Method for detecting KL-6 protein content in exosomes and its use

Abstract

A method for detecting KL-6 protein content in exosomes and its use. The detection method includes binding KL-6 protein in exosomes with magnetic beads by immunomagnetic bead capture, detecting the luminescence intensity of the system by chemiluminescence immunoassay, and finally calculating the protein content of KL-6 in exosomes by calibration curve method. The detection method of KL-6 protein content in exosomes is proposed for the first time, has good stability and feasibility, and has important application value in fields such as basic research related to exosomes. It is the first time that the KL-6 protein content in exosomes has a significant correlation with the status of pulmonary disease, and provides a reference value of KL-6 protein content in exosomes as an evaluation standard for pulmonary disease, and provides a reagent kit for diagnosing pulmonary disease.

Description

[Technical Field]

[0001] The present application belongs to the technical field of protein detection and relates to a method for detecting KL-6 protein content in exosomes and its use, and more particularly to a method for detecting KL-6 protein content in exosomes and a reagent kit for diagnosing lung diseases. [Background technology]

[0002] KL-6 (sialylated carbohydrate antigen, Krebs Von den Lungen-6) was discovered by Kohno in 1985. He prepared several monoclonal antibodies using mice immunized with a human lung adenocarcinoma cell line (VMRC-LCR). The sialylated carbohydrate antigen recognized by the sixth antibody was named KL-6. KL-6 is primarily secreted by proliferating, regenerating, or damaged type II alveolar epithelial cells. In normal lung tissue, KL-6 is expressed in type II alveolar epithelial cells, respiratory bronchiolar epithelial cells, and bronchial gland serous cells. When lung inflammation, particularly type II alveolar-associated pneumonia, occurs, massive death and regeneration of type II alveoli and disruption of the pulmonary interstitial epithelial cell barrier result in the release of large amounts of KL-6 into the blood. Extensive research on KL-6 has demonstrated its positive clinical significance as a serological indicator of interstitial lung diseases (ILDs). Therefore, measuring peripheral blood KL-6 levels can be used to diagnose interstitial pneumonia and determine treatment guidelines. While serum is considered an ideal test sample because it can be collected noninvasively and repeatedly, more than 99% of the proteins contained in serum are irrelevant proteins (e.g., albumin), which can affect detection accuracy.

[0003] Exosomes are small membrane vesicles (30–150 nm) containing complex RNA and proteins, currently referred to as discoid vesicles with diameters of 40–100 nm. Exosomes were first discovered in sheep reticulocytes in 1983, and Johnstone coined the term "exosome" in 1987. Various cells can secrete exosomes under normal and pathological conditions, and exosomes naturally occur in bodily fluids such as blood, saliva, urine, cerebrospinal fluid, and milk. Exosomes are considered specifically secreted membrane vesicles that can mediate intercellular communication. In recent years, research on exosomes has attracted increasing attention. Studies have shown that exosomes contain multiple proteins related to the physiological activities of various cells. However, the content and function of KL-6 protein in exosomes remains unknown, and no methods for detecting KL-6 protein in exosomes have been reported.

[0004] Therefore, the development of a method for detecting the content of KL-6 protein in exosomes has important application value in fields such as exosome-related research and functional research on KL-6 protein. Summary of the Invention [Problem to be solved by the invention]

[0005] The present application provides a method for detecting the KL-6 protein content in exosomes and uses thereof, and specifically provides a method for detecting the KL-6 protein content in exosomes and a reagent kit for diagnosing lung diseases. [Means for solving the problem]

[0006] In aspect 1, the present application provides: Step (1) of mixing and reacting magnetic beads labeled with a capture antibody with a test exosome sample to obtain a magnetic bead-exosome complex; Step (2) of mixing and reacting the magnetic bead-exosome complex obtained in step (1) with a luminescently labeled detection antibody to obtain a magnetic bead-exosome-detection antibody complex; Step (3) of mixing and reacting the magnetic bead-exosome-detection antibody complex obtained in step (2) with a luminescent substrate and detecting the luminescence intensity of the reaction system by chemiluminescence; and step (4) calculating the KL-6 protein content of the test exosome sample from the luminescence intensity detected in step (3) using a calibration curve method. A method for detecting KL-6 protein content in exosomes is provided.

[0007] This application presents the first method for detecting KL-6 protein content in exosomes. The detection principle is shown in Figure 1. In the figure, 1 denotes magnetic beads labeled with a capture antibody, 2 denotes exosomes containing KL-6 protein, and 3 denotes a detection antibody labeled with a luminescent substance. The specific binding between the capture antibody and KL-6 protein and the detection antibody and KL-6 protein forms a magnetic bead-exosome-detection antibody complex. Because the detection antibody is labeled with a luminescent substance, the luminescence intensity of the reaction system can be detected to indirectly reflect the KL-6 protein content in exosomes. The specific value of KL-6 protein content can be calculated using a calibration curve. The detection method described herein has good stability, and the detection results significantly correlate with those of conventional serum detection, demonstrating its high accuracy and feasibility. Its application is of great value in fields such as basic research related to exosomes. Based on this detection method, the present application discovered for the first time that the KL-6 protein content in exosomes is highly correlated with the state of lung disease, a discovery that is of great significance in terms of mechanistic research and clinical diagnosis of lung disease.

[0008] Preferably, the reaction temperature in step (1) is 33 to 40°C, for example, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, etc. The reaction time is 5 to 15 minutes, for example, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes, 15 minutes, etc.

[0009] Preferably, the reaction temperature in step (2) is 33 to 40°C, for example, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, etc. The reaction time is 5 to 15 minutes, for example, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes, 15 minutes, etc.

[0010] Preferably, the reaction temperature in step (3) is 33 to 40°C, for example, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, etc. The reaction time is 5 to 10 minutes, for example, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, etc.

[0011] Preferably, the test exosome sample is prepared from any one of whole blood, plasma, serum, bronchoalveolar lavage fluid, cerebrospinal fluid, and urine.

[0012] Preferably, the method for preparing the test exosome sample includes any one of centrifugation, ultrafiltration, magnetic bead immunoassay, polyethylene glycol precipitation, and reagent kit extraction, and centrifugation is preferred.

[0013] Preferably, the rotation speed of the centrifuge is 10,000 to 20,000 g, for example, 10,000 g, 11,000 g, 12,000 g, 13,000 g, 14,000 g, 15,000 g, 16,000 g, 17,000 g, 18,000 g, 19,000 g, 20,000 g, etc. The centrifugation time is 1 to 2 hours, for example, 1 hour, 1.1 hours, 1.2 hours, 1.3 hours, 1.4 hours, 1.5 hours, 1.6 hours, 1.7 hours, 1.8 hours, 1.9 hours, 2 hours, etc.

[0014] In a preferred embodiment of the present application, the method for detecting the content of KL-6 protein in exosomes comprises the following steps:

[0015] (1) Dilute the test sample by adding 0.5 to 3 times the sample volume of phosphate buffer, and centrifuge at 10,000 to 20,000 g for 1 to 2 hours at 0 to 4°C. Collect the precipitate and resuspend it in phosphate buffer to obtain the test exosome sample.

[0016] Add 400-600 μL of equilibration solution to 0.8-1.2 mg of magnetic beads and mix. Remove the liquid. Add 150-250 μL of magnetic bead activation solution and mix. Incubate at 15-40°C for 20-60 min. Remove the liquid. Add 80-120 μg of capture antibody and incubate at 15-40°C for 2.5-4 h. Remove the liquid. Add 400-600 μL of blocking solution and block at 15-40°C for 2-3 h. Wash. Add magnetic bead storage solution to obtain magnetic beads labeled with the capture antibody.

[0017] Dilute 8 to 12 μL of luminescent substance 8 to 12 times with anhydrous dimethylformamide, then take 10 to 30 μL and mix with 80 to 120 μg of labeled detection antibody, incubate at 15 to 40°C in the dark for 20 to 60 minutes, add 10 to 30 μL of blocking solution, block at 15 to 40°C in the dark for 20 to 60 minutes, add storage solution, and obtain the detection antibody labeled with the luminescent substance.

[0018] (2) Mix 8-12 μL of the test exosome sample with 200-300 μL of magnetic beads labeled with a capture antibody, react for 5-15 minutes at 33-40°C, wash, remove the liquid, add 200-300 μL of detection antibody labeled with a luminescent substance, react for 5-15 minutes at 33-40°C, wash, remove the liquid, add 180-220 μL of luminescent substrate, incubate for 5-10 minutes at 33-40°C, and detect the luminescence intensity of the reaction system at a wavelength of 477 nm.

[0019] (3) Take a KL-6 standard sample with the same volume as the test exosome sample, repeat the procedure in step (2), fit a calibration curve according to the detected luminescence intensity of the standard sample, and calculate the KL-6 content in the test sample using the calibration curve method.

[0020] Specific values of the above 0.5 to 3 times may be 0.5 times, 0.6 times, 0.7 times, 0.8 times, 0.9 times, 1 time, 1.2 times, 1.5 times, 1.7 times, 2 times, 2.2 times, 2.5 times, 2.7 times, 3 times, etc.

[0021] Specific values of the above 0.8 to 1.2 mg may be 0.8 mg, 0.85 mg, 0.9 mg, 0.95 mg, 1 mg, 1.05 mg, 1.1 mg, 1.15 mg, or 1.2 mg.

[0022] Specific values of the above 400 to 600 μL may be 400 μL, 420 μL, 440 μL, 450 μL, 460 μL, 480 μL, 500 μL, 520 μL, 540 μL, 550 μL, 560 μL, 580 μL, 600 μL, etc.

[0023] Specific values of the above 150 to 250 μL may be 150 μL, 160 μL, 170 μL, 180 μL, 190 μL, 200 μL, 210 μL, 220 μL, 230 μL, 240 μL, 250 μL, and the like.

[0024] Specific values of the above 80 to 120 μg may be 80 μg, 85 μg, 90 μg, 95 μg, 100 μg, 105 μg, 110 μg, 115 μg, 120 μg, and the like.

[0025] The specific value of the above 2 to 3 hours may be 2 hours, 2.1 hours, 2.2 hours, 2.3 hours, 2.4 hours, 2.5 hours, 2.6 hours, 2.7 hours, 2.8 hours, 2.9 hours, 3 hours, etc.

[0026] Specific values of the above 8 to 12 μL may be 8 μL, 8.5 μL, 9.5 μL, 10 μL, 10.5 μL, 11 μL, 11.5 μL, 12 μL, and the like.

[0027] The specific numerical value of the above 8 to 12 times may be 8 times, 8.5 times, 9.5 times, 10 times, 10.5 times, 11 times, 11.5 times, 12 times, or the like.

[0028] The specific value of the above 10 to 30 μL may be 10 μL, 12 μL, 15 μL, 18 μL, 20 μL, 22 μL, 25 μL, 28 μL, 30 μL, or the like.

[0029] Specific values of the above 80 to 120 μg may be 80 μg, 85 μg, 90 μg, 95 μg, 100 μg, 105 μg, 110 μg, 115 μg, 120 μg, and the like.

[0030] Specific values of the above 200 to 300 μL may be 200 μL, 210 μL, 220 μL, 230 μL, 240 μL, 250 μL, 260 μL, 270 μL, 280 μL, 290 μL, 300 μL, and the like.

[0031] Specific values of the above 180 to 220 μL may be 180 μL, 185 μL, 190 μL, 195 μL, 200 μL, 205 μL, 210 μL, 215 μL, 220 μL, and the like.

[0032] In a second aspect, the present application provides use of the method for detecting KL-6 protein content in exosomes described in the first aspect in the manufacture of a reagent kit for diagnosing lung diseases.

[0033] In aspect 3, the present application provides: The kit includes magnetic beads labeled with a capture antibody, a detection antibody labeled with a luminescent substance, a luminescent substrate, and a KL-6 standard. A reagent kit for diagnosing lung disease is provided.

[0034] This application creatively provides a reagent kit for diagnosing lung disease, in which a cutoff value (CUTOFF value) is set according to 2.1 times the concentration of KL-6 in exosomes of healthy individuals, i.e., 50 U / mL, depending on the detection results, determining whether the KL-6 detection result in the test sample exosomes is negative or positive, and thereby confirming the patient's lung disease status. Compared with conventional serum detection, this reagent kit has better sensitivity in diagnosing lung disease and reduces false-negative diagnostic results, making it of great practical value.

[0035] Preferably, the capture antibody comprises 8MKL-61.

[0036] Preferably, the detection antibody comprises 8MKL-62.

[0037] Preferably, the reagent kit is used to detect the level of KL-6 protein content in exosomes.

[0038] The magnetic beads labeled with the capture antibody are The antibody is prepared by a preparation method including the steps of mixing the capture antibody with activated magnetic beads, incubating the mixture, and blocking the magnetic beads.

[0039] Preferably, the incubation temperature is 15 to 40°C, for example, 15°C, 17°C, 20°C, 22°C, 25°C, 27°C, 30°C, 32°C, 35°C, 37°C, 40°C, etc. The incubation time is 2.5 to 4 hours, for example, 2.5 hours, 2.6 hours, 2.7 hours, 2.8 hours, 2.9 hours, 3 hours, 3.1 hours, 3.2 hours, 3.3 hours, 3.4 hours, 3.5 hours, 3.6 hours, 3.7 hours, 3.8 hours, 3.9 hours, 4 hours, etc.

[0040] Preferably, the mass ratio of the capture antibody to the magnetic beads is 1:(8 to 12), for example, 1:8, 1:8.5, 1:9, 1:9.5, 1:10, 1:10.5, 1:11, 1:11.5, 1:12, etc.

[0041] Preferably, the detection antibody labeled with a luminescent substance is The luminescent substance is prepared by a preparation method including the steps of mixing a luminescent substance with a labeled detection antibody, incubating the mixture in the dark, and blocking the antibody.

[0042] Preferably, the incubation temperature is 15 to 40°C, for example, 15°C, 17°C, 20°C, 22°C, 25°C, 27°C, 30°C, 32°C, 35°C, 37°C, 40°C, etc. The incubation time is 20 to 60 minutes, for example, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 60 minutes, etc.

[0043] Preferably, the type of lung disease comprises pneumonia or interstitial lung disease.

[0044] In a fourth aspect, the present application provides: Step (1) of mixing and reacting magnetic beads labeled with a capture antibody with a test exosome sample to obtain a magnetic bead-exosome complex; Step (2) of mixing and reacting the magnetic bead-exosome complex obtained in step (1) with a detection antibody labeled with a luminescent substance to obtain a magnetic bead-exosome-detection antibody complex; Step (3) of mixing and reacting the magnetic bead-exosome-detection antibody complex obtained in step (2) with a luminescent substrate and detecting the luminescence intensity of the reaction system by chemiluminescence; and (4) calculating the KL-6 protein content of the test exosome sample from the luminescence intensity detected in step (3) by a calibration curve method using a KL-6 standard. A method of using the reagent kit for diagnosing a lung disease according to embodiment 3 is provided.

[0045] Preferably, the temperature of the reaction described in step (1) is 33 to 40° C., and the time of the reaction is 5 to 15 minutes.

[0046] Preferably, the temperature of the reaction described in step (2) is 33 to 40° C., and the reaction time is 5 to 15 minutes.

[0047] Preferably, the reaction temperature in step (3) is 33 to 40° C., and the reaction time is 5 to 10 minutes.

[0048] Preferably, the test exosome sample is prepared from any one of whole blood, plasma, serum, bronchoalveolar lavage fluid, cerebrospinal fluid, and urine.

[0049] Preferably, the method for preparing the test exosome sample includes any one of centrifugation, ultrafiltration, magnetic bead immunoassay, polyethylene glycol precipitation, and reagent kit extraction, and centrifugation is preferred.

[0050] Preferably, the rotation speed of the centrifuge is 10,000 to 20,000 g, and the centrifugation time is 1 to 2 hours.

[0051] In one preferred embodiment of the present application, a method for using the reagent kit for diagnosing a lung disease comprises the following steps:

[0052] (1) Dilute the test sample by adding 0.5 to 3 times the sample volume of phosphate buffer, and centrifuge at 10,000 to 20,000 g for 1 to 2 hours at 0 to 4°C. Collect the precipitate and resuspend it in phosphate buffer to obtain the test exosome sample.

[0053] Add 400-600 μL of equilibration solution to 0.8-1.2 mg of magnetic beads and mix. Remove the liquid. Add 150-250 μL of magnetic bead activation solution and mix. Incubate at 15-40°C for 20-60 min. Remove the liquid. Add 80-120 μg of capture antibody and incubate at 15-40°C for 2.5-4 h. Remove the liquid. Add 400-600 μL of blocking solution and block at 15-40°C for 2-3 h. Wash. Add magnetic bead storage solution to obtain magnetic beads labeled with the capture antibody.

[0054] Dilute 8 to 12 μL of luminescent substance 8 to 12 times with anhydrous dimethylformamide, then take 10 to 30 μL and mix with 80 to 120 μg of labeled detection antibody, incubate at 15 to 40°C in the dark for 20 to 60 minutes, add 10 to 30 μL of blocking solution, block at 15 to 40°C in the dark for 20 to 60 minutes, add storage solution, and obtain the detection antibody labeled with the luminescent substance.

[0055] (2) Mix 8-12 μL of the test exosome sample with 200-300 μL of magnetic beads labeled with a capture antibody, react for 5-15 minutes at 33-40°C, wash, remove the liquid, add 200-300 μL of detection antibody labeled with a luminescent substance, react for 5-15 minutes at 33-40°C, wash, remove the liquid, add 180-220 μL of luminescent substrate, incubate for 5-10 minutes at 33-40°C, and detect the luminescence intensity of the reaction system at a wavelength of 477 nm.

[0056] (3) Take a KL-6 standard sample with the same volume as the test exosome sample, repeat the procedure in step (2), fit a calibration curve according to the detected luminescence intensity of the standard sample, and calculate the KL-6 content in the test sample using the calibration curve method. [Effects of the Invention]

[0057] Compared to the prior art, the present invention has the following beneficial effects:

[0058] This application proposes, for the first time, a method for detecting KL-6 protein content in exosomes, which can be applied to serum samples. This detection method has high stability. The detection results obtained using this detection method significantly correlate with those obtained using conventional serum detection, demonstrating the high accuracy and feasibility of this detection method. The method for detecting KL-6 protein content in exosomes described herein has important application value in fields such as basic research related to exosomes. This application also discovers, for the first time, that KL-6 protein content in exosomes is significantly correlated with the status of pulmonary disease. This discovery is of great significance in the study of the mechanisms of pulmonary disease and clinical diagnosis. This application also provides a reagent kit for diagnosing pulmonary disease. Based on the detection results, a cutoff value (CUTOFF value) is set at 2.1 times the KL-6 concentration in exosomes of healthy individuals, i.e., 50 U / mL. This kit determines whether the KL-6 detection result in the test sample's exosomes is negative or positive, thereby confirming the patient's pulmonary disease status. The reagent kit of the present application has better sensitivity in diagnosing lung diseases compared with traditional serum detection, reduces false negative diagnostic results, and has important application value. [Brief explanation of the drawings]

[0059] [Figure 1] Figure 1 shows the detection principle of the method for detecting the KL-6 protein content in exosomes. [Figure 2] FIG. 2 is a diagram showing the morphological characterization of exosomes prepared in step (1) in Example 1. [Figure 3] Figure 3 shows a comparison of the detection results between samples from healthy individuals and samples from ILD patients. [Figure 4] FIG. 4 is an analytical diagram of the correlation between the detection results of Example 1 and conventional serum detection results. [Figure 5] FIG. 5 is a comparative analysis diagram between the clinical reference value of KL-6 in serum and the cutoff value of KL-6 in exosomes of the present application. [Explanation of symbols]

[0060] 1: Magnetic beads labeled with a capture antibody; 2: Exosomes containing KL-6 protein; 3: Detection antibody labeled with a luminescent substance. DETAILED DESCRIPTION OF THE INVENTION

[0061] The technical solution of the present application will be further described below through specific embodiments. Those skilled in the art should understand that the above examples are only for understanding the present application and should not be considered as specific limitations of the present application.

[0062] The serum samples of healthy subjects in the following examples were derived from healthy volunteers, and the serum samples of ILD patients were derived from the First Affiliated Hospital of Guangzhou Medical University. All subjects gave informed consent for this experiment before sampling, and serum samples were collected in accordance with hospital standards. [Example]

[0063] This example provides a method for detecting KL-6 protein content in exosomes, which includes the following steps:

[0064] (1) Isolation and extraction of exosomes Sample pretreatment: The serum sample (1 mL) was centrifuged at 4°C at a rotation speed of 10,000 g for 10 minutes to remove impurities from the sample, and the supernatant after centrifugation was transferred to a new centrifuge tube. Pretreatment of supernatant: Four times the sample volume of pre-cooled 0.01 M PBS was added to the clarified supernatant, and the mixture was placed at 4°C and allowed to stand for 2 h. Exosome isolation: The mixture was centrifuged at 10,000 g for 1 hour at 4°C, the supernatant was discarded, and the precipitate was collected. Half the sample volume of pre-chilled 0.01 M PBS was added and mixed uniformly by pipetting to obtain the test exosome sample.

[0065] (2) Preparation of magnetic beads labeled with capture antibodies Pretreatment of magnetic beads: 500 μL of equilibration solution (0.01 M PBS) was added to 1 mg of magnetic beads, mixed uniformly by vortexing, and the liquid was removed using a magnetic rack. 200 μL of magnetic bead activation buffer (10 mg / mL EDC, 10 mg / mL NHS) was added, mixed uniformly by vortexing, and incubated at 25°C for 30 minutes. Antibody coupling: The magnetic bead activation buffer was removed using a magnet rack, and the capture antibody (100 μg of 8MKL-61) was added, mixed uniformly by vortexing, and incubated with shaking at 25°C for 3 hours using a vortex mixer. Blocking of magnetic beads: Uncoupled antibodies were removed using a magnet rack, and 500 μL of blocking solution (5% BSA prepared in 0.01 M PBS) was added. The beads were mixed uniformly by vortexing and then blocked for 2 hours upside down at 25°C using a vertical mixer. After washing, 1 mL of magnetic bead storage solution (3% BSA prepared in 0.01 M PBS) was added and the beads were stored at 4°C.

[0066] (3) Preparation of detection antibodies labeled with luminescent substances 10 μL of AP (alkaline phosphatase) was taken, and 90 μL of anhydrous DMF (anhydrous dimethylformamide) was added. After mixing uniformly, 20 μL to 100 μL was taken and added to the labeled detection antibody (8MKL-62). After mixing uniformly, the mixture was incubated at 25°C in the dark for 30 minutes using a shaker. Antibody blocking: 20 μL of blocking solution (5% lysine) was added to the incubated AP-labeled detection antibody, mixed evenly, and then blocked by shaking for 30 minutes at 25°C in the dark using a shaker. After dialysis, the antibody was resuspended in 1 mL of AP storage solution (0.01 M PBS) and stored at 4°C.

[0067] (4) Detection and calculation of KL-6 content in the test exosome samples 10 μL of the test exosome sample prepared in step (1) was mixed with 180 μL of sample diluent (0.01 M PBS, 0.05% Tween-20) and thoroughly mixed to obtain the test sample test solution. 10 μL of a standard (KL-6 recombinant protein of known concentration) was mixed with 180 μL of sample diluent and thoroughly mixed to obtain the standard test solution. 10 μL of a calibrator (440 U / ml KL-6) was mixed with 180 μL of sample diluent and thoroughly mixed to obtain the low-value calibrator test solution. 10 μL of a calibrator (1040 U / ml KL-6) was mixed with 180 μL of sample diluent and thoroughly mixed to obtain the high-value calibrator test solution. The following procedure was carried out for each of the test sample test solution, the standard test solution (each of which was carried out for six concentrations of KL-6 recombinant protein), the low value calibrator test solution, and the high value calibrator test solution.

[0068] 250 μL of the magnetic beads labeled with the capture antibody prepared in step (2) was taken, 10 μL of the test solution was added, and after thorough homogeneity, the mixture was incubated at 37°C for 10 minutes. The reaction solution was removed by repeated washing, and the magnetic beads that captured exosomes were obtained.

[0069] 250 μL of the AP-labeled antibody prepared in step (3) was mixed with the magnetic beads capturing exosomes, thoroughly mixed, and then incubated at 37°C for 10 minutes. The reaction solution was removed by repeated washing, and the antibody-bound magnetic bead complex was obtained. 200 μL of AMPPD substrate solution was added, thoroughly mixed, and then reacted at 37°C for 5 minutes. The luminescence intensity of the reaction system was detected at a wavelength of 477 nm.

[0070] A calibration curve was fitted based on the detected luminescence intensity of the standard product, and the KL-6 content in the test sample was calculated using the calibration curve. [Example]

[0071] This example provides a method for detecting KL-6 protein content in exosomes, which includes the following steps:

[0072] (1) Isolation and extraction of exosomes Sample pretreatment: The serum sample (1 mL) was centrifuged at 4°C at a rotation speed of 10,000 g for 10 minutes to remove impurities from the sample, and the supernatant after centrifugation was transferred to a new centrifuge tube. Pretreatment of the supernatant: One sample volume of pre-chilled PBS (concentration 0.01 M) was added to the supernatant after removing impurities to obtain a mixed solution. Exosome isolation: The mixture was centrifuged at 20,000 g for 1 hour at 4°C, the supernatant was discarded, and the precipitate was collected. One sample volume of pre-chilled PBS was added, and the mixture was mixed uniformly by pipetting to obtain the test exosome sample.

[0073] (2) Preparation of magnetic beads labeled with capture antibodies Pretreatment of magnetic beads: 400 μL of equilibration solution (0.01 M PBS) was added to 0.9 mg of magnetic beads, vortexed to mix uniformly, and the liquid was removed using a magnetic rack. 160 μL of magnetic bead activation buffer (10 mg / mL EDC, 10 mg / mL NHS) was added, vortexed to mix uniformly, and incubated at 25°C for 50 minutes. Antibody coupling: The magnetic bead activation buffer was removed using a magnet rack, and the capture antibody (100 μg of 8MKL-61) was added, mixed uniformly by vortexing, and incubated with shaking using a vortex mixer at 25°C for 2.5 h. Blocking of magnetic beads: Uncoupled antibodies were removed using a magnetic rack, and 450 μL of blocking solution (5% BSA prepared in 0.01 M PBS) was added. The beads were mixed uniformly by vortexing and then blocked for 3 hours upside down at 25°C using a vertical mixer. After washing, 1 mL of magnetic bead storage solution (3% BSA prepared in 0.01 M PBS) was added and the beads were stored at 4°C.

[0074] (3) Preparation of detection antibodies labeled with luminescent substances 10 μL of AE (acridinium ester) was taken, and 90 μL of anhydrous DMF (anhydrous dimethylformamide) was added. After mixing uniformly, 20 μL to 100 μL was taken and added to the labeled detection antibody (8MKL-62). After mixing uniformly, the mixture was incubated at 25°C in the dark for 40 minutes using a shaker. Antibody blocking: 20 μL of blocking solution (5% lysine) was added to the incubated AE-labeled detection antibody, mixed evenly, and then blocked by shaking for 40 minutes at 25°C in the dark using a shaker. After dialysis, the antibody was resuspended in 1 mL of AE storage solution (0.01 M PBS, pH 7.4) and stored at 4°C.

[0075] (4) Detection and calculation of KL-6 content in the test exosome samples 10 μL of the test exosome sample prepared in step (1) was mixed with 180 μL of sample diluent (0.01 M PBS, 0.05% Tween-20) and thoroughly mixed to obtain a test sample test solution. 10 μL of a standard (KL-6 recombinant protein of known concentration) was mixed with 180 μL of sample diluent and thoroughly mixed to obtain a standard test solution. 10 μL of a calibrator (440 U / ml KL-6) was mixed with 180 μL of sample diluent and thoroughly mixed to obtain a low-value calibrator test solution. 10 μL of a calibrator (1040 U / ml KL-6) was mixed with 180 μL of sample diluent and thoroughly mixed to obtain a high-value calibrator test solution. The following procedure was carried out for each of the test sample test solution, the standard test solution (each of which was carried out for six concentrations of KL-6 recombinant protein), the low value calibrator test solution, and the high value calibrator test solution. 200 μL of the magnetic beads labeled with the capture antibody prepared in step (2) was taken, 10 μL of the test solution was added, and after thorough homogeneity, the mixture was incubated at 37°C for 15 minutes. The reaction solution was removed by repeated washing, and the magnetic beads capturing exosomes were obtained.

[0076] 200 μL of the AE-labeled antibody prepared in step (3) was mixed with the magnetic beads capturing exosomes, thoroughly mixed, and then incubated at 35°C for 8 minutes. The reaction solution was removed by repeated washing, and the antibody-bound magnetic bead complex was obtained. 100 μL of AE pre-excitation solution was added to the mixture, followed immediately by 100 μL of excitation solution. After thorough mixing, the maximum luminescence intensity was measured. A calibration curve was fitted based on the luminescence intensity detected by the standard, and the KL-6 content in the test sample was calculated using the calibration curve. [Example]

[0077] This example provides a method for detecting KL-6 protein content in exosomes, which includes the following steps:

[0078] (1) Isolation and extraction of exosomes Sample pretreatment: The serum sample (1 mL) was centrifuged at 4°C at a rotation speed of 10,000 g for 10 minutes to remove impurities from the sample, and the supernatant after centrifugation was transferred to a new centrifuge tube. Pretreatment of the supernatant: Two times the sample volume of pre-chilled PBS (concentration 0.01 M) was added to the supernatant after removing impurities to obtain a mixed solution. Exosome isolation: The mixture was centrifuged at 12,000 g for 2 hours at 4°C, the supernatant was discarded, and the precipitate was collected. Half the sample volume of pre-chilled PBS was added and mixed evenly by pipetting to obtain the test exosome sample.

[0079] (2) Preparation of magnetic beads labeled with capture antibodies Pretreatment of magnetic beads: 600 μL of equilibration solution (0.01 M PBS) was added to 1.1 mg of magnetic beads, vortexed to mix uniformly, and the liquid was removed using a magnetic rack. 300 μL of magnetic bead activation buffer (10 mg / mL EDC, 10 mg / mL NHS) was added, vortexed to mix uniformly, and incubated at 25°C for 20 minutes. Antibody coupling: The magnetic bead activation buffer was removed using a magnet rack, and the capture antibody (100 μg of 8MKL-61) was added, mixed uniformly by vortexing, and incubated with shaking at 25°C for 3 hours using a vortex mixer. Blocking of magnetic beads: Uncoupled antibodies were removed using a magnetic rack, and 600 μL of blocking solution (5% BSA prepared in 0.01 M PBS) was added. The beads were mixed uniformly by vortexing and then blocked for 2.5 hours upside down at 25°C using a vertical mixer. After washing, 1 mL of magnetic bead storage solution (3% BSA prepared in 0.01 M PBS) was added and the beads were stored at 4°C.

[0080] (3) Preparation of detection antibodies labeled with luminescent substances 10 μL of AP (alkaline phosphatase) was taken, and 100 μL of anhydrous DMF (anhydrous dimethylformamide) was added. After mixing uniformly, 20 μL to 100 μL was taken and added to the labeled detection antibody (8MKL-62). After mixing uniformly, the mixture was incubated at 25°C in the dark using a shaker for 20 minutes. Antibody blocking: 25 μL of blocking solution (5% lysine) was added to the incubated AP-labeled detection antibody, mixed evenly, and then blocked by shaking for 20 minutes at 25°C in the dark using a shaker. The dialyzed AP-labeled antibody was resuspended in AP storage solution and stored at 4°C.

[0081] (4) Detection and calculation of KL-6 content in the test exosome samples

[0082] 10 μL of the test exosome sample prepared in step (1) was mixed with 180 μL of sample diluent (0.01 M PBS, 0.05% Tween-20) and thoroughly mixed to obtain a test sample test solution. 10 μL of a standard (KL-6 recombinant protein of known concentration) was mixed with 180 μL of sample diluent and thoroughly mixed to obtain a standard test solution. 10 μL of a calibrator (440 U / ml KL-6) was mixed with 180 μL of sample diluent and thoroughly mixed to obtain a low-value calibrator test solution. 10 μL of a calibrator (1040 U / ml KL-6) was mixed with 180 μL of sample diluent and thoroughly mixed to obtain a high-value calibrator test solution. The following procedure was carried out for each of the test sample test solution, the standard test solution (each of which was prepared with six concentrations of KL-6 recombinant protein), the low value calibrator test solution, and the high value calibrator test solution.

[0083] 280 μL of the magnetic beads labeled with the capture antibody prepared in step (2) was taken, 10 μL of the test solution was added, and after thorough homogeneity, the mixture was incubated at 37°C for 10 minutes. The reaction solution was removed by repeated washing, and the magnetic beads capturing exosomes were obtained.

[0084] 280 μL of the AP-labeled antibody prepared in step (3) was mixed with the magnetic beads capturing exosomes, thoroughly mixed, and then incubated at 38°C for 6 minutes. The reaction solution was removed by repeated washing, and the antibody-bound magnetic bead complex was obtained. 200 μL of AMPPD substrate solution was added, thoroughly mixed, and then reacted at 36°C for 5 minutes. The luminescence intensity of the reaction system was detected at a wavelength of 477 nm.

[0085] A calibration curve was fitted based on the detected luminescence intensity of the standard product, and the KL-6 content in the test sample was calculated using the calibration curve.

[0086] [Test Example 1] Morphological characterization of exosomes The test exosome samples prepared in step (1) of Example 1 were morphologically characterized using a transmission electron microscope, and the results are shown in Figure 2. As shown in the figure, the extracted exosomes had a typical vesicular structure and a particle size distribution of 40 to 100 nm, which was consistent with the results reported in related literature, demonstrating that exosomes were successfully extracted.

[0087] [Test Example 2] Stability of the method Serum samples were collected from 12 healthy volunteers, 3 mL from each case, and the KL-6 protein content in exosomes was detected using the detection methods of Examples 1 to 3 (each sample volume was 1 mL). The detection results using the three detection methods are shown in Table 1.

[0088] [Table 1]

[0089] The results show that the detection methods of Examples 1 to 3 have good consistency in the detection results for the same samples and have small standard errors, indicating that the detection method of the present application is highly stable and has certain application value.

[0090] [Test Example 3] Comparison of detection results between healthy and ILD patient samples Twelve serum samples from ILD patients were collected, and the KL-6 protein content in exosomes was detected using the detection method of Example 1 (all sample volumes were 1 mL). The detection results are shown in Table 2. The detection results of 12 healthy subject samples obtained in Test Example 2 using the detection method of Example 1 are also shown in Table 2 for comparison.

[0091] [Table 2]

[0092] Overall, the results indicated that the KL-6 protein content in exosomes from ILD patients was significantly higher than that in exosomes from healthy volunteers. This phenomenon was further confirmed by plotting the data in a boxplot (see Figure 3). The KL-6 concentration in exosomes varied from sample to sample. The KL-6 concentration in exosomes from healthy volunteers ranged from 9 to 51 U / mL, with a mean of 24. The KL-6 concentration in exosomes from ILD patients ranged from 51 to 10,000 U / mL, with a mean of 1,147. A cutoff value of 50 U / mL was set at 2.1 times the KL-6 concentration in exosomes from healthy volunteers. This cutoff value allowed for determining whether the KL-6 detection result in the test sample exosomes was negative or positive, and thus for assessing the patient's pulmonary disease status.

[0093] [Test Example 4] Comparison of the results of the present detection method (i.e., reagent kit detection) and conventional serum detection methods The serum samples of 12 ILD patients in Test Example 2 were analyzed using a conventional serum KL-6 detection method. The results were compared with those obtained by the method of the present invention, and a correlation analysis was performed. The results are shown in Figure 4. The results showed a significant correlation between the results obtained by the two methods, with a correlation coefficient of 0.9349 (P<0.0001), demonstrating the accuracy and feasibility of this detection method.

[0094] Currently, the clinically accepted reference value for determining whether a patient has pulmonary disease using conventional serum KL-6 detection methods is 500 U / mL. As shown in Figure 5, the vertical dotted line in the figure represents the serum KL-6 detection level of 500 U / mL, and the horizontal dotted line in the figure represents the cutoff level of 50 U / mL for the detection of KL-6 in exosomes according to the present invention. The four patients indicated by the arrows in the figure had serum KL-6 levels below 500 U / mL, and would therefore be diagnosed as negative using conventional serum KL-6 detection methods. However, these four patients had exosomal KL-6 levels above the cutoff level of 50 U / mL according to the present invention, and would therefore be diagnosed as positive using the present reagent kit. These results demonstrate that the present reagent kit for detecting pulmonary disease has better sensitivity and reduced false-negative diagnostic results compared to conventional serum detection, making it of significant practical value.

[0095] In summary, this application proposes for the first time a method for detecting KL-6 protein content in exosomes, which can be applied to serum samples. This detection method has high stability. The detection results obtained using this method correlated significantly with those obtained using conventional serum detection methods, demonstrating the high accuracy and feasibility of this method. This method for detecting KL-6 protein content in exosomes has important application value in fields such as basic research related to exosomes. This application also discovers for the first time that KL-6 protein content in exosomes is significantly correlated with the status of pulmonary disease. This finding is of great significance for mechanistic research and clinical diagnosis of pulmonary disease. This application also provides a reagent kit for diagnosing pulmonary disease. Based on the detection results, a cutoff value (CUTOFF value) of 2.1 times the KL-6 concentration in exosomes of healthy individuals, i.e., 50 U / mL, is set. This kit determines whether the KL-6 detection result in the test sample's exosomes is negative or positive, thereby confirming the patient's pulmonary disease status. The reagent kit of the present application has better sensitivity in diagnosing lung diseases compared with traditional serum detection, reduces false negative diagnostic results, and has important application value.

[0096] The present application has described the method for detecting KL-6 protein content in exosomes and its use in the above examples, but the applicant declares that the present application is not limited to the above examples, i.e., it does not mean that the present application must be carried out in accordance with the above examples. Those skilled in the art should understand that any modifications to the present application, equivalent substitutions for each ingredient of the product of the present application, addition of auxiliary ingredients, selection of specific forms, etc., are all within the scope of protection and disclosure of the present application.

[0097] Although the preferred embodiments of the present application have been described in detail above, the present application is not limited to the specific contents of the above embodiments. Various simple modifications can be made to the technical solutions of the present application within the scope of the technical ideas of the present application, and all of these simple modifications fall within the scope of the claims of the present application.

[0098] It should be noted that the specific technical features described in the above specific embodiments can be combined in any appropriate manner unless there is a contradiction, and in order to avoid unnecessary duplication, the present application does not specifically describe various possible combinations. The inventions described in the original claims of this application are set forth below. [1] Step (1) of mixing and reacting magnetic beads labeled with a capture antibody with a test exosome sample to obtain a magnetic bead-exosome complex; Step (2) of mixing and reacting the magnetic bead-exosome complex obtained in step (1) with a detection antibody labeled with a luminescent substance to obtain a magnetic bead-exosome-detection antibody complex; Step (3) of mixing and reacting the magnetic bead-exosome-detection antibody complex obtained in step (2) with a luminescent substrate and detecting the luminescence intensity of the reaction system by chemiluminescence; and step (4) calculating the KL-6 protein content of the test exosome sample from the luminescence intensity detected in step (3) using a calibration curve method. Method for detecting KL-6 protein content in exosomes. [2] The reaction temperature in step (1) is 33 to 40°C, and the reaction time is 5 to 15 minutes. [1] Method for detecting KL-6 protein content in exosomes. [3] The reaction temperature in step (2) is 33 to 40°C, and the reaction time is 5 to 15 minutes. [1] Method for detecting KL-6 protein content in exosomes. [4] The reaction temperature in step (3) is 33 to 40°C, and the reaction time is 5 to 10 minutes. [1] Method for detecting KL-6 protein content in exosomes. [5] The test exosome sample is prepared from any one of whole blood, plasma, serum, bronchoalveolar lavage fluid, cerebrospinal fluid, and urine. A method for detecting the content of KL-6 protein in exosomes according to any one of [1] to [4]. [6] The method for preparing the test exosome sample includes any one of centrifugation, ultrafiltration, magnetic bead immunoassay, polyethylene glycol precipitation, and reagent kit extraction, and is preferably centrifugation. Preferably, the rotation speed of the centrifuge is 10,000 to 20,000 g, and the centrifugation time is 1 to 2 hours. A method for detecting the content of KL-6 protein in exosomes according to any one of [1] to [5]. [7] Use of the method for detecting the KL-6 protein content in exosomes according to any one of [1] to [6] in the manufacture of a reagent kit for diagnosing lung diseases. [8] The kit includes magnetic beads labeled with a capture antibody, a detection antibody labeled with a luminescent substance, a luminescent substrate, and a KL-6 standard. A reagent kit for diagnosing lung diseases. [9] the capture antibody comprises 8MKL-61; Preferably, the detection antibody comprises 8MKL-62. [8] A reagent kit for diagnosing lung diseases.

[10] Used to detect the level of KL-6 protein content in exosomes, [8] or [9] Reagent kit for diagnosing lung diseases.

[11] The magnetic beads labeled with the capture antibody are The method includes the steps of mixing the capture antibody with the activated magnetic beads, incubating the mixture, and blocking the magnetic beads. Preferably, the incubation temperature is 15 to 40°C, and the incubation time is 2.5 to 4 hours. Preferably, the mass ratio of the capture antibody to the magnetic beads is 1:(8 to 12). A reagent kit for diagnosing a lung disease according to any one of [8] to

[10] .

[12] The detection antibody labeled with the luminescent substance is The method includes the steps of mixing a luminescent substance with a labeled detection antibody, incubating the mixture in the dark, and blocking the antibody, Preferably, the incubation temperature is 15 to 40°C, and the incubation time is 20 to 60 minutes. A reagent kit for diagnosing a lung disease according to any one of [8] to

[11] .

[13] The type of lung disease includes pneumonia or interstitial lung disease; A reagent kit for diagnosing a lung disease according to any one of [8] to

[12] .

Claims

1. A reagent kit for detecting the KL-6 protein content in serum exosomes, comprising magnetic beads coupled with a capture antibody, a detection antibody labeled with a luminescent substance, a luminescent substrate, and a KL-6 standard, The detection (1) mixing and reacting the magnetic beads coupled with the capture antibody with a test serum exosome sample to obtain a magnetic bead-exosome complex; Step (2) of mixing the magnetic bead-exosome complex obtained in step (1) with the detection antibody labeled with the luminescent substance, and reacting the detection antibody with KL-6 in the serum exosomes through specific binding to obtain a magnetic bead-exosome-detection antibody complex; Step (3) of mixing and reacting the magnetic bead-exosome-detection antibody complex obtained in step (2) with the luminescent substrate and detecting the luminescence intensity of the reaction system by chemiluminescence; and (4) calculating the KL-6 protein content of the test serum exosome sample from the luminescence intensity detected in step (3) using a calibration curve method. Reagent kit.

2. The reaction temperature in step (1) is 33 to 40°C, and the reaction time is 5 to 15 minutes. The reagent kit according to claim 1.

3. The reaction temperature in step (2) is 33 to 40°C, and the reaction time is 5 to 15 minutes. The reagent kit according to claim 1.

4. The reaction temperature in step (3) is 33-40°C, and the reaction time is 5-10 min. The reagent kit according to claim 1.

5. The method for preparing the test serum exosome sample is any one selected from centrifugation, ultrafiltration, magnetic bead immunoassay, polyethylene glycol precipitation, and reagent kit extraction methods. The reagent kit according to claim 1 .

6. The method for preparing the test serum exosome sample is centrifugation; The reagent kit according to claim 5 .

7. The centrifugal rotation speed of the centrifugation method is 10,000 to 20,000 g, and the centrifugation time is 1 to 2 hours. The reagent kit according to claim 6 .

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