Use of substances that detect exosome CA125 in the manufacture of products for diagnosing or assisting in the diagnosis of lung diseases.

Abstract

The present invention belongs to the field of biodetection technology, and more specifically, relates to the use of a substance for detecting exosome CA125 in the manufacture of products for diagnosing or assisting in the diagnosis of lung diseases. The present invention provides an exosome processing solution, and by pre-treating an exosome sample to be measured using the exosome processing solution provided by the present invention, the influence of components in the eluate on exosome protein detection is reduced, the detection time is effectively shortened, stable detection of exosome proteins is achieved, and it is advantageous for realizing fully automated detection of exosome proteins.

Description

Technical Field

[0001] The present invention belongs to the field of biodetection technology, and specifically relates to the use of substances for detecting exosomal CA125 in the manufacture of products for diagnosing or assisting in the diagnosis of lung diseases.

[0002] (Related Application) This application claims the priority of Chinese Patent Application No. 2023105475792 filed on May 15, 2023, the entire content of which is incorporated herein by reference and is cited for all purposes.

Background Art

[0003] Interstitial lung diseases (ILDs) are a heterogeneous group of diseases with progressive dyspnea and dry cough as the main symptoms. The lesion site is in the lung parenchyma, accompanied by extensive inflammation and diffuse fibrosis. Pulmonary fibrosis is one of the common changes in interstitial lung diseases, and specifically appears as alveolar epithelial cell damage, diffuse local alveolitis, pathological interstitial proliferation of fibroblasts, and deposition of extracellular matrix (ECM) in the lung interstitium and alveoli, causing irreversible lung structure reconstruction and function loss, gas exchange impairment, and even respiratory failure. Early diagnosis of interstitial pneumonia and pulmonary fibrosis can guide medication and improve the prognosis. Currently, the commonly used diagnostic methods in clinical practice include pulmonary function tests, chest HRCT, invasive bronchoalveolar lavage fluid tests, and lung biopsies. Therefore, serological tests for early screening and progression monitoring of ILD diseases have become a promising research field in recent years.

[0004] Currently, sialylated glycan antigen (KL-6) is a serum marker already used clinically as an auxiliary diagnostic tool for interstitial lung disease (ILD), but its detection rate and sensitivity are not sufficiently high, and therefore it is not widely used. The sugar antigen CA125 is currently the most widely used tumor serum marker, and it is mainly expressed in coelocellular epithelial tissues that occur in mesothelium, Müllerian epithelium, and Müllerian duct derivatives. When tissue lesions or abnormal proliferation occur in these tissues, a clear increase in CA125 levels is observed. Multiple studies have confirmed that the CA125 positivity rate in the serum of lung cancer patients reaches 60% to 70%. Furthermore, studies have confirmed a significant correlation between serum CA125 and ILD, and that it can distinguish idiopathic pulmonary fibrosis (IPF) from other ILDs.

[0005] Exosomes are extracellular vesicles with a diameter of 30 nm to 150 nm, and an average diameter of 100 nm. They are formed by invagination of the cytoplasmic membrane and fusion with other intracellular vesicles and organelles. Exosomes are rich in proteins, nucleic acids, lipids, and metabolites, and through these substances, they can act as a medium for cell-to-cell communication by regulating downstream signaling pathways in receptor cells.

[0006] Exosomes play a crucial role in disease development and progression, and possess unique advantages and potential value in disease diagnosis and treatment. Proteins within exosomes are also widely studied as potential biomarkers. CA125 is a glycoprotein present on the cell membrane where the MUC16 gene is expressed. Recent studies have revealed that the CA125 content in ovarian cancer exosomes is higher than that of free CA125 in the blood, but the application of CA125 protein on exosomes to the diagnosis of lung diseases has not yet been reported. [Overview of the Initiative] [Problems that the invention aims to solve]

[0007] The first aspect of the present invention is to provide an exosome-treated solution.

[0008] A second aspect of the present invention is to provide the use of the exosome-treated solution of the first aspect of the present invention.

[0009] A third aspect of the present invention is to provide a reagent.

[0010] A fourth aspect of the present invention is to provide a reagent kit.

[0011] The object of the fifth aspect of the present invention is to provide a detection system.

[0012] An object of the sixth aspect of the present invention is to provide a method for detecting the presence or content of exosome proteins.

[0013] An object of the seventh aspect of the present invention is to provide a substance for detecting exosome CA125 for use in the production of products. [Means for solving the problem]

[0014] To achieve the above objectives, the technical solution employed by this invention is as follows.

[0015] A first aspect of the present invention is the provision of an exosome treatment solution comprising a buffering agent, an ionic strength maintaining agent, a blocking agent, a surfactant, a chelating agent, and sugars.

[0016] Preferably, the content of the buffering substance in the exosome-treated solution is 2-4% by weight, and more preferably 2.2-3.8%.

[0017] Preferably, the buffering substance comprises at least one of borax salt, trishydroxymethylaminomethane (Tris), phosphate buffer, acetate buffer, and citrate buffer, and further comprises trishydroxymethylaminomethane (Tris) and phosphate buffer.

[0018] Preferably, the content of the tris(hydroxymethyl)aminomethane (Tris) in the exosome treatment solution is 0.9 to 2.1% by weight percentage, and further 1 to 2%.

[0019] Preferably, the content of the phosphate buffer salt in the exosome treatment solution is 1.1 to 1.9% by weight percentage, and further 1.2 to 1.8%.

[0020] Preferably, the phosphate buffer salt is at least one of sodium phosphate, potassium phosphate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, disodium hydrogen phosphate and sodium dihydrogen phosphate, and further a mixture of disodium hydrogen phosphate and sodium dihydrogen phosphate.

[0021] Preferably, the weight ratio of the disodium hydrogen phosphate to the sodium dihydrogen phosphate is 1:(0.7 to 1.1), and further 1:(0.8 to 1).

[0022] Preferably, the disodium hydrogen phosphate is disodium hydrogen phosphate dodecahydrate.

[0023] Preferably, the sodium dihydrogen phosphate is sodium dihydrogen phosphate dihydrate.

[0024] Preferably, the ionic strength maintainer contains a chloride salt, and further is at least one of potassium chloride, sodium chloride, calcium chloride and magnesium chloride, and further sodium chloride.

[0025] Preferably, the content of the ionic strength maintainer in the exosome treatment solution is 0.6 to 1.2% by weight percentage, and further 0.8 to 1%.

[0026] Preferably, the blocking agent contains at least one of bovine serum albumin (BSA), casein and collagen peptide.

[0027] Preferably, the blocking agent contains at least one of casein, collagen peptide, and BSA.

[0028] Preferably, the blocking agent contains BSA.

[0029] Preferably, the content of the blocking agent in the exosome treatment solution is 0.8 to 2.2% by weight percentage, and further 1 to 2%.

[0030] Preferably, the surfactant contains at least one of an anionic surfactant, a cationic surfactant, an amphoteric ion surfactant, and a nonionic surfactant, and further contains a nonionic surfactant. For example, a nonionic surfactant containing a polyoxyethylene alcohol structure (e.g., alcohol ethoxylate, polyoxyethylene-polyoxyalkylene block copolymer), polyoxyethylene sorbitan fatty acid ester (e.g., TWEEN (registered trademark) series (e.g., Tween-20, Tween-40, Tween-80)), polyoxyethylene octyl phenyl ether (e.g., TRITON (registered trademark) series (e.g., Triton X-100, Triton X-114, Triton X-305, Triton X-405, Triton X-705)), N-D-glucose-N-methylalkane amide (e.g., MEGA series (e.g., MEGA 8, MEGA 10)), and further contains Tween-20.

[0031] Preferably, the content of the surfactant in the exosome treatment solution is 0.05 to 0.6% by weight percentage, and further 0.1 to 0.5%.

[0032] Preferably, the chelating agent contains at least one of ethylenediaminetetraacetic acid and a water-soluble salt of ethylenediaminetetraacetic acid, further contains a water-soluble salt of ethylenediaminetetraacetic acid, and further contains disodium ethylenediaminetetraacetate.

[0033] Preferably, the content of the chelating agent in the exosome-treated solution is 0.03 to 0.25% by weight, and more preferably 0.05 to 0.2%.

[0034] Preferably, the sugars include at least one of sucrose and trehalose, and further include trehalose.

[0035] Preferably, the content of the sugars in the exosome-treated solution is 0.8 to 2.2% by weight, and more preferably 1 to 2%.

[0036] Preferably, the exosome-treated solution further contains a preservative.

[0037] Preferably, the preservative comprises at least one of sodium azide, Proclin 300, 5-bromo-5-nitro-1,3-dioxane (BND), and Proclin 900, and further comprises sodium azide or Proclin 300.

[0038] Preferably, the content of the preservative in the exosome-treated solution is 0.08 to 0.22% by weight, and more preferably 0.1 to 0.2%.

[0039] Preferably, the exosome-treated solution further contains water, and the amount of water used is the remaining amount.

[0040] Preferably, the pH of the exosome-treated solution is 6 to 8, more preferably 7 to 8, and more preferably 7.5.

[0041] Preferably, the exosomes are derived from one or more of the following: whole blood, serum, plasma, bronchoalveolar lavage fluid, or urine.

[0042] The second aspect of the present invention is to provide applications relating to at least one of a1) to a6) of the exosome-treated solution according to the first aspect of the present invention.

[0043] a1) To detect the presence or absence of exosome material, a2) To detect the content of exosome material, a3) Manufacturing products for diagnosing or assisting in the diagnosis of a disease, wherein the disease is diagnosed or assisting in the diagnosis based on the presence or absence of exosome material or its content. a4) To manufacture a product for evaluating the severity of a disease, wherein the severity of the disease is evaluated by the amount of exosome material contained, a5) To manufacture a product that detects the presence or absence of exosome material. a6) To manufacture a product that detects the content of exosome material, The aforementioned substance includes at least one of nucleic acids and proteins.

[0044] Preferably, the nucleic acid includes at least one of DNA and RNA.

[0045] Preferably, the substance contains protein.

[0046] Preferably, the product includes at least one of a reagent, a reagent kit, or a detection system.

[0047] Preferably, a1) and a2) the use described above is for non-diagnostic purposes.

[0048] Preferably, the exosomes are derived from one or more of the following: whole blood, serum, plasma, bronchoalveolar lavage fluid, and urine.

[0049] A third aspect of the present invention is to provide a reagent, which includes the exosome treatment solution of the first aspect of the present invention.

[0050] Preferably, the reagent is used in at least one of b1) to b4), b1) To detect the presence or absence of exosome material, b2) To detect the content of exosome material, b3) Diagnosing or assisting in the diagnosis of a disease, the disease being diagnosed or assisting in the diagnosis based on the presence or absence of exosome material, or its content. b4) To assess the severity of the disease, the severity of the disease shall be assessed by the amount of exosome material present.

[0051] The aforementioned substance includes at least one of nucleic acids and proteins.

[0052] Preferably, the nucleic acid includes at least one of DNA and RNA.

[0053] Preferably, the substance contains protein.

[0054] Preferably, the exosomes are derived from one or more of the following: whole blood, serum, plasma, bronchoalveolar lavage fluid, and urine.

[0055] Preferably, the reagent is used in at least one of b1) to b4) by pre-treating the exosomes.

[0056] A fourth aspect of the present invention is to provide a reagent kit comprising at least one of c1) to c2). c1) Exosome-treated solution according to the first aspect of the present invention, c2) A reagent relating to the third aspect of the present invention.

[0057] Preferably, the reagent kit is used in at least one of the third aspects b1) to b4) of the present invention.

[0058] Preferably, the reagent kit includes at least one of c1) to c2), magnetic beads coated with a capture antibody, and a detection antibody labeled with a luminescent substance. c1) Exosome-treated solution according to the first aspect of the present invention, c2) A reagent relating to a third aspect of the present invention.

[0059] The aforementioned reagent kit is used in at least one of d1) to d4), d1) Detect the presence or absence of exosome proteins. d2) To detect the content of exosome proteins, d3) Diagnosing or assisting in the diagnosis of a disease, the disease being diagnosed or assisting in the diagnosis based on the presence or absence of exosomal proteins or their content, d4) To assess the severity of the disease, the severity of the disease is assessed by the content of exosome proteins, The capture antibody and / or the detection antibody specifically bind to the protein.

[0060] Preferably, the capture antibody and the detection antibody are each for different antigenic epitopes of the protein.

[0061] Preferably, the luminescent substance comprises at least one of acridinium ester, horseradish peroxidase, and alkaline phosphatase, and further comprises acridinium ester.

[0062] Preferably, the reagent kit further comprises at least one of a luminescent substrate and a protein calibrator, and further comprises a luminescent substrate and a protein calibrator.

[0063] Preferably, the luminescent substrate includes at least one of the following: APS-5 or AMPPD buffer (corresponding to alkaline phosphatase), acridinium ester pre-luminescence solution (pre-trigger solution) and luminescence solution (trigger solution) (corresponding to acridinium ester), and luminol or isoluminol buffer (corresponding to horseradish peroxidase).

[0064] Preferably, the protein is CA125, and the reagent kit is used in at least one of e1) to e5). e1) To detect the presence or absence of exosome CA125, e2) To detect the amount of exosome CA125, e3) To diagnose or assist in the diagnosis of lung disease, e4) To assess the severity of lung disease, e5) To diagnose or assist in the diagnosis of pulmonary fibrosis.

[0065] Preferably, the lung disease includes pneumonia or interstitial lung disease.

[0066] Preferably, the exosomes are derived from one or more of the following: whole blood, serum, plasma, bronchoalveolar lavage fluid, and urine.

[0067] A fifth aspect of the present invention is to provide a detection system comprising at least one of f1) to f3) and a chemiluminescence measuring device. f1) Exosome-treated solution according to the first aspect of the present invention, f2) Reagents relating to the third aspect of the present invention, f3) A reagent kit relating to the fourth aspect of the present invention.

[0068] Preferably, the detection system is used in at least one of the third aspects b1) to b4) of the present invention, or at least one of the fourth aspects d1) to d4) of the present invention.

[0069] A sixth aspect of the present invention is to provide a method for detecting the presence or content of exosome proteins. This method includes the step of pre-treating the exosomes with the exosome treatment solution of the first aspect of the present invention.

[0070] Preferably, the step of pre-treating the exosomes with the exosome treatment solution according to the first aspect of the present invention is as follows: the exosome treatment solution and the exosomes are mixed and reacted.

[0071] Preferably, the volume ratio of the exosome treatment solution to the exosomes is 1:(2~30), for example, 1:2, 1:5, 1:9, 1:10, 1:12, 1:15, 1:18, 1:20, 1:22, 1:25, 1:28, or 1:30.

[0072] Preferably, the reaction time is 5 to 120 minutes, and more preferably 5 to 10 minutes, such as 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, or 10 minutes.

[0073] Preferably, the reaction temperature is 25-42°C, more specifically 35-40°C, such as 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, etc.

[0074] Preferably, the exosomes are derived from one or more of the following: whole blood, serum, plasma, bronchoalveolar lavage fluid, and urine.

[0075] Preferably, the method for preparing the exosomes is either a fully automated exosome extraction device or a manual extraction method (e.g., centrifugation, reagent kit method, ultrafiltration, magnetic bead immunoassay, polyethylene glycol precipitation).

[0076] Preferably, the method further, (1) A step of mixing pre-treated exosomes with magnetic beads coated with capture antibodies and reacting them to obtain magnetic bead-exosome complexes coated with capture antibodies, (2) A step of mixing a magnetic bead-exosome complex coated with a capture antibody with a detection antibody labeled with a luminescent substance, reacting them to obtain a complex of magnetic beads coated with a capture antibody, exosomes, and detection antibody labeled with a luminescent substance, (3) The step of mixing a complex of magnetic beads coated with a capture antibody, exosomes, and a detection antibody labeled with a luminescent substance with a luminescent substrate, reacting the mixture, detecting the luminescence intensity, and obtaining the protein content in the exosome sample.

[0077] Preferably, the reaction time in steps (1) and (2) is 5 to 120 minutes, and more specifically 5 to 10 minutes, such as 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, or 10 minutes.

[0078] Preferably, the reaction temperature in steps (1) and (2) is 25 to 42°C, and more preferably 35 to 40°C, such as 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, etc.

[0079] Preferably, the method for obtaining the protein content in the exosome sample in step (3) involves constructing a standard curve using a protein calibrator and then calculating the protein content in the exosome sample based on the detected luminescence intensity.

[0080] Preferably, the magnetic beads coated with the capture antibody, the detection antibody labeled with a luminescent substance, and the luminescent substrate are the magnetic beads coated with the capture antibody, the detection antibody labeled with a luminescent substance, and the luminescent substrate in the fourth aspect of the present invention.

[0081] Preferably, the method for preparing the magnetic beads coated with the capture antibody is a common method in this art, and specifically is as follows: the beads are obtained by mixing activated magnetic beads with the capture antibody, incubating them, and blocking them.

[0082] Preferably, the mass ratio of the activated magnetic beads to the captured antibody is 100:(1-10), further 100:(1-4), and further 100:(2-3).

[0083] Preferably, the method for preparing the activated magnetic beads involves mixing and reacting magnetic beads, EDC, and NHS.

[0084] Preferably, the mass ratio of the magnetic beads, EDC, and NHS is (0.03~0.07):(0.06~0.14):1.

[0085] Preferably, the magnetic beads are a magnetic bead activation buffer containing magnetic beads.

[0086] Preferably, the reaction conditions are such that the reaction is carried out at room temperature for 15 to 120 minutes.

[0087] Preferably, the incubation period is 1 to 4 hours.

[0088] Preferably, the specific method of blocking involves mixing a mixture of activated magnetic beads and capture antibodies after hatching with a blocking agent.

[0089] Preferably, the amount of the mixture of activated magnetic beads and capture antibodies after hatching and the blocking agent used is 1:(2~10) in mass ratio of magnetic beads to blocking agent, and further 1:(2~5).

[0090] Preferably, the blocking is followed by the following steps: removing the supernatant by magnetic separation, and then diluting with a diluent.

[0091] Preferably, the diluent consists of the following components by weight percentage: Tris 0.05-0.1%, NaCl 0.9-3.0%, BSA 1-3%, Tween-20 0.1-0.5%, sodium azide 0.1-0.2%, water (residual volume), and pH 7.5.

[0092] Preferably, the method for preparing the detection antibody labeled with the luminescent substance is a commonly used method in this art, and specifically is as follows: the luminescent substance and the detection antibody are mixed, incubated, quenched, and obtained.

[0093] Preferably, the incubation period is 30 to 120 minutes.

[0094] Preferably, the specific method of quenching involves mixing a mixture of the luminescent substance after hatching and the detection antibody with a quenching agent and allowing it to react.

[0095] Preferably, the reaction time is 30 to 120 minutes.

[0096] Preferably, the following steps are included after the quenching: namely, purification and dilution with a diluent.

[0097] Preferably, the purification method includes dialysis or desalting column treatment.

[0098] Preferably, the diluent consists of the following components by weight percentage: MES 0.05-0.1%, NaCl 0.9-3.0%, BSA 1-3%, Tween-20 0.1-0.5%, sodium azide 0.1-0.2%, water (residual volume), and pH 6.5.

[0099] Preferably, the above method is a non-disease diagnostic method.

[0100] A seventh aspect of the present invention is to provide a substance for detecting exosome CA125 for use in the manufacture of a product. The aforementioned product is used in at least one of g1) to g3), g1) To diagnose or assist in the diagnosis of lung disease, g2) To assess the severity of lung disease, g3) To diagnose or assist in the diagnosis of pulmonary fibrosis.

[0101] Preferably, the lung disease includes pneumonia or interstitial lung disease.

[0102] Preferably, the substance for detecting CA125 includes a substance for detecting CA125 at the gene level and / or protein level, and further includes a substance for detecting CA125 at the protein level.

[0103] Preferably, the product is a reagent, a reagent kit, a detection chip, or a detection system.

[0104] Preferably, the product comprises an exosome-treated solution according to the first aspect of the present invention. [Effects of the Invention]

[0105] The beneficial effects of this invention are as follows:

[0106] The present invention provides an exosome processing solution, and by pre-treating an exosome sample to be measured using the exosome processing solution provided by the present invention, the influence of components in the eluate on exosome protein detection is reduced, the detection time is effectively shortened, stable detection of exosome proteins is achieved, and this is advantageous for realizing fully automated detection of exosome proteins.

[0107] This invention is the first to disclose exosome CA125 as a marker for the diagnosis, auxiliary diagnosis, severity assessment, and / or diagnosis of pulmonary fibrosis of lung diseases, and the method has superior diagnostic efficacy compared to the conventional marker serum CA125. That is, it has higher specificity and sensitivity, and has significant clinical value and potential for widespread adoption. [Brief explanation of the drawing]

[0108] [Figure 1] This figure shows the results of detecting the CA125 protein content on exosomes using the detection methods provided in Comparative Examples 1, 2, and 5 in Example 2. [Figure 2] This is an ROC curve diagram for diagnosing interstitial lung disease using serum CA125 and CA125 on exosomes in Effect Example 3. [Figure 3] This figure shows the results of detecting the CA125 protein content on exosomes using the detection method provided in Example 5 for a healthy human group, an ILD-positive sample that was negative for previously released serum KL-6 detection reagent, and an ILD-positive sample that was positive for previously released serum KL-6 detection reagent in Example 4. [Modes for carrying out the invention]

[0109] The present invention will be described in more detail below with reference to specific examples.

[0110] These examples are for illustrative purposes only and should not be used to limit the scope of the present invention.

[0111] In the following examples, experimental methods where specific conditions are not explicitly stated shall generally follow standard conditions or conditions suggested by the manufacturer. Unless otherwise specified, the materials, reagents, etc., used in these examples are commercially available reagents and materials.

[0112] The abbreviations and important terms used in this embodiment are defined as follows: Tris: Tris-hydroxymethylaminomethane BSA: Bovine serum albumin Tween-20: Tween 20 EDTA-2Na: Disodium ethylenediaminetetraacetate MES: Morpholinoethanesulfonic acid EDC: 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride NHS: N-hydroxysuccinimide BND: 5-bromo-5-nitro-1,3-dioxane.

[0113] Example 1: Exosome-treated solution The exosome-treated solution consists of the following components by weight percentage: Tris 1%, disodium hydrogen phosphate dodecahydrate 0.6%, sodium dihydrogen phosphate dihydrate 0.6%, NaCl 0.8%, BSA 1%, Tween-20 0.1%, EDTA-2Na 0.05%, trehalose 1%, sodium azide 0.1%, water (remainder), and pH 7.5.

[0114] Example 2: Exosome-treated solution The exosome-treated solution consists of the following components by weight percentage: Tris 2%, disodium hydrogen phosphate dodecahydrate 1%, sodium dihydrogen phosphate dihydrate 0.8%, NaCl 1%, BSA 2%, Tween-20 0.5%, EDTA-2Na 0.2%, trehalose 2%, sodium azide 0.2%, water (residual volume), and pH 7.5.

[0115] Example 3: Exosome-treated solution The exosome-treated solution consists of the following components by weight percentage: Tris 1.5%, disodium hydrogen phosphate dodecahydrate 0.8%, sodium dihydrogen phosphate dihydrate 0.7%, NaCl 0.9%, BSA 1.5%, Tween-20 0.3%, EDTA-2Na 0.1%, trehalose 1.5%, sodium azide 0.15%, water (residual volume), and pH 7.5.

[0116] Example 4: Exosome-treated solution The exosome-treated solution consists of the following components by weight percentage: Tris 1%, disodium hydrogen phosphate dodecahydrate 0.6%, sodium dihydrogen phosphate dihydrate 0.6%, NaCl 0.8%, BSA 1%, Tween-20 0.1%, EDTA-2Na 0.05%, trehalose 1%, Proclin 300 0.1%, water (remaining amount), and pH 7.5.

[0117] Example 5: Method for detecting CA125 protein on exosomes A method for detecting the CA125 protein on exosomes, comprising the following steps. (1) Preparation of magnetic bead coatings 1) Magnetic bead activation: Add 10 mg of magnetic beads to 1 mL of magnetic bead activation buffer (aqueous solution containing 1% MES by mass), add EDC solution (final concentration 0.5 mg / mL) and NHS solution (final concentration 1 mg / mL), and allow to react at room temperature for 30 minutes to activate the magnetic beads. Then remove the supernatant by magnetic separation.

[0118] 2) Antibody binding: 10 mg of magnetic beads are resuspended in 1 mL of magnetic bead binding buffer (aqueous solution containing 1% MES by mass), 300 μg of CA125 capture antibody (Beijing Kaijing, catalog number: BXA003) is added, and incubation is performed at room temperature for 2 hours to obtain a solution of magnetic beads-antibody conjugates.

[0119] 3) Blocking: Add 100 μL of blocking agent (1% by mass aqueous solution of bovine serum albumin) to the magnetic bead-antibody conjugate solution and incubate at room temperature for 2 hours.

[0120] 4) Washing: After removing the supernatant by magnetic separation, add 1 mL of magnetic bead washing solution (containing PBS buffer with a mass concentration of 0.1% Proclin 300) and wash. Repeat the washing step twice, then remove the supernatant by magnetic separation to obtain magnetic beads coated with CA125 capture antibody (CA125 magnetic bead coated material).

[0121] 5) Preparation of the working solution for the magnetic bead coating: The CA125 magnetic bead coating is diluted with a magnetic bead coating diluent to obtain a working solution of the magnetic bead coating with a concentration of 0.5 mg / mL. The magnetic bead coating diluent consists of the following components by weight percentage: Tris 0.1%, NaCl 2%, BSA 3%, Tween-20 0.5%, sodium azide 0.1%, water (residual volume), and pH 7.5.

[0122] (2) Preparation of acridinium ester labeled products 1) Preparation of acridinium ester mother liquor: Dissolve acridinium ester in anhydrous DMSO to a final concentration of 5 mg / mL.

[0123] 2) Labeling reaction: Dilute 300 μg of CA125 detection antibody (Beijing Kaijing, catalog number: BXA004) with 0.2 M NaHCO3 (pH=9.0) solution, add 15 μL of acridinium ester mother liquor to bring the total volume to 300 μL, at which point the acridinium ester concentration will be 0.25 mg / mL, wrap in aluminum foil, place the mixture in a constant temperature mixer, and label at room temperature for 30 minutes.

[0124] 3) Quenching reaction: Add 100 μL of label termination buffer (containing a 0.2 M NaHCO3 solution of 10% lysine by mass, pH 9.0) and mix at room temperature for 30 minutes.

[0125] 4) Purification: The labeled antibody is purified using a desalting column, the acridinium ester-labeled protein component is collected, and its concentration is detected to obtain the CA125 detection antibody acridinium ester-labeled product.

[0126] 5) Preparation of the working solution of the luminescent antibody (working solution of the acridinium ester label): The CA125 detection antibody acridinium ester label is diluted with a diluent of the acridinium ester label to obtain a working solution of the acridinium ester label with a concentration of 0.4 μg / mL. The diluent of the acridinium ester label consists of the following components by weight percentage: MES 0.1%, NaCl 2%, BSA 3%, Tween-20 0.1%, sodium azide 0.1%, water (remainder), and pH 6.5.

[0127] (3) Pretreatment of exosome samples 1) Sample preparation: After diluting serum or plasma 1-fold with PBS, filter it using a 0.22 μm filter membrane to remove cell debris and other impurities.

[0128] 2) Exosome extraction: Exosomes are extracted using an automated exosome extraction device.

[0129] Mix 10 μL of the exosome sample with 90 μL of the exosome treatment solution from Example 1 and allow to react at 37°C for 5 minutes.

[0130] (4) Detection and calculation of CA125 content in the exosome sample to be measured 1) Take 20 μL of the pre-treated sample obtained in step (3), add 50 μL of the working solution of the magnetic bead coating, mix, incubate at 37°C for 5 min, then magnetically separate, wash to remove unbound material, remove the supernatant to obtain the magnetic bead coating-exosome complex.

[0131] 2) Add 50 μL of working solution of luminescent antibody (working solution of acridinium ester labeling) to the magnetic bead-exosome complex, mix, incubate at 37°C for 5 minutes, then magnetically separate, wash to remove unbound material, and obtain the magnetic bead-exosome-labeled complex.

[0132] 3) Add 100 μL of acridinium ester pre-luminescence solution (an aqueous solution containing 0.1 wt% 30 wt% hydrogen peroxide and 0.2 mol / L nitric acid) and 100 μL of luminescence solution (an aqueous solution containing 0.1 wt% cetyltrimethylammonium chloride and 0.2 mol / L sodium hydroxide) to the obtained magnetic bead-coated product-exosome-labeled product complex, mix thoroughly, measure the maximum luminescence intensity, fit the luminescence intensity obtained from the detection of the standard to a standard curve, and calculate the CA125 content in the exosome sample to be measured using the standard curve.

[0133] Example 6: Method for detecting CA125 protein on exosomes The detection method in this embodiment is the same as in Example 5, with the only difference being the following: In step (3), the exosome treatment solution from Example 2 is used.

[0134] Example 7: Method for detecting CA125 protein on exosomes The detection method in this embodiment is the same as in Example 5, with the only difference being the following: In step (3), the exosome treatment solution from Example 3 is used.

[0135] Example 8: Method for detecting CA125 protein on exosomes The detection method in this embodiment is the same as in Example 5, with the only difference being the following: In step (3), the exosome treatment solution from Example 4 is used.

[0136] Example 9: Method for detecting CA125 protein on exosomes The detection method in this example is the same as in Example 5, with the only difference being the following: the amount of CA125 capture antibody used in step (1) 2) is 200 μg.

[0137] Example 10: Method for detecting CA125 protein on exosomes The detection method in this embodiment is the same as in Example 5, with the only difference being the following: In step (2) 2), the amount of CA125 detection antibody used is 200 μg.

[0138] Comparative Example 1: Method for detecting CA125 protein on exosomes The detection method in this comparative example is the same as in Example 5, with the only difference being that no pretreatment is performed on the exosome sample, i.e., step (3) is omitted.

[0139] Comparative Example 2: Method for detecting CA125 protein on exosomes The detection method in this comparative example is the same as in Example 5, with the only difference being the following: the exosome treatment solution from Example 1 is replaced with a commercially available PBS buffer (Shanghai Biyuntian Biotechnology Co., Ltd., catalog number: ST447).

[0140] Effect Example 1 The effect of exosome pretreatment on the dilution and recovery of exosome CA125 protein detection results. Exosomes were extracted from serum exosome CA125-positive samples using a reagent kit to obtain exosome stock solution. The stock solution was then diluted 2-fold and 4-fold using PBS buffer, and the CA125 protein content on the exosomes was detected using the detection methods described in Examples 5-10 and Comparative Examples 1-2, respectively, on a fully automated chemiluminescence immunoassay analyzer (model: shine i2910). The dilution recovery rate was calculated based on the luminescence values ​​of the exosome stock solution, 2-fold dilution, and 4-fold dilution. After dilution, the samples were left at room temperature for at least 1 hour before being detected simultaneously with the exosome stock solution.

[0141] The results are shown in Table 1. When exosome proteins were detected after pretreatment with the exosome processing solution provided by the present invention (Examples 5-10), the dilution recovery rate was 92-112%, which is significantly better than when no pretreatment was performed or when pretreatment was performed with PBS buffer (Comparative Examples 1-2). It can be seen that pretreatment of the exosome sample to be measured with the exosome processing solution provided by the present invention and dilution of the exosome sample reduces the interference that high salt and surfactant components in the eluate have on the detection of exosome proteins, which is advantageous for realizing fully automated detection of exosome proteins.

[0142] Table 1: Effect of exosome treatment on exosome CA125 protein detection results [Table 1]

[0143] Effect Example 2 The effect of exosome pretreatment on the significance of differences in exosome CA125 protein detection results. Eight samples of healthy human serum (CN) and eight samples of ILD-positive (ILD) were taken, and the CA125 protein content on exosomes was detected using the detection methods provided by Comparative Example 1, Comparative Example 2, and Example 5. The significance of the differences between the three methods was then evaluated. The results are shown in Figure 1. There was no significant difference in the results using the detection method of Comparative Example 1, there was a significant difference using the detection method of Comparative Example 2 (p<0.05), and there was a very significant difference using the detection method of Example 5 (p<0.001).

[0144] Effect Example 3 Comparison of serum CA125 and exosome CA125 detection results from healthy human samples and ILD patient samples. We took serum samples from 20 healthy individuals, 20 ILD-positive samples that were negative for commercially available serum KL-6 detection reagents, and 20 ILD-positive samples that were positive for commercially available serum KL-6 detection reagents. We detected CA125 in the serum of 60 samples using a serum CA125 reagent kit already approved on the market, and detected the CA125 protein content on exosomes using the detection method provided in Example 5. ROC analysis was performed on the two sets of results, and the results are shown in Figure 2. Based on the clinical diagnosis of interstitial lung disease, we defined the yin and yang of the samples and created ROC curves for each. Among these, the AUC of the ROC curve for CA125 on exosomes (serum-exo) was 0.88, and the AUC of the ROC curve for serum CA125 (serum) was 0.83, indicating that the diagnostic effect of CA125 on exosomes (serum-exo) is superior to that of serum CA125 (serum). Based on AUC, the cutoff value for exosome CA125 is 11,000 with a sensitivity of 90%, while the cutoff value for serum CA125 is 115,000 with a sensitivity of 70%. The sensitivity of exosome CA125 measurements is higher than that of serum CA125 measurements, meaning that exosome CA125 has higher sensitivity as a marker for lung diseases such as ILD.

[0145] Effect Example 4 Comparison of CA125 detection results on exosomes from healthy human samples and ILD patient samples. We took 20 serum samples from healthy individuals, 20 samples that were ILD-positive and negative for previously released serum KL-6 detection reagents, and 20 samples that were ILD-positive and positive for previously released serum KL-6 detection reagents. We detected the CA125 protein content on exosomes using the detection method provided in Example 5. The results are shown in Table 2 and Figure 3. In serum KL-6-positive ILD patients, the luminescence values ​​for CA125 protein detection on exosomes were all significantly higher than those of healthy individuals (P<0.0001), and the luminescence values ​​for CA125 protein detection on serum were all significantly higher than those of healthy individuals (P<0.01), although the significance was not as high as that for CA125 on exosomes. In serum KL-6-negative ILD patients, there was no statistically significant difference between the luminescence values ​​of serum CA125 protein detection in these patients and those of healthy individuals (P=0.942). However, the luminescence values ​​of CA125 protein detection on exosomes tended to be higher than those of exosomes in healthy individuals (P=0.061). This indicates that exosome CA125 detection can distinguish, to a certain extent, between ILD-positive, serum KL-6-negative patient samples and healthy individuals (its effectiveness is superior to serum CA125 detection), demonstrating a better diagnostic effect.

[0146] Table 2 Comparison of CA125 detection results on exosomes from healthy individuals and ILD patients [Table 2]

[0147] Effect Example 5 Use of exosome CA125 in the diagnosis of interstitial lung disease Based on the luminescence values ​​of CA125 on exosomes in healthy individuals and ILD patient samples in Effect Example 3, a preliminary cutoff value of 11000 can be determined for the exosome CA125 detection method. Based on the cutoff, the negative / positive results of the exosome CA125 detection in ILD patient samples were determined, and then the correlation between the negative / positive detection results and the clinical symptoms of ILD patients was evaluated. The results are shown in Table 3. Combining the measurement results in Tables 2 and 3, a preliminary cutoff value of 11000 for exosome CA125 was determined. For ILD patients with negative results (<500 U / mL) from the commercially available serum KL-6 detection reagent kit, the detection rate of exosome CA125 was 35% (7 / 20), indicating that exosome CA125 has a higher interstitial pneumonia detection rate in serum KL-6 negative samples compared to the serum KL-6 detection reagent kit. Furthermore, by comparing the correlation between exosome CA125 and serum KL-6 measurements and the clinical manifestations of ILD-positive patients, we found that among 7 patients who were serum KL-6 negative and CA125 positive, one developed respiratory failure and one had idiopathic interstitial pneumonia. Among 11 patients who were positive for both exosome CA125 and serum KL-6, 8 patients were clinically diagnosed with pulmonary fibrosis, and 3 patients developed severe pneumonia / respiratory failure. This indicates that exosome CA125 can be used as a marker to screen patients with interstitial pneumonia who have already developed pulmonary fibrosis or progressed to severe pneumonia, demonstrating that exosome CA125 has significant potential clinical value in the process of diagnosing and assessing the severity of interstitial lung disease.

[0148] Table 3 Results of using exosome CA125 for the diagnosis of interstitial lung disease [Table 3]

[0149] While the above embodiments represent preferred embodiments of the present invention, the embodiments of the present invention are not limited to these embodiments. Any other modifications, alterations, substitutions, combinations, or simplifications that do not contradict the spirit and principles of the present invention should be considered equivalent means of modification and are all within the scope of protection of the present invention.

Claims

1. An exosome-treated solution containing buffering agents, ionic strength maintaining agents, blocking agents, surfactants, chelating agents, and sugars.

2. The buffering substance comprises at least one of borax salt, trishydroxymethylaminomethane, phosphate buffer, acetate buffer, and citrate buffer. Preferably, the ionic strength maintaining agent includes a chloride salt. Preferably, the blocking agent comprises at least one of bovine serum albumin, casein, and collagen peptide. Preferably, the surfactant includes at least one of an anionic surfactant, a cationic surfactant, an amphoteric surfactant, and a nonionic surfactant. Preferably, the chelating agent comprises at least one of ethylenediaminetetraacetic acid and a water-soluble salt of ethylenediaminetetraacetic acid. Preferably, the sugars include at least one of sucrose and trehalose. Preferably, the exosome treatment solution according to claim 1 further contains a preservative.

3. The aforementioned preservative comprises at least one of sodium azide, Proclin 300, 5-bromo-5-nitro-1,3-dioxane, and Proclin 900. Preferably, the content of the buffering material in the exosome treatment solution is 2 to 4% by weight. Preferably, the buffering material comprises trishydroxymethylaminomethane and a phosphate buffer salt. Preferably, the content of trishydroxymethylaminomethane in the exosome treatment solution is 0.9 to 2.1% by weight. Preferably, the content of the phosphate buffer in the exosome treatment solution is 1.1 to 1.9% by weight. Preferably, the phosphate buffer salt is at least one of sodium phosphate, potassium phosphate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, disodium hydrogen phosphate, and sodium dihydrogen phosphate, and is further a mixture of disodium hydrogen phosphate and sodium dihydrogen phosphate. Preferably, the weight ratio of disodium hydrogen phosphate to sodium dihydrogen phosphate is 1:(0.7 to 1.1), Preferably, the disodium hydrogen phosphate is dodecahydrated disodium hydrogen phosphate. Preferably, the sodium dihydrogen phosphate is dihydrated sodium dihydrogen phosphate. Preferably, the content of the ionic strength maintaining agent in the exosome treatment solution is 0.6 to 1.2% by weight. Preferably, the ionic strength maintaining agent is at least one of potassium chloride, sodium chloride, calcium chloride, and magnesium chloride, and more preferably sodium chloride. Preferably, the content of the blocking agent in the exosome treatment solution is 0.8 to 2.2% by weight. Preferably, the blocking agent comprises at least one of casein and collagen peptide and BSA, and further comprises BSA. Preferably, the content of the surfactant in the exosome treatment solution is 0.05 to 0.6% by weight. Preferably, the surfactant includes a nonionic surfactant, and further includes at least one of a nonionic surfactant containing a polyoxyethylene alcohol structure, a polyoxyethylene sorbitan fatty acid ester system, a polyoxyethylene octylphenyl ether system, or an N-D-glucose-N-methylalkaneamide, and further includes at least one of an alcohol ethoxylate, a polyoxyethylene-polyoxyalkylene block copolymer, Tween-20, Tween-40, Tween-80, Triton X-100, Triton X-114, Triton X-305, Triton X-405, Triton X-705, MEGA 8, or MEGA 10, and further includes Tween-20. Preferably, the content of the chelating agent in the exosome treatment solution is 0.03 to 0.25% by weight. Preferably, the chelating agent comprises a water-soluble salt of ethylenediaminetetraacetic acid, and further comprises disodium ethylenediaminetetraacetic acid. Preferably, the content of the sugars in the exosome-treated solution is 0.8 to 2.2% by weight. Preferably, the sugars include trehalose. Preferably, the content of the preservative in the exosome-treated solution is 0.08 to 0.22% by weight. Preferably, the exosome treatment solution according to claim 1 or 2, characterized in that the preservative comprises sodium azide or proclin 300.

4. The content of the buffering substance in the exosome-treated solution is 2.2 to 3.8% by weight. Preferably, the content of trishydroxymethylaminomethane in the exosome treatment solution is 1-2% by weight. Preferably, the content of the phosphate buffer in the exosome treatment solution is 1.2 to 1.8% by weight. Preferably, the weight ratio of disodium hydrogen phosphate to sodium dihydrogen phosphate is 1:(0.8 to 1), Preferably, the content of the ionic strength maintaining agent in the exosome treatment solution is 0.8 to 1% by weight. Preferably, the content of the blocking agent in the exosome treatment solution is 1 to 2% by weight. Preferably, the content of the surfactant in the exosome treatment solution is 0.1 to 0.5% by weight. Preferably, the content of the chelating agent in the exosome treatment solution is 0.05 to 0.2% by weight. Preferably, the content of the sugars in the exosome-treated solution is 1-2% by weight. Preferably, the content of the preservative in the exosome-treated solution is 0.1 to 0.2% by weight. Preferably, the exosome treatment solution further contains water, and the amount of water used is the remaining amount. Preferably, the pH of the exosome treatment solution is 6 to 8, and more preferably 7 to 8, as described in claim 3.

5. A reagent or reagent kit comprising an exosome treatment solution according to any one of claims 1 to 4, Preferably, the reagent or reagent kit is used in at least one of b1) to b4), b1) To detect the presence or absence of exosome material, b2) To detect the content of exosome material, b3) Diagnosing or assisting in the diagnosis of a disease, the disease being diagnosed or assisting in the diagnosis based on the presence or absence of exosome material, or its content. b4) To evaluate the severity of the disease, the severity of the disease is evaluated by the amount of exosome material, Preferably, the substance comprises at least one of nucleic acids and proteins. Preferably, the substance includes protein, Preferably, the exosomes are derived from one or more of the following: whole blood, serum, plasma, bronchoalveolar lavage fluid, or urine. Preferably, the reagent or reagent kit is characterized in that it is used in at least one of b1) to b4) by pre-treating the exosomes.

6. A detection reagent kit comprising at least one of c1) to c2), a magnetic bead coated with a capture antibody, and a detection antibody labeled with a luminescent substance, c1) Exosome-treated solution according to any one of claims 1 to 4, c2) The reagent according to claim 5, The aforementioned detection reagent kit is used in at least one of d1) to d4), d1) To detect the presence or absence of exosome proteins, d2) To detect the content of exosome proteins, d3) Diagnosing or assisting in the diagnosis of a disease, the disease being diagnosed or assisting in the diagnosis based on the presence or absence of exosome proteins or their content, d4) To evaluate the severity of the disease, the severity of the disease is evaluated by the content of exosome proteins, A detection reagent kit characterized in that the capture antibody and / or the detection antibody specifically bind to the protein.

7. The luminescent substance comprises at least one of acridinium ester, horseradish peroxidase, and alkaline phosphatase. Preferably, the detection reagent kit further comprises at least one of a luminescent substrate and a protein calibrator. Preferably, the protein is CA125, and the reagent kit is used in at least one of e1) to e5). e1) To detect the presence or absence of exosome CA125, e2) To detect the amount of exosome CA125, e3) Diagnosing or assisting in the diagnosis of lung disease, e4) To assess the severity of lung disease, e5) To diagnose or assist in the diagnosis of pulmonary fibrosis, Preferably, the detection reagent kit according to claim 6, wherein the lung disease includes pneumonia or interstitial lung disease.

8. A detection system characterized by including at least one of f1) to f3) and a chemiluminescence measuring device. f1) Exosome-treated solution according to any one of claims 1 to 4, f2) The reagent or the reagent kit described in claim 5, f3) The detection reagent kit according to any one of claims 6 to 7.

9. A method for detecting the presence or content of exosome proteins, comprising the step of pre-treating the exosomes with an exosome treatment solution according to any one of claims 1 to 4.

10. The step of pre-treating the exosomes using the exosome treatment solution specifically involves mixing the exosome treatment solution with the exosomes and allowing them to react. Preferably, the volume ratio of the exosome treatment solution to the exosomes is 1:(2-30), Preferably, the reaction time is 5 to 120 minutes. Preferably, the reaction temperature is 25 to 42°C. Preferably, the exosomes are derived from one or more of the following: whole blood, serum, plasma, bronchoalveolar lavage fluid, or urine. Preferably, the method further includes the following steps: (1) Mix pre-treated exosomes with magnetic beads coated with capture antibodies and react to obtain a magnetic bead-exosome complex coated with capture antibodies. (2) Mix a magnetic bead-exosome complex coated with a capture antibody with a detection antibody labeled with a luminescent substance, react the mixture, and obtain a magnetic bead-exosome-detection antibody complex coated with a capture antibody. (3) Mix a complex of magnetic beads coated with capture antibodies, exosomes, and detection antibodies labeled with a luminescent substance with a luminescent substrate, react the mixture, detect the luminescence intensity, and obtain the protein content in the exosome sample. Preferably, the reaction time in steps (1) and (2) is 5 to 120 minutes. Preferably, the reaction temperature in steps (1) and (2) is 25 to 42°C. Preferably, the method for obtaining the protein content in the exosome sample in step (3) is characterized by constructing a standard curve using a protein calibrator and then calculating the protein content in the exosome sample based on the detected luminescence intensity, according to the method of 9.

11. Use of the exosome treatment solution according to any one of claims 1 to 4 in at least one of a1) to a6), a1) To detect the presence or absence of exosome material, a2) To detect the content of exosome material, a3) Manufacturing products for diagnosing or assisting in the diagnosis of diseases, wherein the diseases are diagnosed or assisting in the diagnosis based on the presence or absence of exosome material or its content. a4) To manufacture a product for evaluating the severity of a disease, wherein the severity of the disease is evaluated by the amount of exosome material contained, a5) To manufacture a product that detects the presence or absence of exosome material, a6) To manufacture a product that detects the content of exosome material, The use of an exosome-treated solution characterized in that the substance contains at least one of nucleic acids and proteins.

12. The nucleic acid includes at least one of DNA and RNA. Preferably, the substance includes protein, Preferably, the product includes at least one of a reagent, a reagent kit, or a detection system. Preferably, the use according to claim 11 is characterized in that the exosomes are derived from one or more combinations of whole blood, serum, plasma, bronchoalveolar lavage fluid, or urine.

13. one of the methods a1) to a2), a1) A method for detecting the presence or absence of exosome material, comprising the step of pre-treating the exosomes with an exosome treatment solution according to any one of claims 1 to 4, a2) A method for detecting the content of exosome material, comprising the step of pre-treating the exosomes with the exosome treatment solution described in any one of claims 1 to 4, The method is characterized in that the substance comprises at least one of nucleic acids and proteins.

14. The nucleic acid comprises at least one of DNA and RNA. Preferably, the substance includes protein, Preferably, the method according to 13, wherein the exosomes are derived from one or more combinations of whole blood, serum, plasma, bronchoalveolar lavage fluid, and urine.

15. The use of a substance in the manufacture of a product for detecting exosome CA125, The above product is used in at least one of g1) to g3), g1) To diagnose or assist in the diagnosis of lung disease, g2) To assess the severity of lung disease, g3) Use characterized by diagnosing or assisting in the diagnosis of pulmonary fibrosis.

16. The aforementioned lung diseases include pneumonia or interstitial lung disease. Preferably, the substance for detecting CA125 includes a substance for detecting CA125 at the gene level and / or protein level, and further includes a substance for detecting CA125 at the protein level. Preferably, the product is a reagent, a reagent kit, a detection chip, or a detection system. Preferably, the use according to claim 15, wherein the product comprises the exosome treatment solution described in any one of claims 1 to 4.

17. One of the methods g1) to g3). g1) A method for diagnosing or assisting in the diagnosis of lung disease, comprising the step of detecting the content, expression level, and / or activity of exosome CA125 in a test sample. g2) A method for assessing the severity of lung disease, comprising the step of detecting the content, expression level, and / or activity of exosome CA125 in a test sample. g3) A method for diagnosing or adjunctally diagnosing pulmonary fibrosis, comprising the step of detecting the content, expression level, and / or activity of exosome CA125 in a test sample.

18. The lung diseases in g1) to g2) include pneumonia or interstitial lung disease. Preferably, the method according to claim 17, characterized in that the content, expression level, and / or activity of exosome CA125 in the test sample are detected using the product described in claim 15 or 16 in g1) to g3).

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