Inflammation marker and application thereof

Abstract

The invention discloses an inflammatory marker and application thereof. The application potential of the TKFC in inflammation detection is disclosed for the first time, the expression condition of the TKFC on a cell membrane based on immune cells is found, the immune cell state and / or the body inflammation condition can be accurately judged, and a new technical means is provided for accurate recognition and monitoring of inflammation.

Description

Technical Field

[0001] This invention relates to the field of biomarker technology, and in particular to an inflammatory marker and its application. Background Technology

[0002] Inflammation is a complex physiological and pathological response of the body to infection, injury, or abnormal stimuli, and it is widely involved in the occurrence, development, and outcome of various diseases. Whether it is an infectious disease (such as pneumonia and sepsis) or a non-infectious disease (such as atherosclerosis, rheumatoid arthritis, and tumors), inflammation plays a crucial role. Therefore, timely and accurate detection of inflammatory status is of great significance for early diagnosis, treatment strategy development, and efficacy monitoring.

[0003] Currently, commonly used clinical indicators for detecting inflammation mainly include white blood cell count and neutrophil percentage in routine blood tests, as well as serum markers such as C-reactive protein (CRP), procalcitonin (PCT), and interleukin-6 (IL-6). While these indicators can provide some information about inflammation, they still have significant limitations: for example, a single indicator often lacks specificity and is difficult to distinguish between infectious and non-infectious inflammation; the dynamic changes of these indicators are lagging, and they cannot reflect the inflammatory process or treatment effect in real time; most markers can be elevated in various inflammatory states, which is not conducive to accurately determining the type of inflammation and immune status.

[0004] Taking interferon-γ (IFN-γ) as an example, its concentration increases significantly during various inflammatory processes, serving as an important molecular marker of inflammatory activation. IFN-γ is mainly secreted by activated T cells and NK cells, amplifying the inflammatory response through positive regulatory circuits, and is significantly elevated in infectious inflammations (such as tuberculosis and influenza) and autoimmune diseases (such as rheumatoid arthritis). However, IFN-γ detection still suffers from problems such as complex operation, high cost, and insignificant changes in some types of inflammation, limiting its clinical application and comprehensive value.

[0005] Therefore, existing technologies still lack an ideal biomarker and corresponding detection method that is highly specific, sensitive, convenient to detect, and can more comprehensively reflect the inflammatory state. To address this technological gap, this invention proposes using TKFC (Triokinase / FMN cyclase) as an inflammatory biomarker for inflammation detection. Although the sequence of TKFC has been previously disclosed, its expression changes during the inflammatory process and its application as an inflammatory biomarker have not been reported to date. Summary of the Invention

[0006] This invention aims to at least solve one of the aforementioned technical problems existing in the prior art. Therefore, the purpose of this invention is to provide an inflammatory biomarker and its application. This invention reveals for the first time the application potential of TKFC in inflammation detection and discovers that the expression of TKFC on the cell membrane of immune cells can accurately determine the state of immune cells and / or the body's inflammatory status, providing a new technical means for the precise identification and monitoring of inflammation.

[0007] In a first aspect, the invention provides the use of a trikinase / FMN cyclase (TKFC) on the cell membrane of immune cells as an immune cell surface marker characterizing the state of immune cells and / or the inflammatory condition of the body.

[0008] In some embodiments of the present invention, the immune cell state includes a resting state and an activated state.

[0009] In some embodiments of the present invention, the term "resting state" refers to a state in which cells are not stimulated by specific antigens, cytokines, or danger signals. This state is characterized by low metabolic activity, low proliferative capacity, and functional quiescence, but possesses a preparatory state with the potential for rapid response. Resting-state cells continuously circulate and patrol within the body, constantly monitoring microenvironment signals through their antigen receptors (such as TCRs and BCRs) and pattern recognition receptors (PRRs), but do not respond to their own normal tissues.

[0010] In some embodiments of the present invention, the term "activated state" refers to the functional execution state in which immune cells, after receiving stimulation from specific antigens, cytokines, or danger signals, undergo a series of intracellular signal transductions, metabolic reprogramming, and gene expression remodeling, thereby acquiring proliferative capacity and specific effector functions.

[0011] In this invention, the transition between the "resting state" and the "activated state" is central to the initiation, execution, and termination of the immune response. This process is also a highly regulated threshold process, requiring stimulation of sufficient intensity and duration to overcome the activation threshold. However, it should be noted that the activated state is not the endpoint. After clearing the antigen, most effector cells are eliminated through activation-induced cell death or apoptosis to maintain homeostasis; a small portion of the activated cells differentiate into memory cells. They re-enter a state of "resting but highly alert."

[0012] In some embodiments of the present invention, the activated state is not uniform. Due to differences in microenvironmental signals (such as IFN-γ+ LPS or IL-4), macrophages can be polarized into the classically activated M1 type (pro-inflammatory, cytotoxic) or the alternatively activated M2 type (anti-inflammatory, repair).

[0013] In some embodiments of the present invention, activated immune cells refer to immune cells activated by cytokines or inflammatory factors.

[0014] In some embodiments of the present invention, the cytokines or inflammatory factors include interferon-γ (IFN-γ).

[0015] In some embodiments of the present invention, the cytokine or inflammatory factor is interferon-γ (IFN-γ).

[0016] In some embodiments of the present invention, the inflammatory condition of the body includes the presence or absence of inflammation and / or the degree of inflammation.

[0017] In some embodiments of the present invention, the immune cells include leukocytes.

[0018] In this invention, leukocytes are a sub-concept relative to immune cells, referring to all nucleated, colorless blood cells present in blood and lymphatic tissues.

[0019] In some embodiments of the present invention, the leukocytes include myeloid cells.

[0020] In some embodiments of the present invention, the white blood cells include granulocytes and monocytes.

[0021] In some embodiments of the present invention, the granulocytes include neutrophils, eosinophils, and basophils.

[0022] In some embodiments of the present invention, the immune cells further include macrophages, dendritic cells, and mast cells.

[0023] In some embodiments of the present invention, the macrophages include macrophages differentiated from monocytes as precursors, as well as tissue macrophages, including but not limited to Kupffer cells of the liver, microglia of the brain, and osteoclasts of bone.

[0024] In some embodiments of the present invention, the dendritic cells include: dendritic cells differentiated from blood mononuclear cells, and dendritic cells directly developed from tissue precursor cells.

[0025] In some embodiments of the present invention, the immune cells are derived from animals.

[0026] In some embodiments of the present invention, the animal is a vertebrate.

[0027] In some embodiments of the present invention, the vertebrates include humans and non-human mammals.

[0028] In some embodiments of the present invention, the non-human mammals include rodents (including mice, rats, hamsters, guinea pigs, minks, etc.), farm animals (including pigs, sheep, dogs, cattle, horses), and non-human primates (including monkeys and orangutans).

[0029] In some embodiments of the present invention, the inflammation includes systemic inflammation and local inflammation.

[0030] In this invention, the term "systemic inflammation" refers to inflammatory diseases that can release large amounts of inflammatory mediators (such as cytokines) into the bloodstream, triggering a strong, systemic response. These include, but are not limited to, sepsis, systemic inflammatory response syndrome, cytokine storms caused by diseases or symptoms (such as severe influenza, severe COVID-19, and complications of CAR-T cell therapy), chronic inflammation associated with rheumatic and immune diseases (such as rheumatoid arthritis, systemic lupus erythematosus, and ankylosing spondylitis), and metabolic diseases (such as obesity and type 2 diabetes). In the art, indicators such as C-reactive protein, procalcitonin, and interleukin-6 are generally used to characterize these systemic inflammations.

[0031] In some embodiments of the present invention, local inflammation includes inflammation occurring in the following sites or systems: Skin and soft tissues, respiratory system, digestive system, musculoskeletal system, nervous system, urogenital system, and sensory organs.

[0032] In some embodiments of the present invention, the inflammation includes, but is not limited to: boils, carbuncles, cellulitis, dermatitis, eczema, wound infection, rhinitis, pharyngitis, tonsillitis, bronchitis, pneumonia, pleurisy, gastritis, enteritis, appendicitis, cholecystitis, pancreatitis, inflammatory bowel disease (such as Crohn's disease, ulcerative colitis), arthritis, tendinitis, bursitis, osteomyelitis, encephalitis, meningitis, radiculitis, cystitis, urethritis, nephritis, prostatitis, pelvic inflammatory disease, conjunctivitis, otitis media, sinusitis, periodontitis, etc.

[0033] In some embodiments of the present invention, the inflammation is pneumonia.

[0034] In some embodiments of the present invention, the inflammation is inflammation caused by a pathogen.

[0035] In some embodiments of the present invention, the pathogen includes at least one of bacteria, fungi, viruses, parasites, mycoplasma, chlamydia, rickettsia, and prions.

[0036] In some embodiments of the present invention, the inflammation is pneumonia caused by a pathogen.

[0037] In some embodiments of the present invention, the inflammation includes bacterial pneumonia, viral pneumonia, fungal pneumonia, and mycoplasmal pneumonia.

[0038] In some embodiments of the present invention, the pathogens causing the bacterial pneumonia include: Streptococcus pneumoniae, Haemophilus influenzae, Staphylococcus aureus, Moraxella catarrhalis, Legionella pneumophila, Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, Escherichia coli, methicillin-resistant Staphylococcus aureus, etc.

[0039] In some embodiments of the present invention, the pathogens of the viral pneumonia include, but are not limited to: influenza virus, respiratory syncytial virus, human metapneumovirus, SARS-CoV-2, adenovirus, rhinovirus, parainfluenza virus, seasonal coronavirus, cytomegalovirus, varicella-zoster virus, herpes simplex virus, measles virus, Hantavirus, etc.

[0040] In some embodiments of the present invention, the pathogens of the fungal pneumonia include, but are not limited to: Candida spp., Aspergillus spp., Pneumocystis jirovecii, Mucorales, Cryptococcus, Histoplasma capsulatum, Coccidioides, Blastomyces, etc.

[0041] In some embodiments of the present invention, the pathogens of the mycoplasma pneumonia include, but are not limited to: Mycoplasma pulmonaryis, Mycoplasma hominis, Mycoplasma genitalium, Mycoplasma fermentans, etc.

[0042] A second aspect of the invention provides the use of a substance that detects TKFCs on the cell membrane of immune cells in the preparation of products for diagnosing inflammation.

[0043] In some embodiments of the present invention, the immune cells are as defined above.

[0044] In some embodiments of the invention, the inflammation is as defined above.

[0045] In some embodiments of the present invention, the product is as defined above.

[0046] In some embodiments of the present invention, the detection includes quantitative and / or qualitative detection.

[0047] In some embodiments of the present invention, the product includes a detection reagent, a detection kit, and a detection chip.

[0048] In some embodiments of the present invention, the substance can be used to detect TKFCs on the cell membrane of immune cells based on at least one of the following methods: Fluorescence microscopy, immunohistochemistry, flow cytometry, Western blotting, enzyme-linked immunosorbent assay (ELISA), mass spectrometry, radioligand binding, and CRISPR-based labeling techniques.

[0049] In some embodiments of the present invention, depending on the specific method chosen, the method may or may not include the step of lysing or breaking down immune cells without affecting the accuracy of the detection.

[0050] In some embodiments of the present invention, the method does not include the step of lysing or breaking down immune cells in order to minimize the interference of TKFCs on non-cell membranes with detection.

[0051] In some embodiments of the present invention, the substance includes antibodies.

[0052] In some embodiments of the present invention, the antibody comprises a monoclonal antibody against TKFC.

[0053] In this invention, there are no limitations on the method for preparing the antibody; conventional antibody preparation methods in the art can be used, including but not limited to hybridoma cells, solid-phase synthesis, and animal immunization. The applicant's prior Chinese patent CN118271448 A (incorporated herein by reference) discloses a method for preparing such monoclonal antibodies. In this invention, the anti-TKFC monoclonal antibody can be any anti-TKFC monoclonal antibody. In this invention, experimental verification shows that even if these anti-TKFC monoclonal antibodies have different antigen-binding sites, they can still be effectively used to characterize the level of TKFC, thereby enabling their detection in this invention.

[0054] In some embodiments of the present invention, the antibody is an antibody that targets the C-terminal epitope of the L domain of the TKFC protein.

[0055] In some embodiments of the present invention, the amino acid sequence of the antigenic peptide of the antibody is shown in SEQ ID NO:1.

[0056] In some embodiments of the present invention, the product further includes substances for detecting immune-active factors and / or immune cell markers.

[0057] In some embodiments of the present invention, the immune-active factors include immune cell cytokines.

[0058] In some embodiments of the present invention, the immune cell cytokines include, but are not limited to: IFN-γ (interferon-γ), TNF-α (tumor necrosis factor-α), IL-1β, IL-6, IL-8, IL-12, and IL-17.

[0059] In some embodiments of the present invention, the immune cell cytokine is IFN-γ.

[0060] In some embodiments of the present invention, the concentration of interferon-γ used is 1-100 ng / mL, and it acts on 1×10 6 One immune cell.

[0061] In some embodiments of the present invention, the concentration of interferon-γ used is 20 ng / mL, acting on 1×10 6 One immune cell.

[0062] In some embodiments of the present invention, the immune cell markers include differentiation cluster (CD) proteins.

[0063] In some embodiments of the present invention, the differentiation cluster (CD) protein includes, but is not limited to, CD14, CD45, etc.

[0064] In some embodiments of the present invention, the test sample of the product includes at least one of peripheral blood, pleural effusion, ascites, cerebrospinal fluid, and other tissue exudates.

[0065] In some embodiments of the present invention, the test sample further includes a tissue or organ containing at least one of the above-mentioned peripheral blood, pleural effusion, ascites, cerebrospinal fluid and other tissue exudates.

[0066] A third aspect of the invention provides the use of a substance for detecting TKFCs on the cell membrane of immune cells in the preparation of products characterizing the state of immune cells.

[0067] In some embodiments of the present invention, the immune cells are as defined above.

[0068] In some embodiments of the present invention, the immune cell state is as defined above.

[0069] In some embodiments of the present invention, the product is as defined above.

[0070] A fourth aspect of the present invention provides a method for characterizing the state of immune cells, comprising: The expression of TKFC on the cell membrane of immune cells was detected, and the state of immune cells was determined by comparing the expression differences of TKFC on resting immune cells.

[0071] In some embodiments of the present invention, the resting immune cells include immune cells derived from healthy individuals or non-inflammatory patients.

[0072] In some embodiments of the present invention, the resting immune cells include those derived from healthy individuals.

[0073] In some embodiments of the present invention, the criteria for determining the state of immune cells are as follows: When the expression level of TKFC on the cell membrane of the immune cell being tested is higher than the baseline level, it indicates that the immune cell being tested is an activated immune cell; when it is equal to or lower than the baseline level of resting immune cells, it indicates that the immune cell being tested is a resting immune cell.

[0074] In some embodiments of the present invention, the statistical mean of various leukocyte indicators isolated from peripheral blood or tissue fluid of healthy individuals is used as the baseline for the expression level of TKFC in the resting state. When the expression level of TKFC on the cell membrane of the immune cell to be tested is higher than the baseline, it indicates that the immune cell to be tested is an activated immune cell; when it is equal to or lower than the baseline, the immune cell to be tested is a resting immune cell.

[0075] In some embodiments of the present invention, the expression baseline of TKFC in resting immune cells was obtained based on a cohort of 100 healthy individuals: the mean resting state value of neutrophils, characterized by the percentage of positive cells, was 7.7%; and the mean resting state value of monocytes, characterized by fluorescence intensity, was 2335.

[0076] Of course, it should be understood that, depending on the chosen detection method, the baseline can also be characterized or replaced by other equivalent indicators, retaining, but not limited to, indicators such as the percentage of positive cells based on flow cytometry and the fluorescence intensity of immunofluorescence.

[0077] In some embodiments of the present invention, when the expression level of TKFC on the cell membrane of the immune cell to be tested is significantly higher than the baseline level, the immune cell to be tested is an activated immune cell.

[0078] In this invention, the term "significant" means that, through statistical algorithms well known to those skilled in the art, the probability that a corresponding effect (e.g., the difference between two sets of data) is caused by random chance is extremely low. This can be achieved through... p value( p The significance of a threshold value is used as the core indicator for measuring significance. p Whether the value is small enough, commonly 0.05 or 0.01.

[0079] A fifth aspect of the present invention provides a method for diagnosing inflammation, comprising: The expression level or content of TKFC on the cell membrane of immune cells in the test sample was detected. By comparing the expression differences of TKFC on resting immune cells, the inflammatory status of the subject was determined.

[0080] In some embodiments of the invention, the inflammatory condition is as defined above.

[0081] In some embodiments of the present invention, the immune cells are as defined above.

[0082] In some embodiments of the present invention, the definition of the sample to be tested is the same as that of the "test sample" described above.

[0083] In some embodiments of the present invention, when the expression level or content of TKFC on the cell membrane of immune cells in the test sample is significantly higher than the expression level or content of TKFC on resting immune cells, it is determined that the subject has inflammation.

[0084] In some embodiments of the present invention, as described above, the present invention obtains a baseline of TKFC expression levels in resting immune cells based on a cohort of 100 healthy individuals: wherein the mean resting state value of neutrophils, characterized by the percentage of positive cells, is 7.7%; and the mean resting state value of monocytes, characterized by fluorescence intensity, is 2335.

[0085] In some embodiments of the present invention, when the percentage of neutrophil-positive cells in the test sample is significantly greater than 7.7%, the subject is considered to have inflammation; or when the TKFC fluorescence intensity on the monocyte cell membrane in the test sample is greater than 2335, the subject is considered to have inflammation.

[0086] In some embodiments of the present invention, the source of resting immune cells is the same as described in the above aspects.

[0087] The beneficial effects of this invention are: 1. This invention is the first to discover that TKFC on the cell membrane of immune cells is related to the state of immune cells and the inflammation of the body, and can be used as a biomarker to characterize the state of immune cells and the inflammation of the body, thereby providing a new technical means for judging the state of immune cells and / or the inflammation of the body.

[0088] 2. Based on the correlation between TKFC on the cell membrane of immune cells and the state of immune cells and the body's inflammatory status, anti-TKFC antibodies can be used to achieve accurate diagnosis of immune cell status and / or the body's inflammatory status. Taking monocytes and neutrophils as examples, their AUC values ​​are 0.9146 and 0.8771, respectively, with sensitivities of 86.00% and 77.90%, and specificities of 81.40% and 93.00%. Attached Figure Description

[0089] Figure 1 The competitive inhibition curve of the anti-TKFC monoclonal antibody KA-002 is shown.

[0090] Figure 2 The competitive inhibition curve of the anti-TKFC monoclonal antibody KA-007 is shown.

[0091] Figure 3The effect of interferon-γ stimulation on TKFC translocation in HL60 cells.

[0092] Figure 4 The results are from flow cytometry analysis of immune cells in patients with inflammation and healthy individuals.

[0093] Figure 5 Differences in TKFC expression on the cell membrane surface of monocytes and neutrophils in patients with inflammation and healthy individuals.

[0094] Figure 6 Flow cytometry plots showing changes in TKFC expression levels on the surface of peripheral blood leukocytes in a mouse model of endotoxemia induced by a single LPS injection at 16 h, 40 h, 64 h, and 7 D post-injection.

[0095] Figure 7 Differences in TKFC expression on the cell membrane surface of monocytes and neutrophils from pneumonia patients with different causes and healthy individuals.

[0096] Figure 8 For based on Figure 7 ROC curve of the test results. Detailed Implementation

[0097] The present invention will be further described in detail below through specific embodiments. Unless otherwise specified, the raw materials, reagents, or apparatus used in the embodiments and comparative examples are all available from conventional commercial sources or can be obtained by existing technical methods. Unless otherwise specified, the test or experimental methods are conventional methods in the art.

[0098] In this invention, unless the context clearly indicates otherwise, references to numerical values ​​or parameters should be understood as approximate values, even if words such as “about,” “approximately,” or “substantially” are not explicitly used. For a specific numerical point (e.g., “10%”), it should be interpreted as covering a reasonable range around that numerical point, such as ±10%, ±5%, ±1%, or other reasonable deviations that would be understood by those skilled in the art.

[0099] Any numerical value or range disclosed in this invention is intended to explicitly encompass all subranges therebetween. For example, the range “1-10” includes not only the endpoints 1 and 10, but also all intermediate subranges such as 2 to 8, 3 to 7, 4 to 6, etc., and all point values ​​within these ranges such as 1, 2, 3, 4, etc.

[0100] Example 1 In this embodiment, a monoclonal antibody against Triokinase / FMN cyclase (TKFC) was prepared. Specifically, in this embodiment, the antigenic peptide KS41 of the monoclonal antibody was synthesized using a CEM fully automated microwave peptide synthesizer based on a solid-phase synthesis method. Its amino acid sequence is: KNMEAGAGRASYISSARLEQPDPGAVAAAAILRAILEVLQS (SEQ ID NO: 1). Then, based on this antigenic peptide KS41, an anti-TKFC monoclonal antibody was obtained by immunizing animals.

[0101] The specific preparation method is as follows: (1) Animal immunization: The antigenic peptide KS41 (1 mg / mL) was mixed with Freund's complete adjuvant at a 1:1 ratio and emulsified to obtain a mixture. Using Balb / C mice as experimental subjects, the mixture was injected subcutaneously into the abdomen and paw pads of Balb / C mice at multiple sites during the initial immunization. The initial dose of antigenic peptide was 100 μg per mouse (based on the amount of antigenic peptide KS41), with a total dose of 0.2 mL per mouse.

[0102] Twenty-one days after the first immunization, a second immunization was administered. During the second immunization, Freund's complete adjuvant was replaced with Freund's incomplete adjuvant, and the injection dose of the mixture was 50 μg / 0.1 mL / animal.

[0103] Following the second immunization, immunizations were performed every two weeks, using Freund's incomplete adjuvant for emulsification. Blood was collected after the fourth immunization, and serum was used for ELISA testing. The specific steps were as follows: 2 μg / mL of antigenic peptide KS41 was used as the coating antigen, added to 100 μL / well of a 96-well plate, and incubated overnight at 4°C. After washing three times with PBST, 200 μL / well of 5% (w / v, water as solvent) skim milk powder was added, and the plate was blocked at 37°C for 2 h. After washing three times with PBST, the serum was serially diluted and added to 100 μL / well, and incubated at 37°C for 1 h. After washing three times with PBST, 100 μL / well of horseradish peroxidase-labeled goat anti-mouse secondary antibody (purchased from Beyotime) was added, and the plate was incubated at 37°C for 1 h. After washing three times with PBST, 100 μL / well of TMB chromogenic buffer (purchased from Merk Inc.) was added, and the plate was developed for 15 min. After stopping the cell fusion process by adding 50 μL of stop solution (2 M sulfuric acid) per well, the OD value was measured at 405 nm using a microplate reader. Subsequent cell fusion procedures could proceed once the titer reached 1:20000.

[0104] Three days before cell fusion, 50 μg of the antigen peptide KS41 was injected intraperitoneally again for a sprint immunization.

[0105] (2) Cell fusion: After euthanizing the immunized mice, the spleens were harvested, ground, and sieved to remove macromolecular impurities. The spleen cells were then washed multiple times with 1640 medium, and their counts were performed. Simultaneously, healthy SP2 / 0 cells (mouse myeloma cells) in logarithmic growth phase were added to the immunized mouse spleen cells at ratios of 1:5-10. After thorough mixing and centrifugation, the supernatant was discarded. 1 mL of 50% (w / v) polyethylene glycol (PEG, wt 1450, purchased from Merk Inc.) solution was added to the cell pellet. The centrifuge tube was gently rotated to ensure full and uniform contact of the cells with the PEG, and the mixture was allowed to stand for 1 min. 1, 2, 3, 4, 5, and 10 mL of 1640 medium were added every 2 min to terminate the PEG reaction. The mixture was allowed to stand for 10 min. The mixture was then centrifuged at 800 rpm for 5 min, and the cell pellet was collected. Cells were resuspended in HAT medium, and then the cell suspension was seeded into 96-well plates for further culture. When the cells (hybridoma cells) grew to occupy approximately 1 / 10 of the well area, the supernatant was collected for ELISA. The specific experimental steps were the same as the ELISA assay described above, except that an equal volume of cell supernatant was used instead of serum. Positive hybridoma cells were obtained through screening.

[0106] The obtained positive hybridoma cells were subcloned four times to obtain a cell line that stably secretes the desired monoclonal antibody. The cell line was acclimatized with serum-free medium, and the culture supernatant was collected. The antibody in the supernatant was enriched and purified using protein A / G magnetic beads to obtain the anti-TKFC monoclonal antibody.

[0107] In this embodiment, two monoclonal antibodies that bind to different sites of KS41 were randomly selected and named KA-002 and KA-007, respectively.

[0108] Antibody titers of KA-002 and KA-007 were determined.

[0109] Specifically, antibody titers were determined using ELISA: The antibody was coated overnight at 4°C with the antigenic peptide KS41 at a concentration of 2 μg / mL (using carbonate buffer (CBS) as the solvent). Then, it was blocked with 5% skim milk powder and incubated at 37°C for 2 hours. The antibody was washed three times with PBST. KA-002 and KA-007 were serially diluted with PBS to concentrations of 4 μg / mL, 2 μg / mL, 1 μg / mL, 0.5 μg / mL, 0.25 μg / mL, 0.125 μg / mL, and 0.0625 μg / mL. PBS was used as a negative control, and three replicates were set up. The serially diluted monoclonal antibodies were added to the wells and incubated at 37°C for 1 hour. The antibody was washed three times with PBST. HRP-labeled goat anti-mouse secondary antibody (purchased from Beyotime) diluted 10000× was added, and the antibody was incubated at 37°C for 1 hour. The antibody was then washed three times with PBST. Add TMB chromogenic substrate (purchased from Merk Inc.) and incubate at 37°C for 10 min. Terminate the reaction by adding 2 M sulfuric acid. Detect the OD value at 405 nm using a microplate reader. For the indirect ELISA method, an OD value ≥ 0.2 is used as the positive criterion, and the highest antibody dilution detected as positive is the antibody titer.

[0110] The results are shown in the table below.

[0111] Table 1 Antibody titers of anti-TKFC monoclonal antibodies

[0112] It was found that the titer of KA-002 was 0.0625 μg / mL, and the titer of KA-007 was 0.125 μg / mL. Both anti-TKFC monoclonal antibodies showed good binding affinity to the antigenic peptide KS41.

[0113] To further determine the coating concentration of antigenic peptide KS41 and the concentration of the two anti-TKFC monoclonal antibodies used in subsequent experiments, the inventors conducted an antibody competitive inhibition experiment based on the checkerboard titration method.

[0114] The specific experimental steps are as follows: The antigenic peptide KS41 was coated overnight at 4°C at a concentration of 1 μg / mL (using carbonate buffer (CBS) as the solvent). Then, it was blocked with 5% skim milk powder and incubated at 37°C for 2 h. The mixture was washed three times with PBST. The antigenic peptide KS41 was serially diluted with PBS to concentrations of 1.77 μmol / L, 0.88 μmol / L, 0.44 μmol / L, 0.22 μmol / L, 0.11 μmol / L, and 0.055 μmol / L. KA-002 and KA-007 were diluted with PBS to a concentration of 2 μg / mL. The diluted KA-002 and KA-007 were then mixed with different concentrations of antigenic peptide KS41. The mixtures were added to the wells, with three replicates per group. The mixtures were incubated at 37°C for 1 h. The mixtures were washed three times with PBST. Add 10000× diluted HRP-labeled goat anti-mouse secondary antibody (purchased from Beyotime), incubate at 37℃ for 1 h, and wash 3 times with PBST. Add TMB chromogenic substrate (purchased from Merk Inc.), and incubate at 37℃ for 10 min. Terminate the reaction with 2 M sulfuric acid. Detect the OD value at 405 nm using a microplate reader. Plot a competitive inhibition curve based on the OD value to determine the IC50. 50 value.

[0115] The results are as follows Figure 1 and Figure 2 As shown.

[0116] It can be observed that, The competition curves of the two anti-TKFC monoclonal antibodies showed a concentration-dependent inhibitory trend, with KA-002 exhibiting a higher IC50 value. 50 The IC50 of KA-007 is 0.512 μmol / L. 50 The concentration was 0.768 μmol / L, and both anti-TKFC monoclonal antibodies could bind efficiently to KS41.

[0117] Example 2 In this embodiment, based on the anti-TKFC monoclonal antibody obtained in the above embodiments, a method for detecting TKFC expression on the surface of leukocytes using flow cytometry is established.

[0118] The specific testing steps include: (1) Construction of fluorescently labeled anti-TKFC monoclonal antibody: Using Alexa Flour ®647 (purchased from Thermo Fisher Scientific Inc.) was used to modify the anti-TKFC monoclonal antibody (KA-002 in this example). The specific modification method was as follows: 1 mL of 2.54 mg / mL anti-TKFC monoclonal antibody was dialyzed with 0.01 M phosphate buffer for 4 h, and then 165 μL of Alexa Flour was added. ® 647-DMSO solution (prepared using DMSO as solvent, containing Alexa Flour at a final concentration of 10 mg / mL) ® (A mixture of 647) was reacted for 1 h, followed by dialysis with PBS for 48 h to obtain Alexa Flour. ® 647-modified anti-TKFC monoclonal antibody.

[0119] (2) Flow cytometry detection: Collect 50 μL of whole blood and mix it with 0.4% ammonium chloride (prepared with pure water and sterilized by filtration through a 0.1 μm membrane) at a volume ratio of approximately 1:20. Lyse the cells at room temperature for 10 min, centrifuge at 1500 rpm for 5 min, and collect the cell pellet. Resuspend the pellet in PBS, centrifuge again at 1500 rpm for 5 min, and wash twice with PBS to obtain white blood cells (WBCs). Incubate with Fc blocking agent (purchased from BioLegend, Inc.) for 5 min to complete the blocking. Add the Alexa Flour obtained in the above steps to the blocked white blood cells. ® The 647-modified anti-TKFC monoclonal antibody was incubated at 4 °C in the dark for 30 min. 1 mL of PBS was added, and the cells were centrifuged at 1500 rpm for 5 min to obtain a cell pellet. The pellet was then centrifuged again at 1500 rpm for 5 min, and washed once with PBS. After resuspending the cells in 100 μL of PBS, the cells were analyzed by flow cytometry.

[0120] Example 3 In this embodiment, based on the method in Example 2, the phenomenon that TKFC translocates from the cytoplasm to the cell membrane surface under the stimulation of interferon-γ was verified.

[0121] Specifically, in this embodiment, HL60 cells (human acute promyelocytic leukemia cells) were used as the experimental subject to represent human neutrophils.

[0122] Following the method described in Example 2 above, construct the Alexa Flour. ® KA-002 modified with 647. According to 1×10 5HL60 cells were seeded into culture dishes at a concentration of 20 ng / mL, and IFN-γ was added to a final concentration of 20 ng / mL. The cells were cultured in a cell culture incubator for 24 h. After centrifugation at 1500 rpm for 5 min, the cell pellet was collected. The cells were washed once with PBS. Cells were then collected and incubated with Fc blocking agent (purchased from BioLegend, Inc.) for 10 min to complete the blocking process. Alexa Flour was then added to the blocked cells. ® KA-002 cells modified with 647 were incubated at 4 °C in the dark for 30 min. 1 mL of PBS was added, and the cells were centrifuged at 1500 rpm for 5 min to obtain a cell pellet. The pellet was then centrifuged again at 1500 rpm for 5 min, and washed once with PBS. The cells were resuspended in 100 μL of PBS and analyzed by flow cytometry.

[0123] HL60 cells without IFN-γ were used as a control.

[0124] The results are as follows Figure 3 As shown.

[0125] It was observed that after HL60 cells were stimulated with interferon-γ, TKFCs translocated from the cytoplasm to the cell membrane and were detected by the KA-002 antibody. This indicates that TKFCs translocate from the cytoplasm to the cell membrane during inflammation, and the occurrence of inflammation can be characterized by detecting TKFCs on the cell membrane.

[0126] A retest using KA-007 revealed that the transposition of TKFC could also be detected.

[0127] Example 4 In this embodiment, based on the method in Example 2, a comparative study was conducted on the expression of TKFC from inflammatory patients and healthy individuals on the cell membrane surface of monocytes and neutrophils.

[0128] Specifically, peripheral blood leukocytes from healthy individuals and pneumonia patients were used as experimental subjects, representing non-inflammatory and inflammatory states, respectively. The expression differences of TKFC on the cell membrane surface of monocytes and neutrophils were detected by flow cytometry according to the method in Example 2.

[0129] The specific experimental method is as follows: Peripheral blood samples were collected from three pneumonia patients (inflammation patient group) and three healthy individuals (healthy group). The samples were mixed with 0.4% ammonium chloride (prepared with pure water, filtered through a 0.1 μm filter for sterilization) at a volume ratio of approximately 1:20. The cells were lysed at room temperature for 10 min, centrifuged at 1500 rpm for 5 min, and the cell pellet was collected. The cells were resuspended in PBS, centrifuged again at 1500 rpm for 5 min, and washed twice with PBS to obtain white blood cells (WBCs). Fc blocking agent (purchased from BioLegend, Inc.) was added and incubated for 5 min to complete the blocking. Alexa Flour was then added to the blocked white blood cells. ® KA-002 cells modified with 647 were incubated at 4 °C in the dark for 30 min. 1 mL of PBS was added, and the cells were centrifuged at 1500 rpm for 5 min to obtain a cell pellet. The pellet was then centrifuged again at 1500 rpm for 5 min, and washed once with PBS. The cells were resuspended in 100 μL of PBS and analyzed by flow cytometry.

[0130] The results are as follows Figure 4 and Figure 5 As shown.

[0131] It was found that the expression of TKFC on the cell membrane surface of monocytes and neutrophils in the inflammatory patient group was significantly higher than that in the healthy group. This indicates that the translocation of intracellular TKFC to the cell membrane surface under inflammatory conditions was sensitively detected by KA-002. A retest with KA-007 confirmed that this phenomenon could also be detected.

[0132] Example 5 In this embodiment, the relationship between TKFC expression on leukocytes and the degree of inflammation in an LPS-induced endotoxemia mouse inflammation model was investigated. Specifically, in this embodiment, an LPS-induced endotoxemia mouse inflammation model was used to simulate the changes following human inflammatory infection, and KA-002 was used to track changes in TKFC expression on the surface of leukocytes. In this embodiment, only neutrophils were characterized because monocytes accounted for a very low percentage (only 2-8%), and even less after LPS injection, making statistical analysis difficult; therefore, they were not considered.

[0133] The specific experimental method is as follows: Kunming mice (KM mice) were randomly divided into an LPS group and a NC group. Mice in the LPS group were intraperitoneally injected with LPS at a dose of 2.5 mg / kg. Blood samples were collected at 16 h, 40 h, 64 h, and 168 h (i.e., 7 days) after injection. Mice in the NC group were fed synchronously with the LPS group but did not receive any drug treatment, and blood samples were collected at the same time points.

[0134] Whole blood tests were performed on both groups of mice. Specifically, following the method in Example 2, whole blood samples were collected from mice and divided into tubes at a volume of 50 μL / tube, and Alexa Flour prepared with PBS containing 10% FBS was added. ® KA-002 cells modified with 647 were mixed with whole blood in an equal volume and incubated at 4°C for 30 min. The mixture was then inoculated with 0.4% ammonium chloride (prepared with pure water and sterilized by 0.1 μm filter) at a volume ratio of approximately 1:20, lysed at room temperature for 10 min, centrifuged at 1500 rpm for 5 min, and the cell pellet was collected. The cells were resuspended in PBS and centrifuged at 1500 rpm for 5 min. After washing the cells twice with PBS and centrifuging at 1500 rpm for 5 min, the cell pellet was collected, resuspended in 100 μL of PBS, and analyzed by flow cytometry.

[0135] The results are as follows Figure 6 As shown.

[0136] As shown in the figure, the cell population represents neutrophils, and P7 represents the percentage of KA-002-positive neutrophils. TKFC expression in the LPS group exhibited a clear time-series dynamic response. Specifically, at 16 h after LPS treatment, the percentage of KA-002 positivity in the LPS group was significantly higher than that in the NC group; at 40 h, although its expression level decreased compared to 16 h, it still remained significantly higher than that in the NC group; at 64 h, the TKFC positivity rate remained significantly higher than that in the NC group; at 168 h, its expression decreased, showing no significant difference compared to the NC group, and correspondingly, the LPS-induced inflammation had subsided by this time. A retest with KA-007 yielded similar results. Therefore, changes in TKFC expression at the cell membrane can reflect changes in the degree of inflammation in the body, and it can be detected in both humans and non-human animals, indicating the state of inflammation.

[0137] Example 6 In this embodiment, the difference in the expression level of TKFC on the surface of leukocytes in patients with inflammation and healthy individuals was further investigated.

[0138] In this embodiment, the inclusion and exclusion criteria for pneumonia patients are as follows: Pneumonia patients meeting the following inclusion criteria were recruited and enrolled: ① Meets the diagnostic criteria for CAP in the Chinese Guidelines for the Diagnosis and Treatment of Pneumonia in Adults (2016 Edition); ② Pulmonary infection is diagnosed by combining the patient's imaging examinations (such as chest CT, etc.), laboratory tests (complete blood count, etiological examination, etc.), symptoms (cough, sputum, fever, etc.), and signs; ③ Age ≥ 18 years old.

[0139] The exclusion criteria are: ① Patients who do not meet the above inclusion criteria after diagnosis; ②Those with a history of lung cancer, pulmonary interstitial fibrosis, pulmonary tuberculosis, etc.; ③ Individuals with combined severe heart, liver, kidney, or systemic diseases, or hematological diseases; ④ Individuals with concurrent infectious diseases such as tuberculosis; ⑤ Subjects who are uncooperative, or subjects whom the researchers judge to be unlikely to complete the study.

[0140] In this embodiment, a total of 86 patients with pneumonia were collected, including 51 cases of pneumonia caused by bacterial infection, 22 cases of pneumonia caused by viral infection, 9 cases of pneumonia caused by mycoplasma infection, and 4 cases of pneumonia caused by fungal infection. A healthy control group of 100 individuals was also included.

[0141] Following the method described in the above embodiments, with Alexa Flour ® Whole blood was counterstained with an antibody combination of 647-modified KA-002, anti-CD45 antibody (purchased from BioLegend, Inc.), and anti-CD14 antibody (purchased from BioLegend, Inc.), and analyzed by flow cytometry. Specifically, 50 μL of anticoagulated whole blood sample was taken, 1 mL of hemolysin was added, and the mixture was incubated at room temperature for 10 min. Leukocytes were then separated by centrifugation and washing. Fc blocking buffer (purchased from BioLegend, Inc.) was added, and the mixture was incubated for 5 min, followed by the addition of Alexa Flour. ® 647-modified KA-002 and fluorescein-labeled CD antibody were added to a final volume of 100 μL with 1×PBS, mixed well, and incubated at 4°C in the dark for 30 min. After incubation, the cells were washed with 1×PBS and analyzed by flow cytometry.

[0142] Data management and analysis were performed using GraphPad Prism software. Measurement data were described as (x±s), and inter-group comparisons were conducted using t-tests. Count data were described as [n(%)], and χ² tests were used. 2 The study analyzed inter-group and inter-sample differences in TKFC expression in monocytes and neutrophils from cases and healthy individuals with different infection sources. Receiver operating characteristic (ROC) curves were plotted, and the area under the curve (AUC) and optimal cutoff value were calculated to determine specificity and sensitivity, thereby evaluating the predictive value of leukocyte membrane TKFC as a biomarker for inflammatory infections. p <0.05 is statistically significant.

[0143] The results are shown in the table below. Figure 7-8 As shown.

[0144] Table 2. Core Table of Diagnostic Efficacy of ROC Curve for Inflammatory Indicators

[0145] It was found that the expression level of TKFC on the surface of monocytes and neutrophils in patients with inflammation was significantly higher than that in healthy individuals, and the difference was statistically significant. In this invention, based on TKFC on the surface of leukocyte cell membranes as a marker, the AUC values ​​for monocytes and neutrophils in the ROC curve were 0.9146 and 0.8771, respectively. Among them, the cut-off value of the positive percentage of neutrophils was 13.98%, with a sensitivity of 86.00% (95% confidence interval: 77.63%-92.13%) and a specificity of 81.40% (95% confidence interval: 71.55%-88.98%). The cut-off value of the mean fluorescence intensity of monocytes was 3148, with a sensitivity of 77.90% (95% confidence interval: 68.12%-85.43%) and a specificity of 93.00% (95% confidence interval: 86.33%-96.20%). A retest using KA-007 revealed that it also had an extremely high AUC value, thus proving that both indicators have high diagnostic ability and application value for inflammation.

[0146] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. The use of trikinase / FMN cyclase (TKFC) on the cell membrane of immune cells as a marker on the surface of immune cells to characterize the state of immune cells and / or the inflammatory condition of the body; in, The immune cell states include resting state and activated state; The inflammation status of the body includes the presence or absence of inflammation and / or the degree of inflammation.

2. The use according to claim 1, characterized in that, The immune cells include white blood cells; Preferably, the leukocytes include myeloid cells; Preferably, the white blood cells include monocytes and granulocytes.

3. The use according to claim 1, characterized in that, The immune cells also include macrophages, dendritic cells, and mast cells.

4. The use according to claim 2 or 3, characterized in that, The immune cells are derived from animals; Preferably, the animal is a vertebrate; Preferably, the vertebrates include humans and non-human mammals.

5. The use according to claim 1, characterized in that, The inflammation includes systemic inflammation and local inflammation.

6. The use according to claim 1, characterized in that, The inflammation described is caused by a pathogen; Preferably, the pathogen includes at least one of bacteria, fungi, viruses, parasites, mycoplasma, chlamydia, rickettsia, and prions.

7. The use of substances that detect TKFC on the cell membrane of immune cells in the preparation of products for diagnosing inflammation; Preferably, the detection includes quantitative and / or qualitative detection; Preferably, the product includes a detection reagent, a detection kit, and a detection chip.

8. The use according to claim 7, characterized in that, The substance enables the detection of TKFCs on the cell membrane of immune cells using at least one of the following methods: Fluorescence microscopy, immunohistochemistry, flow cytometry, Western blotting, enzyme-linked immunosorbent assay (ELISA), mass spectrometry, radioligand binding, and CRISPR-based labeling techniques. Preferably, the method does not include the step of lysing or breaking down immune cells.

9. The use according to claim 7, characterized in that, The substance includes antibodies; Preferably, the antibody comprises a monoclonal antibody against TKFC.

10. The use according to any one of claims 7-9, characterized in that, The product also includes substances for detecting immune-active factors and / or immune cell markers; Preferably, the immune-active factors include immune cell cytokines; Preferably, the immune cell cytokines include interferon-γ; Preferably, the immune cell markers include differentiation cluster (CD) proteins.

11. The use according to claim 10, characterized in that, The test samples for the product include at least one of peripheral blood, pleural effusion, ascites, cerebrospinal fluid, and other tissue exudates.

12. Use of substances that detect TKFC on the cell membrane of immune cells in the preparation of products characterizing the state of immune cells.

13. A method for characterizing the state of immune cells, comprising: The expression of TKFC on the cell membrane of immune cells was detected, and the state of immune cells was determined by comparing the expression differences of TKFC on resting immune cells. Preferably, the resting immune cells include immune cells derived from healthy individuals or non-inflammatory patients.

14. The method according to claim 13, characterized in that, The criteria for determining the state of immune cells are as follows: When the expression level of TKFC on the cell membrane of the immune cell to be tested is higher than the baseline level, the immune cell to be tested is an activated immune cell; When the level is equal to or lower than the baseline level of resting immune cells, the immune cells to be tested are resting immune cells. Preferably, the baseline includes the expression level of TKFC on the cell membrane of leukocytes from non-inflammatory patients; Preferably, the non-inflammatory patient includes a healthy person.

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