A kit and application of KL-6 protein on exosomes for auxiliary diagnosis of interstitial lung disease

Abstract

The present invention belongs to the technical field of protein detection, and specifically relates to a KL-6 protein detection kit on exosomes for auxiliary diagnosis of interstitial lung disease; the detection method is: after the exosome sample is treated with a sample treatment solution, the exosomes containing KL-6 protein in the sample react with a magnetic bead coating working solution and a luminescent object labeled antibody working solution to form an immune complex, and the luminescence intensity is detected by chemiluminescence and the concentration value is calculated; the present invention provides for the first time a method for diagnosing interstitial pneumonia by combining blood-extracted exosome protein KL-6 with CA125 or using KL-6 alone, which reduces the influence of free KL-6 protein and other interfering substances in serum, has higher specificity and sensitivity than traditional serum detection kits, and has important clinical value and promotion prospects in auxiliary diagnosis of interstitial lung disease.

Description

Technical Field

[0001] The present invention belongs to the technical field of protein detection, and specifically relates to a kit, a detection method and an application of KL-6 protein on exosomes for auxiliary diagnosis of interstitial lung diseases. Background Art

[0002] Interstitial lung disease (ILD) is a general term for a clinical-pathological entity consisting of different types of diseases with diffuse lung parenchyma, alveolar inflammation and interstitial fibrosis as basic pathological lesions, and clinical manifestations of active dyspnea, diffuse infiltration shadows on chest X-rays, restrictive ventilation disorders, decreased diffusion (DLCO) function and hypoxemia. Early diagnosis and treatment of interstitial pneumonia can improve the prognosis of patients. Therefore, in addition to the clinical diagnosis of ILD, such as lung CT, lung function, blood gas analysis, bronchoscopy and lung biopsy, serological tests related to the development and prognosis of ILD have also become a research hotspot.

[0003] Siallylated carbohydrate antigen-6 (KL-6) is considered to be a relatively promising serological indicator. Currently, many studies believe that KL-6 has a significant correlation with the occurrence, progression, drug efficacy and prognosis of ILD lesions. However, serum KL-6 detection still shows problems of insufficient clinical sensitivity and specificity. Therefore, the development of interstitial pneumonia detection reagents with higher sensitivity and specificity is an important clinical need.

[0004] Exosomes refer to extracellular vesicles with a diameter of 40nm-160nm and an average diameter of 100nm, which are formed by fusion with other intracellular vesicles and organelles after invagination of the cytoplasmic membrane. A variety of cells can secrete exosomes under normal and pathological conditions, but the relationship between proteins on exosomes and interstitial pneumonia is still insufficient; KL-6 is a single transmembrane protein expressed by the MUC1 gene. In theory, membrane proteins can be retained on exosomes during the formation of exosomes and secreted into the circulatory system through exosomes, which has important potential clinical value in serological diagnosis. However, the detection of the expression level of these proteins on exosomes and their correlation with interstitial pneumonia have not been reported.

[0005] Therefore, establishing a method to accurately detect lung disease-related proteins on exosomes to determine the disease type and progression status of interstitial pneumonia has important clinical and research value. Summary of the invention

[0006] The purpose of the present invention is to provide a KL-6 protein detection kit on exosomes and a detection method thereof, which can be used independently or in combination for auxiliary diagnosis of interstitial lung diseases.

[0007] In the first aspect, the present application provides an application of KL-6 protein on exosomes in the preparation of a kit for diagnosing lung diseases, using the following technical solution:

[0008] Application of KL-6 protein on exosomes in the preparation of a kit for diagnosing lung diseases.

[0009] Furthermore, KL-6 on exosomes is used as an interstitial lung disease marker for the diagnosis of interstitial pneumonia or other lung diseases.

[0010] KL-6 protein is mainly expressed on the surface of type II pneumopneumocytes. When interstitial pneumonia occurs, the basement membrane of the lung is damaged. Type II pneumopneumocytes proliferate to repair the damage, resulting in increased production of KL-6 and entering the blood through the damaged basement membrane. It exists in the blood in two forms: free KL-6 protein and extracellular vesicle (mainly exosome) membrane-borne KL-6 protein. After research, it was found that the detection of KL-6 protein on exosomes in the blood has a higher clinical compliance rate. The detection of exosome KL-6 reduces the impact of free KL-6 protein in serum (present in both healthy and diseased people) and other endogenous serum interferences.

[0011] In the second aspect, the present application provides an application of KL-6 and CA125 on exosomes in combination for preparing a kit for diagnosing lung diseases, using the following technical solution:

[0012] A combined use of KL-6 and CA125 on exosomes in the preparation of a kit for diagnosing lung diseases.

[0013] Furthermore, a protein combination of KL-6 and CA125 on exosomes is used as an interstitial lung disease marker for the diagnosis of interstitial pneumonia or other lung diseases.

[0014] By adopting the above technical solution, high expression of CA125 on exosomes is associated with interstitial pneumonia fibrosis or severe pneumonia. The combined detection of exosome KL-6 and exosome CA125 can effectively diagnose patients in the progressive stage of fibrosis, which helps to provide effective treatment earlier and avoid delays in treatment.

[0015] In a third aspect, the present application provides a kit for diagnosing lung diseases, which adopts the following technical solution:

[0016] A kit for diagnosing lung diseases, comprising a sample processing solution, magnetic beads coated with capture antibodies, detection antibodies labeled with luminescent substances, luminescent substrates and protein calibrators; wherein the capture antibodies include KL-6; and the detection antibodies include KL-6.

[0017] A kit for diagnosing lung diseases comprises a sample processing solution, magnetic beads coated with capture antibodies, detection antibodies labeled with luminescent substances, luminescent substrates and protein calibrators; wherein the capture antibodies comprise CA125; and the detection antibodies comprise CA125.

[0018] By adopting the above technical solution, using exosome KL-6 for independent detection, or using exosome KL-6 and exosome CA125 for combined detection, patients in the progressive stage of fibrosis can be effectively diagnosed, which helps to provide effective treatment earlier and avoid delays in treatment.

[0019] Preferably, the sample processing liquid is a lysis type sample processing liquid, which includes the following raw materials in weight percentage: 0.5-2% disodium hydrogen phosphate dodecahydrate, 0.5-2% sodium dihydrogen phosphate dihydrate, 0.5-3% NaCl, 0.5-3% BSA, 0.05-1% Triton X-100, 0.1-2% anionic surfactant, 0.1-5% zwitterionic surfactant, 0.1-2% Tween-20, 0.02-0.1% EDTA-2Na, 0.05-0.2% sodium azide, and the rest is water, pH 7.4.

[0020] The anionic surfactant is at least one of sodium dodecyl sulfate (SDS), sodium dodecyl sarcosine (NLS), and lithium dodecyl sulfate (SLS).

[0021] The zwitterionic surfactant is at least one of CHAPS, sodium lauroamphoacetate, sodium cocoamphoacetate, CHAPSO, SB3-14, SB3-12, SB3-10, and SB3-8.

[0022] Preferably, the sample processing liquid is a non-lytic sample processing liquid, which includes the following raw materials in weight percentage: 0.5-2% disodium hydrogen phosphate dodecahydrate, 0.5-2% sodium dihydrogen phosphate dihydrate, 0.5-3% NaCl, 0.5-3% BSA, 0.2-2% zwitterionic surfactant, 0.1-2% Tween-20, 0.02-0.1% EDTA-2Na, 0.05-0.2% sodium azide, and the rest is water, pH 7.4.

[0023] Wherein, the zwitterionic surfactant is at least one of SB3-14, sodium lauroamphoacetate, sodium cocoamphoacetate, SB3-12, SB3-10, and SB3-8.

[0024] In a fourth aspect, the present application provides a method for detecting the protein content on exosomes using the above-mentioned kit, using the following technical solution:

[0025] The above kit is used for a method for detecting the protein content on exosomes, and the steps are as follows: before detecting the exosome sample to be tested, the exosome sample to be tested is first pretreated, and the exosome sample to be tested is mixed with a sample treatment solution to obtain a pretreated exosome sample to be tested.

[0026] After the exosome sample is pretreated with the sample treatment solution, the exosomes containing KL-6 (or CA125) protein in the sample react with the magnetic bead coating working solution and the luminescent marker antibody working solution to form an immune complex, and the luminescence intensity is detected by chemiluminescence and the concentration value is calculated.

[0027] In a specific embodiment, the kit is used for a method for detecting the content of KL-6 (or CA125) protein on exosomes, and the specific steps are as follows:

[0028] (1) Pre-treating the exosome sample to be tested: the exosome sample to be tested is mixed with a sample treatment solution for reaction;

[0029] (2) mixing the pretreated exosome sample to be tested obtained in step (1) with the magnetic bead coating working solution to obtain a magnetic bead-exosome / antigen complex;

[0030] (3) mixing the magnetic bead-exosome / antigen complex obtained in step (2) with the detection antibody of the luminescent substance labeled antibody working solution to obtain a magnetic bead-exosome / antigen-detection antibody complex;

[0031] (4) The magnetic bead-exosome / antigen-detection antibody complex obtained in step (3) is mixed with a luminescent substrate to react, and the luminescence intensity is detected.

[0032] (5) Using the standard curve method, the protein content in the exosome sample to be tested is calculated according to the luminescence intensity detected in step (4).

[0033] In a specific implementation scheme, in step (1), the volume ratio of the exosome sample to be tested to the sample processing solution is 1:(2-30).

[0034] For example, in step (1), the volume ratio of the exosome sample to be tested to the sample processing solution is 1:2, 1:5, 1:10, 1:12, 1:15, 1:18, 1:19, 1:20, 1:22, 1:25, 1:28 or 1:30.

[0035] In a specific embodiment, the reaction time of the pretreatment in step (1) is 1-120 min. Preferably, the reaction time is 1-10 min.

[0036] For example, the reaction time is 1 min, 2 min, 3 min, 4 min, 5 min, 6 min, 7 min, 8 min, 9 min or 10 min, etc.

[0037] In a specific embodiment, the reaction temperature of the pretreatment in step (1) is 25-42°C; preferably, the reaction temperature is 35-40°C.

[0038] For example, the reaction temperature is 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, etc.

[0039] In a specific embodiment, the reaction time in step (2) and step (3) is 1-120 min, preferably, the reaction time is 5-15 min.

[0040] For example, the reaction time is 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, 11 min, 12 min, 13 min, 14 min, and 15 min.

[0041] In a specific embodiment, the reaction temperature of step (2) and step (3) is 25-42°C, preferably, the reaction temperature is 35-40°C.

[0042] For example, the reaction temperature is 35°C, 36°C, 37°C, 38°C, 39°C, or 40°C.

[0043] In the above steps, a non-lytic sample treatment solution is used, and the treated sample reacts with the magnetic bead coating to obtain a magnetic bead-exosome KL-6 protein complex; a lytic sample treatment solution is used, and the treated sample reacts with the magnetic bead coating to obtain a magnetic bead-KL-6 antigen complex.

[0044] When the non-lysing sample treatment solution in this application is used to treat the sample, the mixed sample must be incubated at the same time. The exosome sample is pretreated with a lysing sample pretreatment solution, and the next step can be performed without incubation.

[0045] In the present application, the exosome sample to be tested can be prepared from any one of whole blood, serum, plasma, bronchoalveolar lavage fluid or urine.

[0046] The preparation method of the exosome sample to be tested in the present application is any one of a fully automatic exosome extraction instrument or a manual extraction method (such as centrifugation method, kit method, ultrafiltration method, magnetic bead immunoassay), preferably, a fully automatic exosome extraction instrument or a kit method is used.

[0047] In some common exosome extraction methods (such as the kit method, magnetic bead immunoassay, and a fully automatic instrument extraction method), an eluent is often required to elute the exosomes from the adsorption carrier. These eluents are mainly solutions containing high concentrations of salt ions or surfactants, which cause the exosomes to dehydrate and easily coagulate. At the same time, the conformation of the protein on the exosomes may also change. These factors will lead to poor accuracy and repeatability of exosome protein detection results and cannot be used for clinical diagnosis. Studies have found that after the exosome samples are diluted with sample diluent, the detection signal-to-noise ratio can be effectively improved, but a longer reaction time is required and it cannot be used for automated detection. On this basis, by combining a sample processing solution in a specific concentration range, the reaction time can be further reduced, the detection efficiency can be effectively improved, the detection signal-to-noise ratio can be improved, and fully automated detection of exosome proteins can be achieved.

[0048] By pre-treating the exosome samples with sample processing fluid, the detection accuracy can be further improved, and no additional incubation time is required, which can greatly shorten the detection time.

[0049] In a specific embodiment, the preparation method of capture antibody-coated magnetic beads (also called magnetic bead coating) is as follows:

[0050] Activation of magnetic beads: Add 5-20 mg of magnetic beads (magnetic bead concentration 5-20 mg / mL) to 1 mL of magnetic bead activation buffer (containing an aqueous solution of 1% MES by mass), add 25 μL of 20 mg / mL EDC solution (final concentration 0.5 mg / mL) and 25 μL of 40 mg / mL NHS solution (final concentration 1.0 mg / mL), react at room temperature for 15-120 min to activate the magnetic beads, and then remove the supernatant by magnetic separation.

[0051] Antibody coupling: Use 1 mL of magnetic bead coupling buffer (containing an aqueous solution of 1% MES by mass concentration) to resuspend the above-activated magnetic beads (magnetic bead concentration 5-20 mg / mL), add 100-300 μg of KL-6 capture antibody to be coated (final concentration is about 100-300 μg / mL), incubate at room temperature for 1-2 hours to obtain a solution containing magnetic bead-antibody conjugates.

[0052] Blocking: Add 100 μL of blocking agent (aqueous solution containing 1% bovine serum albumin by mass concentration) to the above solution containing magnetic beads-antibody conjugates, the volume ratio of the solution containing magnetic beads-antibody conjugates to the blocking agent is 1:0.1, and incubate at room temperature for 1-4 hours. After magnetic separation and removal of the supernatant, add 1 mL of magnetic bead diluent to obtain magnetic beads coated with capture antibodies; wherein the formula of the magnetic bead diluent is a PBS buffer containing 1% BSA by mass concentration and 0.1% proclin 300 by mass concentration.

[0053] Preparation of magnetic bead coating working solution: Dilute the capture antibody-coated magnetic beads using magnetic bead coating diluent to a solution of target concentration. The concentration after dilution is in the range of 0.1-1.5 mg / mL, and the particularly preferred concentration is 0.3-0.8 mg / mL.

[0054] The main components of the magnetic bead coating diluent are: TRIS 0.05-0.1%, NaCl 0.9-3.0%, BSA 1-3%, Tween-20 0.1-0.5%, sodium azide 0.1%, and the rest is water, pH 7.5.

[0055] The preparation method of the luminescent substance-labeled detection antibody (also called a marker) is:

[0056] Preparation of acridinium ester stock solution: Dissolve acridinium ester in anhydrous DMSO to prepare acridinium ester stock solution (final concentration 2.5 mg / ml).

[0057] Preparation of acridinium ester working solution: Take 10 μL of acridinium ester mother solution (final concentration 2.5 mg / ml), add 90 μL of anhydrous DMSO and dilute 10 times to prepare acridinium ester working solution (final concentration 0.25 mg / ml).

[0058] Labeling reaction: dilute 200-400 μg of KL-6 detection antibody to 300 μL (final antibody concentration 0.66-1.33 mg / mL) with 0.2 M NaHCO3 (pH = 9.0) solution, add 10 μL of acridinium ester working solution (final concentration is about 0.008 mg / ml), wrap with tin foil, and label at room temperature for 0.5-2h.

[0059] Quench the reaction: add 100 μL of labeling termination buffer and mix at room temperature for 30-120 minutes; the buffer solution is a 0.2 M NaHCO3 solution containing 10% lysine by mass, pH 9.0.

[0060] Purification: Use a desalting column to purify the labeled antibody, collect the acridine labeled protein fraction and adjust the concentration to obtain the luminescent labeled detection antibody.

[0061] Preparation of luminescent labeled antibody working solution (also called marker working solution): dilute the luminescent labeled detection antibody with a marker diluent to a solution of target concentration, the concentration range after dilution is 0.05-1.00 μg / mL, and the particularly preferred concentration is 0.1-0.6 μg / mL.

[0062] The main components of the marker diluent are: MES 0.05-0.1%, NaCl 0.9-3.0%, BSA 1-3%, Tween-20 0.1-0.5%, sodium azide 0.1%, and the rest is water, pH 6.5.

[0063] In the method for preparing the detection antibody labeled with a luminescent substance, the luminescent substance may be any one of acridine ester, alkaline phosphatase, and horseradish peroxidase.

[0064] In summary, the present application includes at least one of the following beneficial technical effects:

[0065] 1. The present application provides a method for auxiliary diagnosis of interstitial lung disease by using protein KL-6 on exosomes. Compared with serum KL-6 detection, the KL-6 protein on exosomes reduces the influence of free KL-6 protein and other interfering substances in serum. Compared with traditional serum KL-6 detection kits, it has higher specificity and sensitivity and has good promotion prospects.

[0066] 2. This application provides two sample pretreatment methods: non-lytic and lytic, which reduce the impact of components in the eluent on the detection of exosome proteins, effectively shorten the detection time, and facilitate the realization of fully automated detection of exosome proteins;

[0067] 3. This application provides for the first time a method for the auxiliary diagnosis of interstitial lung diseases using a combination of exosome proteins KL-6 and CA125, which has important clinical value in the auxiliary diagnosis of interstitial lung diseases;

[0068] 4. This application achieves stable detection of proteins on exosomes by establishing a method for detecting KL-6 protein on exosomes, which is of great value for the study of exosome proteins. BRIEF DESCRIPTION OF THE DRAWINGS

[0069] Figure 1 It is a bar chart comparing the test results of healthy person samples and ILD patient samples in test example 2.

[0070] Figure 2 : is the ROC curve diagram of serum KL-6 and exosome KL-6 in test example 3. DETAILED DESCRIPTION

[0071] To make the application easier to understand, the application will be further described in detail below in conjunction with the embodiments, which are only illustrative and are not limited to the scope of application of the application. Within the scope of the technical concept of the application, the technical scheme of the application can be subjected to multiple simple modifications, and these simple modifications all belong to the protection scope of the application. The raw materials or components used in the application can be obtained by commercial means or conventional methods unless otherwise specified.

[0072] Definitions of abbreviations and key terms in this embodiment:

[0073]

[0074]

[0075] Example 1

[0076] A method for detecting KL-6 protein on exosomes comprises the following steps:

[0077] (1) Preparation steps of magnetic bead coating:

[0078] Activation of magnetic beads: Add 10 mg of magnetic beads (magnetic bead concentration 10 mg / mL) to 1 mL of magnetic bead activation buffer (containing an aqueous solution of 1% MES by mass), add 25 μL of 20 mg / mL EDC solution (final concentration 0.5 mg / mL) and 25 μL of 40 mg / mL NHS solution (final concentration 1.0 mg / mL), react at room temperature for 30 minutes to activate the magnetic beads, and then remove the supernatant by magnetic separation.

[0079] Antibody coupling: Use 1 mL of magnetic bead coupling buffer (containing an aqueous solution of 1% MES by mass concentration) to resuspend the above-activated magnetic beads (magnetic bead concentration 10 mg / mL), add 200 μg of KL-6 capture antibody to be coated (final concentration is about 200 μg / mL), incubate at room temperature for 2 h, and obtain a solution containing magnetic bead-antibody conjugates.

[0080] Blocking: Add 100 μL of blocking agent (aqueous solution containing 1% bovine serum albumin by mass concentration) to the above solution containing magnetic beads-antibody conjugates, the volume ratio of the solution containing magnetic beads-antibody conjugates to the blocking agent is 1:0.1, and incubate at room temperature for 2 hours. After magnetic separation and removal of the supernatant, add 1 mL of magnetic bead diluent to obtain capture antibody-coated magnetic beads (KL-6 magnetic bead coating); wherein the formula of the magnetic bead diluent is a PBS buffer containing 1% BSA by mass concentration and 0.1% proclin 300 by mass concentration.

[0081] Preparation of magnetic bead coating working solution: dilute the KL-6 magnetic bead coating with magnetic bead coating diluent to obtain a magnetic bead coating working solution with a concentration of 0.5 mg / mL; wherein the formula of the magnetic bead coating diluent is TRIS 0.1%, NaCl 2%, BSA 3%, Tween-20 0.5%, sodium azide 0.1%, and the rest is water, pH 7.5.

[0082] (2) Preparation steps of marker-detection antibody complex:

[0083] Preparation of acridinium ester stock solution: Dissolve acridinium ester in anhydrous DMSO to prepare acridinium ester stock solution (final concentration 2.5 mg / ml).

[0084] Preparation of acridinium ester working solution: Take 10 μL of acridinium ester mother solution (final concentration 2.5 mg / ml), add 90 μL of anhydrous DMSO and dilute 10 times to prepare acridinium ester working solution (final concentration 0.25 mg / ml).

[0085] Labeling reaction: dilute 300 μg of KL-6 detection antibody to 300 μL (final antibody concentration 1 mg / mL) with 0.2 M NaHCO3 (pH = 9.0) solution, add 10 μL of acridinium ester working solution (final concentration is about 0.008 mg / ml), wrap with tin foil, and label at room temperature for 0.5 h.

[0086] Quench the reaction: add 100 μL of labeling termination buffer and mix at room temperature for 30 minutes; the buffer solution is a 0.2 M NaHCO3 solution containing 10% lysine by mass, pH 9.0.

[0087] Purification: Use a desalting column to purify the labeled antibody, collect the acridine labeled protein fraction and adjust the concentration to obtain the luminescent labeled detection antibody (KL-6 detection antibody acridine lipid label).

[0088] Preparation of luminescent labeled antibody working solution (acridinium lipid marker working solution): dilute the luminescent labeled detection antibody (KL-6 detection antibody acridinium lipid marker) with an acridinium lipid marker diluent to obtain an acridinium lipid marker working solution with a concentration of 0.4 μg / mL; wherein the formula of the acridinium lipid marker diluent is: MES 0.1%, NaCl 2%, BSA 3%, Tween-20 0.1%, sodium azide 0.1%, and the rest is water, pH 6.5.

[0089] (3) Exosome sample pretreatment steps:

[0090] After extracting exosomes using the kit method, the exosome samples were pretreated: 10 μL of exosome sample was mixed with 190 μL of sample treatment solution and then incubated at 37 °C for 5 min.

[0091] The sample processing liquid is a non-lytic sample processing liquid, which includes the following raw materials in weight percentage: 0.6% disodium hydrogen phosphate dodecahydrate, 0.6% sodium dihydrogen phosphate dihydrate, 0.9% NaCl, 1% BSA, 0.4% SB3-14, 0.1% Tween-20, 0.05% EDTA-2Na, 0.1% sodium azide, and the rest is water, pH 7.4.

[0092] (4) Detection and calculation of KL-6 content in the exosome sample to be tested:

[0093] Take 20 μL of the pretreated sample, add 50 μL of the magnetic bead coating working solution, mix well and incubate at 37°C for 5 min, then perform magnetic separation, wash to remove unbound substances, and remove the supernatant to obtain the magnetic bead-exosome complex.

[0094] Add 50 μL of luminescent labeled antibody working solution (acridinium lipid labeling working solution) to the reaction cup containing the magnetic bead-exosome complex, mix well and incubate at 37°C for 5 min, then perform magnetic separation, wash to remove unbound substances, and remove the supernatant to obtain the magnetic bead-exosome-detection antibody complex.

[0095] Add 100 μL of AE pre-stimulation solution and 100 μL of stimulation solution to the reaction cup containing the magnetic beads-exosomes-detection antibody complex, mix thoroughly and measure the maximum luminescence intensity. Fit the luminescence intensity detected by the standard sample into a standard curve, and calculate the KL-6 content in the sample to be tested through the standard curve.

[0096] Example 2

[0097] The main difference from Example 1 is that the sample processing liquid used is different.

[0098] The sample processing liquid is a non-lytic sample processing liquid, which includes the following raw materials in weight percentage: 1.0% disodium hydrogen phosphate dodecahydrate, 1.0% sodium dihydrogen phosphate dihydrate, 0.5% NaCl, 2% BSA, 0.5% sodium lauroamphoacetate, 0.2% Tween-20, 0.05% EDTA-2Na, 0.1% sodium azide, and the rest is water, pH 7.4.

[0099] Example 3

[0100] The main difference from Example 1 is that the sample processing liquid used is different.

[0101] The sample treatment solution is a non-lytic sample treatment solution, which includes the following raw materials in weight percentage: 0.5% disodium hydrogen phosphate dodecahydrate, 0.5% sodium dihydrogen phosphate dihydrate, 0.5% NaCl, 0.5% BSA, 0.2% sodium cocoamphoacetate, 0.5% Tween-20, 0.02% EDTA-2Na, 0.05% sodium azide, and the rest is water, pH 7.4.

[0102] Example 4

[0103] The main difference from Example 1 is that the sample processing liquid used is different.

[0104] The sample treatment solution is a non-lytic sample treatment solution, which includes the following raw materials in weight percentage: 2% disodium hydrogen phosphate dodecahydrate, 2% sodium dihydrogen phosphate dihydrate, 0.8% NaCl, 3% BSA, 1% sodium cocoamphoacetate, 0.5% Tween-20, 0.1% EDTA-2Na, 0.2% sodium azide, and the rest is water, pH 7.4.

[0105] Example 5

[0106] A method for detecting KL-6 protein on exosomes comprises the following steps:

[0107] (1) Preparation steps of magnetic bead coating:

[0108] Activation of magnetic beads: Add 10 mg of magnetic beads (magnetic bead concentration 10 mg / mL) to 1 mL of magnetic bead activation buffer (containing an aqueous solution of 1% MES by mass), add 25 μL of 20 mg / mL EDC solution (final concentration 0.5 mg / mL) and 25 μL of 40 mg / mL NHS solution (final concentration 1.0 mg / mL), react at room temperature for 30 minutes to activate the magnetic beads, and then remove the supernatant by magnetic separation.

[0109] Antibody coupling: Use 1 mL of magnetic bead coupling buffer (containing an aqueous solution of 1% MES by mass concentration) to resuspend the above-activated magnetic beads (magnetic bead concentration 10 mg / mL), add 200 μg of KL-6 capture antibody to be coated (final concentration is about 200 μg / mL), incubate at room temperature for 2 h, and obtain a solution containing magnetic bead-antibody conjugates.

[0110] Blocking: Add 100 μL of blocking agent (aqueous solution containing 1% bovine serum albumin by mass concentration) to the above solution containing magnetic beads-antibody conjugates, the volume ratio of the solution containing magnetic beads-antibody conjugates to the blocking agent is 1:0.1, and incubate at room temperature for 2 hours. After magnetic separation and removal of the supernatant, add 1 mL of magnetic bead diluent to obtain capture antibody-coated magnetic beads (KL-6 magnetic bead coating); wherein the formula of the magnetic bead diluent is a PBS buffer containing 1% BSA by mass concentration and 0.1% proclin 300 by mass concentration.

[0111] Preparation of magnetic bead coating working solution: dilute the KL-6 magnetic bead coating with magnetic bead coating diluent to obtain a magnetic bead coating working solution with a concentration of 0.5 mg / mL; wherein the formula of the magnetic bead coating diluent is TRIS 0.1%, NaCl 2%, BSA 3%, Tween-20 0.5%, sodium azide 0.1%, and the rest is water, pH 7.5.

[0112] (2) Preparation steps of marker-detection antibody complex:

[0113] Preparation of acridinium ester stock solution: Dissolve acridinium ester in anhydrous DMSO to prepare acridinium ester stock solution (final concentration 2.5 mg / ml).

[0114] Preparation of acridinium ester working solution: Take 10 μL of acridinium ester mother solution (final concentration 2.5 mg / ml), add 90 μL of anhydrous DMSO and dilute 10 times to prepare acridinium ester working solution (final concentration 0.25 mg / ml).

[0115] Labeling reaction: dilute 300 μg of KL-6 detection antibody to 300 μL (final antibody concentration 1 mg / mL) with 0.2 M NaHCO3 (pH = 9.0) solution, add 10 μL of acridinium ester working solution (final concentration is about 0.008 mg / ml), wrap with tin foil, and label at room temperature for 0.5 h.

[0116] Quench the reaction: add 100 μL of labeling termination buffer and mix at room temperature for 30 minutes; the buffer solution is a 0.2 M NaHCO3 solution containing 10% lysine by mass, pH 9.0.

[0117] Purification: Use a desalting column to purify the labeled antibody, collect the acridine labeled protein fraction and adjust the concentration to obtain the luminescent labeled detection antibody (KL-6 detection antibody acridine lipid label).

[0118] Preparation of luminescent labeled antibody working solution (acridinium lipid marker working solution): dilute the luminescent labeled detection antibody (KL-6 detection antibody acridinium lipid marker) with an acridinium lipid marker diluent to obtain an acridinium lipid marker working solution with a concentration of 0.4 μg / mL; wherein the formula of the acridinium lipid marker diluent is: MES 0.1%, NaCl 2%, BSA 3%, Tween-20 0.1%, sodium azide 0.1%, and the rest is water, pH 6.5.

[0119] (3) Exosome sample pretreatment steps:

[0120] After extracting exosomes using the kit method, the exosome samples were pretreated: 10 μL of exosome sample was mixed with 190 μL of sample treatment solution, mixed and reacted at 37°C for 5 min, and vortexed to mix thoroughly.

[0121] Among them, the sample processing liquid is a lysis type sample processing liquid, which includes the following raw materials in weight percentage: 0.6% disodium hydrogen phosphate dodecahydrate, 0.6% sodium dihydrogen phosphate dihydrate, 0.9% NaCl, 1% BSA, 1% TritonX-100, 0.2% sodium dodecyl sulfate SDS, 1% CHAPS, 0.1% Tween-20, 0.05% EDTA-2Na, 0.1% sodium azide, and the rest is water, pH 7.4.

[0122] (4) Detection and calculation of KL-6 content in the exosome sample to be tested:

[0123] Take 20 μL of the pretreated sample, add 50 μL of the magnetic bead coating working solution, mix well and react at 37°C for 5 minutes, then separate by magnetic separation, wash to remove unbound substances, and remove the supernatant to obtain the magnetic bead-antigen complex.

[0124] Add 50 μL of luminescent labeled antibody working solution (acridinium lipid labeling working solution) to the reaction cup containing the magnetic bead-antigen complex, mix well and react at 37°C for 5 minutes, then separate by magnetic separation, wash to remove unbound substances, and remove the supernatant to obtain the magnetic bead-antigen-detection antibody complex.

[0125] Add 100 μL of AE pre-stimulation solution and 100 μL of stimulation solution to the reaction cup containing the magnetic bead-antigen-detection antibody complex, mix thoroughly and measure the maximum luminescence intensity. Fit the luminescence intensity detected by the standard sample into a standard curve, and calculate the KL-6 content in the sample to be tested through the standard curve.

[0126] Example 6

[0127] The main difference from Example 5 is that the sample processing liquid used is different.

[0128] The sample processing solution is a lysis type sample processing solution, which includes the following raw materials in weight percentage: 0.5% disodium hydrogen phosphate dodecahydrate, 0.5% sodium dihydrogen phosphate dihydrate, 0.5% NaCl, 2% BSA, 0.5% Triton X-100, 0.1% sodium dodecyl sulfate SDS, 0.5% SB3-8, 0.2% Tween-20, 0.05% EDTA-2Na, 0.1% sodium azide, and the rest is water, pH 7.4.

[0129] Example 7

[0130] The main difference from Example 5 is that the sample processing liquid used is different.

[0131] The sample processing liquid is a lysis type sample processing liquid, which includes the following raw materials in weight percentage: 0.5% disodium hydrogen phosphate dodecahydrate, 0.5% sodium dihydrogen phosphate dihydrate, 0.5% NaCl, 0.5% BSA, 0.05% TritonX-100, 0.2% sodium lauryl sarcosine (NLS), 0.5% sodium lauroamphoacetate, 0.1% Tween-20, 0.02% EDTA-2Na, 0.05% sodium azide, and the rest is water, pH 7.4.

[0132] Example 8

[0133] The main difference from Example 5 is that the sample processing liquid used is different.

[0134] The sample processing solution is a lysis type sample processing solution, which includes the following raw materials in weight percentage: 1% disodium hydrogen phosphate dodecahydrate, 1% sodium dihydrogen phosphate dihydrate, 0.9% NaCl, 3% BSA, 1% Triton X-100, 0.2% sodium lauryl sarcosine (NLS), 1% CHAPS, 2% Tween-20, 0.1% EDTA-2Na, 0.2% sodium azide, and the rest is water, pH 7.4.

[0135] Example 9

[0136] The main difference from Example 1 is that the capture antibody and labeling antibody used are different.

[0137] The capture antibody used in this example is CA125, and the labeled antibody is CA125.

[0138] Comparative Example

[0139] Comparative Example 1:

[0140] The difference between this comparative example and Example 1 is that no pretreatment is performed on the sample.

[0141] Comparative Example 2:

[0142] The difference between this comparative example and Example 1 is that PBS buffer is used to pretreat the sample.

[0143] The PBS buffer solution includes the following raw materials in weight percentage: NaCl 0.8%, KCl 0.02%, potassium dihydrogen phosphate 0.144%, dipotassium hydrogen phosphate 0.024%, and the rest is water, with a pH of 7.4.

[0144] Comparative Example 3:

[0145] The difference between this comparative example and Example 1 is that a commercially available sample diluent is used to pretreat the exosome sample.

[0146] The manufacturer of the sample diluent is Aimejie Technology Co., Ltd., product number: ICT-647.

[0147] Comparative Example 4:

[0148] The difference between this comparative example and Example 5 is that no pretreatment is performed on the sample.

[0149] Comparative Example 5:

[0150] The difference between this comparative example and Example 5 is that PBS buffer is used to pretreat the sample.

[0151] The PBS buffer solution includes the following raw materials in weight percentage: NaCl 0.8%, KCl 0.02%, potassium dihydrogen phosphate 0.144%, dipotassium hydrogen phosphate 0.024%, and the rest is water, with a pH of 7.4.

[0152] Comparative Example 6:

[0153] The difference between this comparative example and Example 5 is that a commercially available sample diluent is used to pretreat the exosome sample.

[0154] The manufacturer of the sample diluent is Aimejie Technology Co., Ltd., product number: ICT-647.

[0155] Test Case

[0156] Test Example 1: Effects of different pretreatment methods on the detection results of exosome KL-6 protein

[0157] After the serum KL-6 positive sample was extracted with a kit for exosomes, it was diluted 2-fold and 4-fold with PBS buffer, and then the detection methods provided in Examples 1-8 and Comparative Examples 1-6 were used to detect the KL-6 protein content on the exosomes on a fully automatic 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 the sample was diluted, it was placed at room temperature for more than 1 hour and then detected simultaneously with the exosome stock solution.

[0158] Table 1 Effects of different pretreatment methods on the detection results of exosomal KL-6 protein

[0159]

[0160]

[0161] In combination with Examples 1-4, Comparisons 1-3, and Table 1, it can be seen that the dilution recovery of the reagent detection results for detecting exosome proteins using the pretreatment method of Examples 1-4 is significantly better than that of Comparative Examples 1-3, indicating that the dilution recovery of the reagent detection results for detecting exosome proteins using the pretreatment method of the present application is significantly better than the method without pretreatment or using a conventional diluent or using a commercially available sample diluent; in the present application, by combining a non-lysing sample treatment solution in a specific concentration range, the incubation time can be further shortened, the detection efficiency and signal-to-noise ratio can be effectively improved, and fully automated detection of exosome proteins can be achieved.

[0162] In combination with Examples 5-8, Comparisons 4-6, and Table 1, it can be seen that the dilution recovery of the reagent detection results for detecting exosome proteins using the pretreatment method of Examples 5-8 is significantly better than that of Comparative Examples 4-6, indicating that the dilution recovery of the reagent detection results for detecting exosome proteins using the pretreatment method of the present application is significantly better than the method without pretreatment or using a conventional diluent or using a commercially available sample diluent; in the present application, by combining a lysis-type sample processing solution in a specific concentration range, the incubation time can be further shortened, the detection efficiency and signal-to-noise ratio can be effectively improved, and fully automated detection of exosome proteins can be achieved.

[0163] When the non-lysing sample treatment solution in this application is used to treat the sample, the mixed sample must be incubated at the same time. The exosome sample is pretreated with a lysing sample pretreatment solution, and the next step can be performed without incubation.

[0164] Test Example 2: Comparison of test results between healthy samples and ILD patient samples

[0165] 20 samples of ILD positive and marketed serum KL-6 detection reagent positive results, 20 samples of ILD positive and marketed serum KL-6 detection reagent negative results, and 20 serum samples of healthy people were taken, and the detection method provided in Example 1 was used to detect the KL-6 protein content on exosomes. ILD positivity was determined based on high-resolution CT (HRCT) and other clinical results. The test results are shown in Table 2.

[0166] Table 2 Comparison of test results between healthy subjects and ILD patients

[0167]

[0168]

[0169] Data analysis: combined with Table 2, Figure 1 It can be seen that the luminescence value of KL-6 protein detection on exosomes of ILD-positive patients, whether they are commercially available serum KL-6 positive or commercially available serum KL-6 negative, is significantly higher than the luminescence value of KL-6 protein detection on exosomes of healthy people. There are very significant differences between ILD-positive serum KL-6-positive patient samples and healthy people samples, and between ILD-positive serum KL-6-negative patient samples and healthy people samples (P<0.0001).

[0170] The above results show that for ILD-positive patients who cannot be effectively detected by serum KL-6 detection, the exosomal KL-6 detection method can identify the vast majority of them. The detection of interstitial pneumonia by exosome KL-6 has significantly higher sensitivity and specificity than that by serum KL-6 detection.

[0171] Test Example 3: Comparison of clinical compliance rates between serum KL-6 and exosome KL-6

[0172] Using the detection method provided in Example 1, serum samples and serum-extracted exosome samples of ILD-positive patients and healthy people (a total of 60 cases) were simultaneously detected, and the positive and negative samples were defined based on the clinical diagnosis results of ILD, and ROC curves of serum KL-6 and exosome KL-6 detection results were prepared respectively, and the cutoff values ​​were determined according to the maximum value of Youden Index (sensitivity + specificity -1). ILD positive and negative were determined based on high-resolution CT (HRCT) and other clinical results. The test results are shown in the attached Figure 2 .

[0173] Data analysis: According to the attached Figure 2 The results show that the AUC (Area Under Curve) value of the exosome KL-6 test result is higher than that of the serum KL-6 test result, indicating that the exosome KL-6 test has a higher clinical compliance rate than the serum KL-6 test in the diagnosis of ILD. At the same time, the difference in AUC between the exosome KL-6 test and the serum KL-6 test is mainly reflected in the specificity. The exosome KL-6 test can reduce the influence of non-exosome-derived KL-6 proteins in the sample and other interferences in the blood, and has higher specificity (lower cutoff) than the serum KL-6 test.

[0174] Test Example 4: Combination of exosome KL-6 and exosome CA125 for diagnosis of interstitial lung disease Based on the ROC curve of the exosome KL-6 detection results in Test Example 2, the cutoff of the exosome KL-6 detection kit was preliminarily determined. The CA125 exosome detection kit was prepared using the detection method provided in Example 9, and the samples in Test Example 2 were tested to determine the preliminary cutoff. The positive and negative results of the exosome CA125 and exosome KL-6 detection results of ILD patient samples were determined according to the cutoff, and then the correlation between the positive and negative results of the exosome KL-6 and exosome CA125 detection results in Test Example 2 and the clinical symptoms of ILD patients was evaluated. The results are shown in Table 3.

[0175] Table 3 Diagnosis results of exosome KL-6 and exosome CA125 combined for interstitial lung disease

[0176]

[0177]

[0178] Results analysis: According to the ROC curve, the exosomal CA125 cutoff value was preliminarily determined to be 11053, and the exosomal KL-6 cutoff value was preliminarily determined to be 39102. There were 14 samples with positive exosomal KL-6 and positive exosomal CA125, accounting for 82% (14 / 17) of all idiopathic pulmonary fibrosis (IPF) or severe pneumonia. There were 16 samples with positive exosomal KL-6 and negative exosomal CA125, of which 3 samples were diagnosed as IPF or severe disease, accounting for 18.7% (3 / 17) of all non-IPF or severe disease.

[0179] The above results show that the combined diagnosis of exosomal KL-6 and exosomal CA125 can effectively identify most IPF or severe pneumonia. The combined detection of exosomal KL-6 and exosomal CA125 may have important clinical value in distinguishing interstitial pneumonia and assessing the severity of interstitial pneumonia.

[0180] It should be noted that the embodiments described above are only used to explain the present application and do not constitute any limitation to the present application. The present application is described with reference to typical embodiments, but it should be understood that the words used therein are descriptive and explanatory words, rather than restrictive words. The present application may be modified as specified within the scope of the claims of the present application, and the present invention may be revised without departing from the scope and spirit of the present application. Although the present application described therein relates to specific methods, materials and embodiments, it does not mean that the present application is limited to the specific examples disclosed therein. On the contrary, the present application can be extended to all other methods and applications with the same functions.

Claims

1. Use of a detection reagent for KL-6 protein on exosomes in the preparation of a kit for diagnosing interstitial lung disease, characterized in that: The kit includes a sample processing solution; the sample processing solution is a lysis type sample processing solution or a non-lysis type sample processing solution; The lysis type sample processing solution includes the following raw materials in weight percentage: disodium hydrogen phosphate dodecahydrate 0.5-2%, sodium dihydrogen phosphate dihydrate 0.5-2%, NaCl 0.5-3.0%, BSA 0.5-3%, Triton X-100 0.05-1%, anionic surfactant 0.1-2%, zwitterionic surfactant 0.1-5%, Tween-20 0.1-2%, EDTA-2Na 0.02-0.1%, sodium azide 0.05-0.2%, and the rest is water; The anionic surfactant is sodium lauryl sulfate or sodium lauryl sarcosinate; the zwitterionic surfactant is CHAPS, sodium lauroamphoacetate or SB3-8.

2. The use according to claim 1, characterized in that: The non-lysing sample treatment solution includes the following raw materials in weight percentage: 0.5-2% disodium hydrogen phosphate dodecahydrate, 0.5-2% sodium dihydrogen phosphate dihydrate, 0.5-3% NaCl, 0.5-3% BSA, 0.2-2% zwitterionic surfactant, 0.1-2% Tween-20, 0.02-0.1% EDTA-2Na, 0.05-0.2% sodium azide, and the rest is water; The zwitterionic surfactant is SB3-14, sodium lauroamphoacetate or sodium cocoamphoacetate.

3. A kit for diagnosing interstitial lung disease, characterized in that: The kit comprises the sample processing solution according to claim 1 or 2, magnetic beads coated with capture antibodies, detection antibodies labeled with luminescent substances, luminescent substrates and protein calibrators; wherein the capture antibody is a KL-6 capture antibody; and the detection antibody is a KL-6 detection antibody.

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