, or nasopharyngeal wash, biopsy fluid or material, amniotic fluid, amniotic fluid, cord blood, lymphatic fluid, cavity fluid, pus, microbiota obtained from a subject, meconium, breast milk, or other excretion or exudate sample.

[0058] "Threshold" refers to the 99th percentile concentration of normal human. Based on different sources of reagents, 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 both monoclonal 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 a marker of the present application. These antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies. In preferred embodiments, the antibodies are labeled with a detectable marker, such as a fluorescent marker, a chemiluminescent marker, etc. In some embodiments, the antibodies of the present application are directed to the amino acid sequence of GSDMD-CT other than the N-terminus, and these antibodies can also be composed into antibody pairs (such as antibody sandwich assay) to detect the part and full length of GSDMD.

[0060] Example 1 Establishment of detection method (optimization screening of magnetic microparticle chemiluminescence method)

[0061] Following methods known to those skilled in the art, several paired antibodies were screened using an ELISA double-antibody sandwich method. These antibodies were then coated with magnetic microparticles for detection of antibody-conjugated acridinium esters. GSDMD and / or fragments were used as calibrators and quality controls. Different pairings were used to screen for antibody pairs consistent with actual clinical cases, and the most sensitive pair of monoclonal antibodies (generated from cell lines MD2025 33# and MD2106 47#, see Figures 2, 9-12 and Table 1) was selected. Based on this, a kit with a functional sensitivity of 0.5 pg / ml and a linear range up to 6000 pg / ml was developed using methods known to those skilled in the art, including sample loading volume, conjugated antibody concentration, reaction time, temperature, reaction sequence, different reaction buffers, and stability (see Figure 1). The following examples all used monoclonal antibodies (for simplicity, they can be directly referred to as monoclonal antibodies MD2025 33# and MD2106 47#) for detection.

[0062] Table 1 Antibody Information

[0063] Example 2: Detection of the concentration of GSDMD or its fragments in samples from healthy individuals and patients with chest pain.

[0064] During hospitalizations at Tangshan Workers' Hospital and Hebei Provincial People's Hospital, hundreds of samples were collected from clinical patients and health centers, including samples from myocardial infarction, heart failure, lung cancer, pulmonary nodules, and healthy controls. This study was conducted in accordance with the principles of the Declaration of Helsinki and approved by the medical ethics committees of Tangshan Workers' Hospital and Hebei Provincial People's Hospital. Serum and / or plasma samples were collected from healthy individuals and patients suffering from various chest pain conditions (including myocardial infarction, heart failure, and lung cancer) to determine the immunoreactivity of GSDMD or its fragments. The immunoreactivity of GSDMD or its fragments was surprisingly increased in disease states compared to healthy individuals (see Figure 3). The following examples are only a few specific examples from many typical cases.

[0065] Example 3: Detection of 4 cases with complete onset cycles of chest pain

[0066] Collecting patient samples of complete onset cycle in hospital clinical patients (2024), including complete samples collected from patient admission due to chest pain to diagnosis, admission treatment, post-treatment detection, and during discharge, and detecting GSDMD or its fragments by serum and / or plasma detection. It is found that the patient has a higher concentration level of GSDMD or its fragments when admitted to the hospital due to chest pain, and the level of GSDMD or its fragments increases due to ischemia-reperfusion injury caused by short-term treatment, and if no heart disease occurs during the treatment of the patient, the level of GSDMD or its fragments will decrease soon, while other indicators such as high-sensitivity troponin are still in a tailing state; if the heart disease occurs again during the treatment, the level of GSDMD or its fragments will increase correspondingly, while other indicators such as high-sensitivity troponin do not change accordingly. At the same time, combined with imaging techniques such as cardiac imaging, it is found that the level of GSDMD or its fragments is positively correlated with the area of cardiac infarction, and the cutoff value is about 20 pg / ml (see Table 2), wherein the 99th percentile concentration of high-sensitivity troponin (Hs-cTnI) is 0.1 ng / ml, and the critical value is 0.5 ng / ml.

[0067] Table 2 Continuous monitoring of patients with cardiac infarction using different markers from onset-treatment-reinfarction-treatment discharge

[0068] Table 2 lists the detection and analysis of GSDMD and high-sensitivity troponin of samples of 4 patients with cardiac infarction at different times. (1) The GSDMD and high-sensitivity troponin values of the 4 patients are significantly higher when the heart infarction occurs, so they can predict the occurrence of heart infarction, and high-sensitivity troponin is a specific protein of myocardium, so simultaneous detection of GSDMD and high-sensitivity troponin is more accurate for predicting heart infarction; (2) As can be seen from the data of the 4 patients in Table 2, GSDMD and high-sensitivity troponin will increase in the early stage of cardiac infarction, so they can predict early cardiac infarction. However, GSDMD can decrease to normal value more quickly after the patient recovers. High-sensitivity troponin has a long half-life and a serious tailing, so GSDMD is more meaningful for monitoring the treatment of the patient. (3) In case 2 and case 3, the patient has heart discomfort during the treatment period. Since high-sensitivity troponin has a long half-life, it cannot reflect the latest state of the patient, and the prediction ability of high-sensitivity troponin for reinfarction or cardiac insufficiency is poor, but GSDMD is more sensitive in case 2 and case 3, and can well monitor the occurrence of discomfort in the patient.

[0069] Example 4 Detection of lung cancer patients and lung nodule patients

[0070] The samples of hospital clinical lung cancer patients were collected and compared with Pro-GRP, CEA, SCC, NSE, CYFRA21-1 and other cancer indicators, and the diagnostic efficiency of plasma GSDMD or its fragments for lung cancer was analyzed by ROC curve. It was found that the area under the ROC curve of plasma GSDMD or its fragments for lung cancer diagnosis was 0.972, which was better than that of carcinoembryonic antigen (CEA) 0.809, squamous cell carcinoma antigen (SCC) 0.717 and neuron-specific enolase (NSE) 0.774 for lung cancer diagnosis efficiency, indicating that GSDMD or its fragments were potential biomarkers for lung cancer (see Table 3, Figure 8).

[0071] Table 3 Diagnostic efficiency of GGSDMD, Pro-GRP, CEA, SCC, NSE, CYFRA21-1 for lung cancer ROC

[0072] The samples of clinical lung cancer patients, patients hospitalized due to lung nodules and healthy people for physical examination were collected. Whether the lung cancer samples or the lung nodule samples, there were significant differences (P<0.0001) in t-test statistical analysis with healthy controls, which could be used as potential monitoring markers for the development of lung nodules to lung cancer (see Figure 13). The present application further clarifies that serum / plasma GSDMD can be used as a new marker to realize early diagnosis and efficacy evaluation of chest pain diseases. Before the present application, the relationship between serum / plasma GSDMD level and chest pain diseases was still unclear. The present application first found that the level of GSDMD in heart failure and myocardial infarction patients was significantly increased, and was positively correlated with the severity of the disease, which was a reliable biomarker for differential diagnosis of cardiovascular diseases. At the same time, the level of GSDMD was significantly related to the treatment efficacy of the above cardiovascular diseases. By quantitative analysis of serum / plasma GSDMD level, objective basis can be provided for early judgment of common cardiovascular diseases, and objective basis can be provided for formulating reasonable treatment plan for treatment of cardiovascular diseases.

[0073] The present application has described the idea and implementation effect evidence of the invention as much as possible. The scope of the present application is limited by the appended claims, and those skilled in the art can clearly understand the scope defined by the claims in combination with the present specification and general common knowledge in the art. Those skilled in the art can make any modification or change to the technical solutions of the present application without departing from the spirit and scope of the present application, and such modification and change also fall within the scope of the present application.

Claims

1. A hybridoma cell line, characterized in that: CGMCC No. 45331.

2. A monoclonal antibody or antigen-binding fragment thereof that specifically binds to GSDMD-CT, characterized in that, It contains the antigenic complementarity determining region CDR1, CDR2 and CDR3 of the heavy chain variable region, and the antigenic complementarity determining region CDR1, CDR2 and CDR3 of the light chain variable region; wherein the amino acid sequences of the antigenic complementarity determining region 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 antigenic complementarity determining region 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.

12.

3. A hybridoma cell line, characterized by: CGMCC No. 45332.

4. A monoclonal antibody or antigen-binding fragment thereof that specifically binds to GSDMD-CT, characterized in that, It contains the antigenic complementarity determining region CDR1, CDR2 and CDR3 of the heavy chain variable region, and the antigenic complementarity determining region CDR1, CDR2 and CDR3 of the light chain variable region; wherein the amino acid sequences of the antigenic complementarity determining region 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 antigenic complementarity determining region 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.

14.

5. The monoclonal antibody or antigen-binding fragment thereof according to claim 2 or 4, characterized in that, The antigen binding fragment includes Fab fragment, Fab', F(ab')2 fragment; Fv fragment, single-chain scFv, single-chain scFv Fc fragment or single-chain antibody ScAb.

6. A cell line for producing the monoclonal antibody or antigen binding fragment thereof according to any one of claims 2, 4, 5.

7. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the monoclonal antibody or antigen binding fragment thereof according to any one of claims 2, 4, 5.

8. An expression cassette, recombinant vector, or recombinant microorganism, comprising, The nucleic acid molecule encodes the monoclonal antibody or antigen binding fragment thereof according to any one of claims 2, 4, 5.

9. A kit, or a composition, or a system, or a device, characterized in that, The nucleic acid molecule encodes the monoclonal antibody or antigen binding fragment thereof according to any one of claims 2, 4, 5. The nucleic acid molecule encodes the monoclonal antibody or antigen binding fragment thereof according to any one of claims 2, 4, 5.

10. Use of a product, characterized in that, The product comprises: a partner of GSDMD or a kit containing the partner, the cell strain of claim 1 or 3, or the monoclonal antibody or antigen-binding fragment thereof of any one of claims 2, 4, 5, or the cell line of claim 6, or the nucleic acid molecule of claim 7, or the expression cassette, recombinant vector, or recombinant microorganism of claim 8, and / or the kit, composition, system, or device of claim 9; and the use is one of: (a) a use in preparing a preparation for determining the degree of pyroptosis of a cell; (b) a use in preparing a preparation for diagnosing inflammation of a patient; (c) a use in preparing a preparation for evaluating the degree of inflammation of an inflammatory disease or the prognosis of an inflammatory disease of a patient; (d) a use in preparing a preparation for monitoring the progression of an inflammatory disease of a patient; (e) a use in preparing a preparation for diagnosing or monitoring the progression of a chest pain disease of a patient; (f) a use in preparing a preparation for determining the severity (such as the area of infarction, the size of damage, etc.) of a chest pain disease of a patient; (g) a use in preparing a preparation for predicting the prognosis of a chest pain disease of a patient; (h) a use in screening a drug for treating a chest pain related disease.

11. Use according to claim 10, characterized in that, The method of using the preparation comprises the steps of: (i) detecting one or more proteins or nucleic acids in a sample of a patient and quantifying the level thereof, (ii) comparing the quantified level with a normal threshold or the level of the corresponding protein or nucleic acid in a control population; the protein comprises a marker of GSDMD; or the nucleic acid comprises a nucleic acid encoding GSDMD.

12. The use according to claim 11, characterized in that, The method of using the preparation comprises determining the level of GSDMD or a fragment thereof from a sample of a patient; and early diagnosis, severity assessment, recurrence diagnosis, efficacy monitoring, and prognosis assessment of a chest pain disease by the level of GSDMD or a fragment thereof.

13. The use according to claim 12, characterized in that, The method of using the preparation further comprises combining other diagnostic or assessment means, such as the level of one or more other markers or one or more other diagnostic or therapeutic results.

14. The use according to claim 13, characterized in that, The one or more other markers comprise D-dimer, CRP, PCT, SAA, IL-6, Pro-GRP, CEA, SCC, NSE, CYFRA21-1, troponin, high-sensitivity troponin, NTpro-BNP or BNP, myeloperoxidase, neopterin, GDF-15, ST2, CK-MB, myoglobin, lactate dehydrogenase, cystatin C, C-reactive protein, and various mature, precursor, and prohormone forms of peptides, such as natriuretic peptides, adrenomedullin, endothelin, or vasopressin.

15. The use according to claim 14, characterized in that, The one or more other diagnostic or therapeutic results comprise electrocardiogram, chest CT, or angiography.

16. Use according to any one of claims 10 to 15, characterized in that, The comparison comprises comparing the level of GSDMD or the fragment thereof with a threshold level, comparing the level of GSDMD or the fragment thereof with the pre- and post- myocardial ischemia reperfusion value, comparing the level of GSDMD or the fragment thereof with the pre- and post- treatment value, comparing the level of GSDMD or the fragment thereof with the patient's angiographic stenosis or occlusion area (heart damage area), or comparing the level of GSDMD or the fragment thereof with the patient's clinical condition.

17. The use according to claim 16, characterized in that, The threshold value refers to the 99% normal person percentile concentration, for example, 20 ng / L.

18. The use of any one of claims 10-17, wherein the inflammatory disease comprises a condition caused by or associated with pyroptosis; or the inflammatory disease comprises an inflammatory disease caused by infection; preferably, the inflammatory disease caused by infection comprises inflammation caused by bacterial infection or inflammation caused by viral infection, or the inflammatory disease caused by infection comprises pneumonia, peritonitis, cholangitis, urinary system infection, cellulitis, meningitis, abscess, trauma, multiple injuries, burn, sepsis, septicemia and septic shock.

19. Use according to any one of claims 10 to 18, characterized in that 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 lung infection, pulmonary embolism, lung nodule or lung tumor.

20. Use according to any one of claims 10 to 19, characterized in that The patient is a human or an animal; or the sample is serum, plasma, urine, blood, cerebrospinal fluid, cell culture fluid, or cell lysate. The threshold value refers to the 99% normal person percentile concentration, for example, 20 ng / L.

18. The use of any one of claims 10-17, wherein the inflammatory disease comprises a condition caused by or associated with pyroptosis; or the inflammatory disease comprises an inflammatory disease caused by infection; preferably, the inflammatory disease caused by infection comprises inflammation caused by bacterial infection or inflammation caused by viral infection, or the inflammatory disease caused by infection comprises pneumonia, peritonitis, cholangitis, urinary system infection, cellulitis, meningitis, abscess, trauma, multiple injuries, burn, sepsis, septicemia and septic shock. 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 lung infection, pulmonary embolism, lung nodule or lung tumor. The patient is a human or an animal; or the sample is serum, plasma, urine, blood, cerebrospinal fluid, cell culture fluid, or cell lysate.

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