Application of urinary exosome proteins as diagnostic markers for viral pneumonia

Abstract

This invention provides an application of urinary exosome proteins as diagnostic biomarkers for viral pneumonia. The urinary exosome proteins include one or more combinations of ANPEP, ASAH1, COL11A1, EHD4, HEXB, LGALS3BP, SERPINA1, and SERPING1. By using extracted urinary exosomes for the detection of protein-based biomarkers related to viral pneumonia, effective diagnosis of viral pneumonia can be achieved.

Description

Technical Field

[0001] This invention relates to the field of in vitro diagnostic reagents, and more particularly to the application of a urinary exosome protein as a diagnostic marker for viral pneumonia. Background Technology

[0002] Viral pneumonia is an inflammation of the lungs caused by upper respiratory tract viral infection that spreads downwards, generally presenting with symptoms such as dry cough and fever. Mycoplasma pneumonia is a lower respiratory tract infection caused by Mycoplasma pneumoniae, and its most common clinical manifestations are fever and cough. Because viral pneumonia and mycoplasma pneumonia have similar symptoms, auxiliary examinations are needed in clinical practice to aid in diagnosis.

[0003] Diagnostic methods for viral pneumonia include clinical manifestations, chest X-ray, and etiological examination. Currently, the diagnosis of viral pneumonia primarily relies on clinical manifestations and chest X-ray findings. Increased lung markings and pulmonary infiltrates can serve as diagnostic criteria, but definitive diagnosis requires etiological examination to rule out other pathogens. Clinical experience suggests that interstitial lung infiltrates indicate viral infection, while alveolar infiltrates indicate bacterial infection. However, chest X-rays are not very sensitive in differentiating between bacterial and bacterial pulmonary pneumonia, as they cannot differentiate between pneumonias caused by different pathogens. Definitive diagnosis depends on etiological examination, including virus isolation, antigen-antibody detection, serological tests, and complete blood count (CBC). Virus isolation requires collecting lower respiratory tract secretions, which is difficult to sample, has a high chance of contamination, and is difficult to detect at low viral infection levels. Specific antigen-antibody detection and serological tests require multiple blood draws, are time-consuming, and can only serve as retrospective diagnoses, lacking early diagnostic value. CBC parameters are based on empirical judgment and are neither sensitive nor specific.

[0004] Currently, initial treatment for viral pneumonia is empirical in both developed and developing countries. As for commonly used methods for detecting viral pneumonia, clinical manifestations and chest X-ray findings rely heavily on experience for judgment, lacking formal evidence and thus hindering widespread adoption. Furthermore, pathogen testing is difficult to sample, prone to contamination, cumbersome, and time-consuming, and therefore does not play a significant role in the diagnosis of viral pneumonia.

[0005] In view of this, the present invention is hereby proposed. Summary of the Invention

[0006] The purpose of this invention is to provide an application of urinary exosome proteins, which rapidly extract urinary exosomes using a kit, and then use the extracted urinary exosomes for the detection of protein-based biomarkers for viral pneumonia, thereby achieving effective diagnosis of viral pneumonia.

[0007] To achieve the above objectives, the present invention employs the following technical solution:

[0008] This invention relates to the application of a quantitative detection reagent for urinary exosome proteins in the preparation of a kit for the diagnosis of viral pneumonia.

[0009] The inventors extracted urinary exosomes using a precipitation method kit. Through principal component analysis of urinary exosome proteomics, they identified eight proteins that showed significant differences between the viral and mycoplasma pneumonia groups and the healthy group. These proteins are ANPEP, ASAH1, COL11A1, EHD4, HEXB, LGALS3BP, SERPINA1, and SERPING1.

[0010] ANPEP protein is a membrane-bound zinc-dependent glycoprotein involved in cell recognition and processing of various peptides. It plays a role in angiogenesis, promoting cholesterol crystallization, and amino acid transport. Located in the microvilli membrane of the small intestine and kidney, it is used for the final digestion of peptides produced by gastric and trypsin hydrolysis of proteins. It can also act as a receptor for human coronavirus 229E / HCoV-229E, which can cause respiratory symptoms in humans.

[0011] ASAH1 protein can bind to calcium-induced differentiation of epidermal keratinocytes, indirectly regulate tumor necrosis factor-induced apoptosis, and also act as a regulator of steroid production. In addition, it can enhance the catalytic efficiency of C12-ceramide, allowing the synthesis of ceramide from fatty acids and sphingosine. Ceramide can regulate the moisture balance of the stratum corneum and maintain skin hydration. Furthermore, ASAH1 protein is also closely related to human metabolism.

[0012] COL11A1 protein is a major component of the basement membrane reticular structure. After being synthesized intracellularly, it directly participates in the formation of the extracellular matrix through procollagen. It can control the intracellular assembly of procollagen molecules and the extracellular assembly of collagen fibrils, playing a key role in tissue growth and repair, and participating in proteoglycan metabolism.

[0013] EHD4 is an ATP and membrane-bound protein that controls membrane remodeling or microtubule formation during ATP hydrolysis. It plays a role in early endosome transport and can participate in the cellular endocytosis cycle, meaning it can influence the process by which extracellular substances enter the cell through membrane invagination and internalization.

[0014] HEXB protein is an enzyme found in lysosomes that hydrolyzes non-reducing n-acetyl-d-hexosamine and / or sulfated n-acetyl-d-hexosamine of glycoconjugates. When HEXB protein is deficient, Gm2 ganglion lipidosis occurs, more accurately known as acute early infantile form (Sandhoff's disease). This leads to symptoms such as decreased muscle tone, inability to sit or stand, blindness, seizures, and mild hepatosplenomegaly that gradually appear in infants around 6 months of age.

[0015] LGALS3BP protein is a galactoglobulin-3 binding protein that can promote integrin-mediated cell adhesion and stimulate the host's defense against viruses and tumor cells. It is also associated with immune responses related to natural killer and lymphokine-activated cytotoxicity.

[0016] SERPINA1 protein is a serine protease inhibitor whose main target is elastase. It can also irreversibly inhibit trypsin, chymotrypsin, and plasminogen activator. It is produced in lymphocytes and monocytes in the liver, bone marrow, lymphoid tissues, and Paneth cells in the intestine. It is associated with chronic obstructive pulmonary disease, emphysema, and chronic liver disease. It can also protect the respiratory tract from the proteolytic damage of human leukocyte elastase (hLE).

[0017] SERPING1 protein is a serine protease inhibitor that plays an important role in regulating key physiological pathways such as complement activation, blood coagulation, fibrinolysis, and kinin production. Furthermore, it can visualize platelet cytoplasmic calcium levels. 2+ Elevated levels of disease and reactions, and untreated pneumonia can lead to diseases such as myocarditis, while platelet cytoplasmic calcium... 2+ Changes in heart function can indicate whether there are abnormalities in the heart's diastolic function, and this can be used to infer whether someone has viral pneumonia.

[0018] Experimental studies have shown that the levels of the above eight proteins are significantly higher in patients with viral pneumonia than in healthy individuals and patients with mycoplasma pneumonia, and that they can be formulated into a diagnostic kit for viral pneumonia.

[0019] All of the aforementioned proteins are present in urine. Using urine as the test sample is easy to obtain, non-invasive, and has good patient compliance. Furthermore, exosomes are widely present in urine. Exosomes are membrane-bound vesicles released into the extracellular matrix by different types of cells, and their function is to participate in intercellular molecular transport. It is well known that exosomes from different sources differ in composition and function due to variations in the proteins and other components they carry, thus performing different biological functions. Simultaneously, these differences are dynamically regulated by the extracellular matrix and microenvironment. This means that exosomes play different roles in various diseases and can serve as biomarkers associated with viral pneumonia, providing rich, stable, and specific biological information.

[0020] Preferably, the quantitative detection reagent is one of the following: a specific antibody, aptamer, receptor, ligand, and polypeptide of the urinary exosome protein.

[0021] Preferably, the quantitative detection reagent is a specific antibody against the urinary exosome protein.

[0022] Preferably, the specific antibody is one of polyclonal antibodies, monoclonal antibodies, antibody fragments, synthetic antibodies, single-chain antibodies, and IgG type antibodies. In this invention, there are no special limitations on the source of the antibody; commercially available products are acceptable.

[0023] Preferably, the specific antibody is an IgG antibody.

[0024] Preferably, the quantitative detection method for urinary exosome proteins includes one of the following: proteomic profiling, protein microarray, Western blotting, ELISA, Luminex, flow cytometry, immunohistochemistry, immunofluorescence, immunochemiluminescence, colloidal gold, and chemical detection.

[0025] This invention also relates to a kit for the diagnosis or auxiliary diagnosis of viral pneumonia, comprising the specific antibodies defined above. This kit enables the extraction of high yields of exosomes from a subject's urine sample, which can be directly used for subsequent diagnostic applications.

[0026] Preferably, the kit further includes at least one of a solid support, blocking solution, chromogenic agent, calibrator of fusion antigen, and washing buffer.

[0027] Preferably, the solid support includes one of agar balls, magnetic beads, particulate matter, and solid-phase chips.

[0028] Preferably, the kit also includes reagent A, reagent B, agar balls, biotin-labeled reagent, Glycine-HCl, and buffer solution.

[0029] The inventors have discovered that known viral pneumonia-related disease biomarkers can be detected and / or quantified by exosomes enriched in urine, and the results can be used for the differential diagnosis of viral pneumonia-related diseases.

[0030] The inventors have discovered that viral pneumonia protein biomarkers, such as ANPEP, ASAH1, COL11A1, EHD4, HEXB, LGALS3BP, SERPINA1, and SERPING1, can be detected in extracted urinary exosomes. Protein biomarkers exposed on the exosome surface can be detected directly without lysis. For protein biomarkers contained within exosomes, lysis of the exosomes is required before detection. Western blotting, ELISA, Luminex, and flow cytometry are preferred methods for detecting protein biomarkers. Specifically, proteomic techniques, Western blotting, ELISA, Luminex, and flow cytometry can be used to identify viral pneumonia-related protein biomarkers.

[0031] It is important to understand that the term "marker" as used herein refers to a molecule intended to be used as a target for analyzing patient laboratory samples. Examples of such molecular targets are proteins or peptides. Proteins or peptides used as markers in this invention are expected to include naturally occurring variants of said protein as well as fragments of said protein or said variants, particularly immunologically detectable fragments.

[0032] In this invention, urinary exosomes are collected using a kit-based method. Protein expression products are detected using mass spectrometry, immunohistochemistry, immunofluorescence, Western blotting, protein microarray, ELISA, flow cytometry, immunochemiluminescence, colloidal gold, and chemical methods. Transmission electron microscopy (TEM), nanoparticle tracking detection (NTA), and Western blotting are used to identify the exosomes. Results show that the urinary exosomes obtained using this method have similar quality to total exosomes obtained using other exosome separation techniques (such as ultracentrifugation). Currently, traditional physical methods (such as ultracentrifugation and ultrafiltration) yield exosomes with low purity and low yield, while the method of this invention does not involve ultracentrifugation, is easy to operate, and yields high purity and high efficiency, making it highly practical.

[0033] Compared with the prior art, the advantages of the present invention are:

[0034] (1) This invention uses urinary exosomes as the test sample, which is non-invasive and harmless to patients;

[0035] (2) The exosome enrichment method of the present invention is simple to operate, does not involve expensive precision instruments, has greater universality, and can be met by general testing laboratories, making it more suitable for promotion in clinical applications.

[0036] (3) The urinary exosome proteins ANPEP, ASAH1, COL11A1, EHD4, HEXB, LGALS3BP, SERPINA1, and SERPING1 used in this invention can clearly distinguish between viral pneumonia and mycoplasma pneumonia as well as the differences between healthy individuals, which helps medical personnel to make early judgments and diagnose and treat viral pneumonia. Attached Figure Description

[0037] By reading the limitations of this invention. Furthermore, throughout the drawings, the same reference numerals denote the same parts. In the drawings:

[0038] Figure 1 This is a flowchart of the experiments in Examples 1-7 of the present invention;

[0039] Figure 2 The results of transmission electron microscopy (TEM) of urinary exosomes provided in Example 3;

[0040] Figure 3 The dispersibility, particle size, and distribution of urinary exosomes provided in Example 4;

[0041] Figure 4 Individual ROC curves for the eight specific proteins provided in Example 6 (children with viral pneumonia vs. healthy children);

[0042] Figure 5 Individual ROC curves for the eight specific proteins provided in Example 6 (children with viral pneumonia vs. children with mycoplasma pneumonia);

[0043] Figure 6 The ROC curves for the eight specific proteins provided in Example 6 (children with viral pneumonia vs. healthy children);

[0044] Figure 7 ROC curves of the eight specific proteins provided in Example 6 (children with viral pneumonia vs. children with mycoplasma pneumonia);

[0045] Figure 8 Individual ROC curves for the eight specific proteins provided in Example 7 (patients with viral pneumonia vs. healthy volunteers);

[0046] Figure 9 The ROC curves for the eight specific proteins provided in Example 7 (patients with viral pneumonia vs. healthy volunteers). Detailed Implementation

[0047] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0048] In some embodiments, the isolated material is primarily exosomes, but also includes a small amount of other types of extracellular vesicles such as microvesicles. The total exosomes described in the examples are almost entirely exosomes, but may inevitably include a very small amount of other sizes of vesicles such as microvesicles.

[0049] In some embodiments, recognition and binding agents of ANPEP, ASAH1, COL11A1, EHD4, HEXB, LGALS3BP, SERPINA1, and SERPING1 in the art include, but are not limited to, DNA, RNA, antibodies, antibody fragments, exogenous lectins, chemical compounds, ligands, and combinations thereof.

[0050] In some embodiments, the antibodies described in this invention can be anti-animal antibodies, particularly primate antibodies. ANPEP, ASAH1, COL11A1, EHD4, HEXB, LGALS3BP, SERPINA1, and SERPING1 antibodies can be prepared by methods known in the art or obtained commercially.

[0051] It should also be noted that common exosome biomarkers include CD9, CD63, etc.

[0052] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0053] Unless otherwise specified, all materials and reagents used in the following examples were purchased from commercial reagents.

[0054] The corresponding reagents are not limited to the companies and models listed below; corresponding models from other companies or self-synthesized reagents can also be used.

[0055] Unless otherwise specified, the buffer solutions in these embodiments of the invention are PBS buffer and DPBS balanced salt solution. The elution buffer is glycine-HCl (pH 3.0).

[0056] like Figure 1 As shown, the present invention provides a flowchart of an implementation scheme. The implementation scheme of the present invention will be described in detail below with reference to the embodiments.

[0057] Example 1: Clinical Sample

[0058] (1) Clinical data

[0059] Pathogen diagnosis is performed through routine laboratory tests, including nasal and pharyngeal swabs to detect mycoplasma antibodies, mycoplasma, and RNA or DNA of various viruses.

[0060] The included population consisted of children with an average age of 5 years. Seven children with viral pneumonia, four children with mycoplasma pneumonia, and 20 healthy children were selected. Healthy controls were children without any respiratory illness or fever symptoms.

[0061] (2) Sample collection

[0062] Collect 20-30 ml of urine sample from each child and store at -80°C.

[0063] Example 2: Extraction of Urinary Exosomes

[0064] Exosomes were extracted from the samples collected in Example 1 using the Exosome Isolation Q3 kit (EIQ3-03001, Wayen Biotechnologies, China). For the extraction of exosomes from the urine of healthy children, four urine samples were pooled together to form one pool sample, for a total of five pools of urine samples from healthy children. A total of 16 urine exosomes were obtained, comprising 7 children with viral pneumonia, 4 children with mycoplasma pneumonia, and 5 healthy children. The specific procedures are as follows:

[0065] (1) Thaw the frozen urine sample in a 37°C water bath until completely thawed, then place it on ice;

[0066] (2) Urine samples were centrifuged at 4℃, 3000×g for 15 min;

[0067] (3) Transfer the supernatant to a new centrifuge tube and place it on ice;

[0068] (4) Take 20 ml of the centrifuged urine sample, add 7.5 ml of extraction reagent A, and invert the sample to mix it thoroughly.

[0069] (5) Add 670 μl of extraction reagent B to the mixture from step (4) and invert the mixture to mix it thoroughly.

[0070] (6) Let the mixture from step (5) stand and incubate at 4°C overnight (12-16h);

[0071] (7) After incubation, centrifuge at 4℃, 3000×g for 60min;

[0072] (8) Take 1 ml of supernatant into a 1.5 ml EP tube, and then completely remove any other residual supernatant;

[0073] (9) Repeatedly blow the bottom of the tube with the residual liquid taken out in step 8) to fully resuspend the precipitate, and transfer the suspension to a 1.5ml EP tube;

[0074] (10) Centrifuge the resulting suspension at 4℃, 10000×g for 10 min, and discard the supernatant;

[0075] (11) Resuspend the precipitate in 1×PBS and repeatedly pipette until homogeneous;

[0076] (12) Centrifuge the suspension from step (11) at 4°C, 10000×g for 5 min, and transfer the supernatant to a new 1.5ml EP tube;

[0077] (13) The supernatant obtained in step (12) is the PBS suspension of urine exosomes.

[0078] In the above steps, extraction reagent A and extraction reagent B used in steps (4) and (5) are both components of the EIQ3 exosome extraction kit developed by Huaying Biotechnology.

[0079] Example 3: Characterization of urinary exosomes

[0080] This invention uses transmission electron microscopy to examine the size and morphology of urinary exosomes obtained in Example 2. The extracted exosomes exhibit a typical "cup-shaped" structure. Nanoparticle tracking analysis (NTA) was used to determine the exosome dispersion, particle size, and distribution; the exosome particle diameter was between 50-150 nm. BCA was used to detect the exosome concentration; there was no difference in concentration among the three groups of urinary exosomes. The specific operating steps are as follows:

[0081] (1) Electron microscopy (TEM) analysis of exosomes

[0082] 1) Take 5 μL of the urine exosome sample from Example 2 and add it to a copper grid. Incubate at room temperature for 5 min.

[0083] 2) After incubation, use absorbent paper to blot away excess liquid on one side;

[0084] 3) Add one drop of 2% uranium acetate to the copper mesh and incubate at room temperature for 1 minute;

[0085] 4) After incubation, use absorbent paper to blot away excess liquid on one side;

[0086] 5) Dry at room temperature for about 20 minutes, then observe on the instrument (Tecnai G2 Spirit BioTwin, FEI), with the accelerating voltage set to 80kV.

[0087] (2) Exosome particle detection

[0088] 1) Take the frozen sample, thaw it in a 25°C water bath, and place it on ice;

[0089] 2) Turn on the switch on the back of the host and click to turn on ZetaView on the computer connected to the host;

[0090] 3) Software initialization;

[0091] 4) Inject 10ml of ultrapure water into the one-way valve on the right side of the main unit using a disposable syringe, and the software will detect the cleanliness of the sample cell.

[0092] 5) Prepare a PS100 standard suspension, slowly inject PS100 into the sample cell, and start laser and camera focusing after the particles stabilize;

[0093] 6) Measure the blank control group;

[0094] 7) Set camera parameters;

[0095] 8) Once the video has stabilized, click the "Run Video Acquisition" button;

[0096] 9) Enter the sample measurement interface, set the parameters, and select the appropriate SOP;

[0097] 10) Click Experiment Parameters, confirm all, and start the test;

[0098] 11) Inject 3-5 ml of the diluted sample suspension through the one-way valve using a syringe;

[0099] 12) After the particles stabilize, set the corresponding parameters, confirm all parameters, and then start the test;

[0100] 13) After the software testing and analysis are completed, the software will automatically obtain and save the particle size distribution report.

[0101] (3) Detection of exosomal protein concentration

[0102] Exosomal protein concentration was detected using the BCA Protein Assay Kit (Enhanced Version) (P0009, Beyotime):

[0103] 1) Preparation of protein standards

[0104] a) Add 1.2 ml of protein standard preparation solution to a tube of protein standard (30 mg BSA), dissolve it completely, and prepare a 25 mg / ml protein standard solution;

[0105] b) Take an appropriate amount of 25 mg / ml protein standard solution and dilute it with 1×PBS to a final concentration of 0.5 mg / ml;

[0106] 2) BCA work is also equipped

[0107] Based on the number of samples, prepare an appropriate amount of BCA working solution by adding 1 volume of BCA reagent B to 50 volumes of BCA reagent A (50:1) and mix thoroughly.

[0108] 3) Protein concentration detection

[0109] a) Add the standard to the standard wells of a 96-well plate at concentrations of 0 μl, 1 μl, 2 μl, 4 μl, 8 μl, 12 μl, 16 μl, and 20 μl, respectively, and then add 1×PBS to bring the total volume to 20 μl. This corresponds to standard concentrations of 0 mg / ml, 0.025 mg / ml, 0.05 mg / ml, 0.1 mg / ml, 0.2 mg / ml, 0.3 mg / ml, 0.4 mg / ml, and 0.5 mg / ml, respectively. Perform two replicates for each concentration.

[0110] b) Add 2 μl of sample to the wells of the 96-well plate, and add 1×PBS to bring the total to 20 μl;

[0111] c) Add 200 μl of BCA working solution to both the standard wells and the sample wells, and incubate at 37°C for 25 min;

[0112] d) Measure the absorbance at a wavelength of 570 nm using an enzyme-linked immunosorbent assay (ELISA) reader;

[0113] e) Calculate the protein concentration of the sample based on the standard curve and the sample dilution factor.

[0114] Summarize the experimental results. Figure 2 This is the result of transmission electron microscopy of urinary exosomes.

[0115] Example 4: Urinary Exosome Proteomics Analysis

[0116] The urine exosomes obtained in Example 2 were analyzed by label-free quantitative proteomics using liquid chromatography-tandem mass spectrometry (LC-MS).

[0117] (1) Protein sample pretreatment

[0118] 1) Add an equal volume of protein lysis buffer (7M urea, 2% SDS, add 1× protease inhibitor cocktail before use);

[0119] 2) Use an ultrasonic cell disruptor, lyse on ice for 2 seconds, pause for 5 seconds, for a total of 1 minute, and then lyse on ice for 2 hours after completion;

[0120] 3) Centrifuge the lysis product at 4°C and 13,000 rpm for 20 min, and transfer the supernatant to a new 1.5 ml EP tube;

[0121] 4) Add 6 times the volume of 100% acetone and let it precipitate overnight at -20°C;

[0122] 5) Centrifuge to obtain precipitate, wash the precipitate twice with 500 μl of pre-cooled washing buffer (ethanol:acetone:acetic acid = 50:50:0.1), centrifuge at 13000 rpm for 15 min at 4℃, redissolve the precipitate with (6M guanidine hydrochloride, 300 mM TEAB) and determine the sample concentration again.

[0123] 6) Store in a refrigerator at 4℃ until needed, and then dilute a portion and determine the concentration using the BCA method.

[0124] (2) FASP (Filter Aided Proteome Preparation) enzymatic hydrolysis

[0125] 1) Take the same mass of protein solution sample as the exosome sample and dilute it to 100 μl with 25 mM ammonium bicarbonate;

[0126] 2) Reductive alkylation: Add 1M DTT to the protein solution (add at a ratio of 2 μl 1M DTT / 100 μl protein, final concentration 20 mM), mix well, and incubate at 57°C for 1 h. Add 10 μl 1M Iodoacetimide / 100 μl solution (final concentration 90 mM, solid powder, dissolved in 25 mM ammonium bicarbonate, freshly prepared and used immediately), mix well, and incubate at room temperature in the dark for 40 min.

[0127] 3) Add the reduced alkylated protein to a 10K ultrafiltration tube, centrifuge at 12,000 rpm, and discard the solution at the bottom of the collection tube;

[0128] 4) Add ammonium bicarbonate (dissolution buffer) to the ultrafiltration tube and wash 4 times;

[0129] 5) Add Trypsin prepared with Dissolution Buffer and incubate overnight at 37°C;

[0130] 6) Collect the enzymatically digested peptides by centrifugation, then concentrate and dry them by centrifugation.

[0131] (3) Desalination

[0132] 1) Dissolve the dried mixed peptides in a 0.1% trifluoroacetic acid (TFA) solution;

[0133] 2) Activate the desalination column using 100% acetonitrile;

[0134] 3) Equilibrate the desalting column using 0.1% TFA solution;

[0135] 4) Add the reconstituted sample to the desalting column and centrifuge;

[0136] 5) Add 0.1% TFA solution to wash the desalting column;

[0137] 6) Add 50% acetonitrile solution, centrifuge, elute the peptides, and collect the elution solution using a new EP tube;

[0138] 7) Centrifuge, concentrate, and dry the elution solution to remove acetonitrile.

[0139] (4) LC-MS analysis

[0140] After vacuum drying, the sample was reconstituted with 0.1% FA, and 1-2 μg of sample was loaded onto the spectrometer. Separation was performed using an EASY-nLC1200 (Thermo Scientific, USA) with an analytical gauge (C18, 2 μm, 50 μm × 15 cm) at a flow rate of 300 nl / min. The mass spectrometer was in QE-HF-X (Thermo Scientific, USA) mode. Tandem mass spectrometry was performed in data-dependent acquisition (DDA) mode. The full scan resolution was 60,000 (FWHM), the mass-to-charge ratio range was set to m / z 350-2000, and the collision energy was set to 28% in HCD fragmentation mode.

[0141] Experimental results are as follows Figure 3 As shown, a total of 1621 proteins were identified, including the typical exosome marker proteins CD9, CD63, CD81, ALIX, and TSG101.

[0142] Example 5: Identification of Specific Differential Proteins in Viral Childhood Pneumonia

[0143] Principal component analysis (PCA) of urinary exosome proteomics obtained in Example 2 showed significant differences between the children with viral pneumonia, the children with mycoplasma pneumonia, and the healthy children.

[0144] Statistical comparisons identified 260 proteins that differed among children with viral pneumonia, children with mycoplasma pneumonia, and healthy children. Further analysis based on protein function and fold change identified eight proteins as specific biomarkers for viral pneumonia in children. These eight proteins are: ANPEP, ASAH1, COL11A1, EHD4, HEXB, LGALS3BP, SERPINA1, and SERPING1.

[0145] Example 6: Validation of Potential Urinary Exosomal Protein Markers for the Diagnosis of Viral Pneumonia in Children

[0146] The population included in the validation included 12 children with viral pneumonia, 17 children with mycoplasma pneumonia, and 20 healthy children. The diagnostic criteria were consistent with those in Example 1.

[0147] Urine samples were collected, and exosomes were extracted using the Exosome Isolation Q3 kit. The procedure was the same as in Example 2. A total of 49 exosome samples were obtained.

[0148] These proteins were detected in the exosomes of the aforementioned validation population using multiple reaction monitoring (MRM) targeted proteomics technology. The specific steps are as follows:

[0149] (1) The protein sample pretreatment, FASP digestion and desalting methods are the same as in Example 4.

[0150] (2) Screening of target protein quantitative characteristic peptides

[0151] The eight viral pneumonia-specific differentially expressed proteins identified in Example 5—ANPEP, ASAH1, COL11A1, EHD4, HEXB, LGALS3BP, SERPINA1, and SERPING1—were detected.

[0152] (3) MRM mode detection of target peptides

[0153] 1) Liquid phase conditions

[0154] Phase A: 2% ACN, 0.1% FA

[0155] Phase B: 98% ACN, 0.1% FA

[0156] Flow rate: 5 μl / min

[0157] Gradient conditions: 0–5 min, 95% A, 5% B; 5–105 min, 70%, 30% B; 105–115 min, 20% A, 80% B; 115–120 min, 98% A, 2% B.

[0158] 2) Mass spectrometry conditions

[0159] The ion source was an electrospray ionization (ESI) source; detection was performed using positive ions; the scanning mode was multiple reaction monitoring (MRM); the jet voltage was 5500 eV; the temperature was 150 °C; the curtain gas (CUR, N2) pressure was 30 psi; the collision gas pressure (CAD, N2) was in High mode; the auxiliary gas GAS1 pressure was 20 psi; the auxiliary gas GAS2 pressure was 15 psi; and the scan time was 10 ms. The declustering voltage (DP) and collision energy (CE) are detailed in the file: 0_CE&DP optimization parameters.xlsx (to be provided as an attachment for published papers); a scheduled MRM acquisition method was used, with a total scan time of 1.7 s and an MRM detection window of 300 s.

[0160] (4) Evaluation of Results

[0161] The efficacy of eight proteins in diagnosing viral pneumonia in children was evaluated using receiver operating characteristic (ROC) curves. The horizontal axis (1-Specificity) represents the false positive rate (FPR), while the vertical axis (Sensitivity) represents the true positive rate (TPR).

[0162] A larger area under the ROC curve (AUC) indicates better diagnostic accuracy; the value ranges from 0.5. <AUC<1。

[0163] ROC curve analysis showed that all eight proteins had high diagnostic accuracy for viral childhood pneumonia. Figure 4 Children with viral pneumonia vs. healthy children; Figure 5 Children with viral pneumonia vs. children with mycoplasma pneumonia).

[0164] When eight proteins were used in combination for diagnosis, the ROC curve is shown below. As can be seen from the figure, the AUC reached 1.0, which can completely distinguish viral pneumonia in children. Figure 6 Children with viral pneumonia vs. healthy children; Figure 7 Children with viral pneumonia vs. children with mycoplasma pneumonia).

[0165] Example 7: Potential Urinary Exosomal Protein Markers for the Diagnosis of Viral Pneumonia in Adults

[0166] The population included in the validation included 20 adult patients with viral pneumonia and 20 healthy volunteers. The diagnostic criteria were consistent with those in Example 1.

[0167] Urine samples were collected, and exosomes were extracted using the Exosome Isolation Q3 kit. The procedure was the same as in Example 2. A total of 40 exosome samples were obtained.

[0168] These proteins were detected in the exosomes of the aforementioned validation population using multiple reaction monitoring (MRM) targeted proteomics technology. The specific steps were the same as in Example 6.

[0169] The efficacy of eight proteins in diagnosing viral pneumonia in adults was evaluated using receiver operating characteristic (ROC) curves. ROC curve analysis showed that all eight proteins had high diagnostic accuracy for viral pneumonia in adults. Figure 8(Viral pneumonia patients vs. healthy volunteers). When using a combination of 8 proteins for differentiation, the AUC of the ROC curve reached 1.0, which can completely distinguish viral pneumonia in adults. Figure 9 (Viral pneumonia patients vs. healthy volunteers).

[0170] As Figure 1 As shown, in this invention, Example 1 selected samples, including children with viral pneumonia, children with mycoplasma pneumonia, and healthy children for the experiment; Example 2 extracted urinary exosomes; Example 3 analyzed the dispersibility, particle size, and distribution of the collected exosomes; Example 4 analyzed the proteomics of urinary proteins; Example 5 found that eight proteins in viral pneumonia had significant specificity; and Examples 6 and 7 verified and detected these eight proteins.

[0171] In summary, based on the experimental procedures and results of Examples 1-7 above, it is evident from Examples 1-5 that, when comparing healthy children with children suffering from viral pneumonia and mycoplasma pneumonia, the eight proteins ANPEP, ASAH1, COL11A1, EHD4, HEXB, LGALS3BP, SERPINA1, and SERPING1 showed specificity. Furthermore, as verified by Example 6, [the results were further confirmed]. Figure 4 It can be seen that the AUC of all proteins in children with viral pneumonia is greater than 0.5. Except for the ASAH1 protein, whose AUC is close to 0.7 (0.686), the AUC of the other 7 proteins is ≥0.7. The experimental accuracy is high, indicating that any of these eight proteins can distinguish children with viral pneumonia from healthy children. Figure 5 The results showed that the AUC of all eight proteins was greater than 0.5, indicating that any one of the eight proteins could differentiate between viral and mycoplasma pneumonia in children. Meanwhile, as... Figure 6 and Figure 7 The combined AUC of these eight proteins reached 1, indicating that their combined use can differentiate viral pneumonia in children from mycoplasma pneumonia in children and healthy children. This suggests that the combined use of these eight proteins can be considered an ideal diagnostic indicator. Furthermore, as verified in Example 7, these eight proteins, used alone or in combination, can differentiate viral pneumonia in adults from healthy adults, suggesting that the use of any one of these eight proteins can be considered an ideal diagnostic indicator. Therefore, it can be fully demonstrated that the eight proteins ANPEP, ASAH1, COL11A1, EHD4, HEXB, LGALS3BP, SERPINA1, and SERPING1 can clearly and rapidly differentiate viral pneumonia, facilitating prompt treatment.

[0172] Finally, it is understood that the above embodiments are merely exemplary embodiments used to illustrate the principles of the present invention, and the present invention is not limited thereto. For those skilled in the art, various modifications and improvements can be made without departing from the principles and essence of the present invention, and these modifications and improvements are also considered to be within the scope of protection of the present invention.

Claims

1. The application of a combined quantitative assay for the detection of the following urinary exosome proteins in the preparation of a kit for the diagnosis of viral pneumonia, wherein the urinary exosome proteins include EHD4, ANPEP, ASAH1, COL11A1, HEXB, LGALS3BP, SERPINA1, and SERPING1.

2. The application according to claim 1, characterized in that, The quantitative detection reagent is one of the specific antibodies, aptamers, receptors, and ligands of the urinary exosome proteins.

3. The application according to claim 2, characterized in that, The quantitative detection reagent is a specific antibody against the urinary exosome protein.

4. The application according to claim 3, characterized in that, The specific antibody is one of the following: polyclonal antibody, monoclonal antibody, antibody fragment, synthetic antibody, single-chain antibody, and IgG antibody.

5. The application according to claim 4, characterized in that, The specific antibody is an IgG type antibody.

6. The application according to claim 1, characterized in that, The quantitative detection methods for urinary exosome proteins include one of the following: proteomic profiling, protein microarray, Western blotting, ELISA, Luminex, flow cytometry, immunohistochemistry, immunofluorescence, immunochemiluminescence, colloidal gold, and chemical detection.

7. A reagent kit for diagnosing viral pneumonia, characterized in that, It includes the specific antibody as defined in any one of claims 2-4.

8. The reagent kit according to claim 7, characterized in that, It also includes at least one of a solid support, blocking solution, colorimetric reagent, calibrator of fusion antigen, and washing buffer.

9. The reagent kit according to claim 8, characterized in that, The solid support includes one of the following: agar balls, magnetic beads, particulate matter, and solid-phase chips.

Images