WO2023082452A1 - 一种外泌体上kl-6蛋白含量的检测方法及其应用 - Google Patents

一种外泌体上kl-6蛋白含量的检测方法及其应用 Download PDF

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WO2023082452A1
WO2023082452A1 PCT/CN2021/143493 CN2021143493W WO2023082452A1 WO 2023082452 A1 WO2023082452 A1 WO 2023082452A1 CN 2021143493 W CN2021143493 W CN 2021143493W WO 2023082452 A1 WO2023082452 A1 WO 2023082452A1
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exosome
exosomes
detection
mix
antibody
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French (fr)
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苏金
李军旗
魏新茹
肖云鹏
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广州呼吸健康研究院
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54326—Magnetic particles
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/12—Pulmonary diseases
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/70—Mechanisms involved in disease identification
    • G01N2800/7095—Inflammation

Definitions

  • This application belongs to the technical field of protein detection, and relates to a method for detecting KL-6 protein content on exosomes and its application, specifically to a method for detecting KL-6 protein content on exosomes and a method for diagnosing lung diseases kit.
  • KL-6 sialoligated sugar chain antigen, Krebs Von den Lungen-6
  • VMRC-LCR human lung adenocarcinoma cell line
  • KL-6 sialoligating sugar chain antigen recognized by the No. 6 antibody
  • KL-6 is mainly secreted by proliferating, regenerating or damaged alveolar type II epithelial cells. In normal lung tissue, KL-6 is expressed on type II alveolar epithelial cells, respiratory bronchiole epithelial cells and bronchial gland serous cells.
  • KL-6 interstitial lung diseases
  • ILDs Interstitial Lung Diseases
  • serum is considered to be an ideal test sample because it can be collected non-invasively and repeatedly.
  • proteins contained therein are irrelevant proteins (such as albumin, etc.). From the perspective of detection, these proteins are impurities and will affect the detection accuracy.
  • Exosomes refer to small membrane vesicles (30-150nm) that contain complex RNA and proteins. Today, they specifically refer to discoid vesicles with a diameter of 40-100nm. In 1983, exosomes were first discovered in sheep reticulocytes, and Johnstone named them "exosomes" in 1987. A variety of cells can secrete exosomes under normal and pathological conditions, and exosomes naturally exist in body fluids, such as blood, saliva, urine, cerebrospinal fluid, and breast milk. Exosomes are regarded as specifically secreted membrane vesicles that can participate in cell-to-cell communication, and research interest in exosomes has grown in recent years.
  • exosomes contain a variety of proteins related to various cellular physiological activities.
  • content and function of KL-6 protein on exosomes are still unclear.
  • the KL-6 protein on exosomes The detection method has not been reported yet.
  • the present application provides a method for detecting KL-6 protein content in exosomes and its application, specifically providing a method for detecting KL-6 protein content in exosomes and a kit for diagnosing lung diseases.
  • the present application provides a method for detecting the KL-6 protein content on exosomes, the detection method comprising the following steps:
  • step (2) Mix the magnetic bead-exosome complex obtained in step (1) with the detection antibody labeled with a luminescent substance, and react to obtain a magnetic bead-exosome-detection antibody complex;
  • step (3) Mix the magnetic bead-exosome-detection antibody complex obtained in step (2) with a luminescent substrate, react, and use chemiluminescence to detect the luminous intensity of the reaction system;
  • This application is the first to propose a detection method for KL-6 protein content on exosomes.
  • the detection principle is shown in Figure 1, in which 1—magnetic beads labeled with capture antibody; 2—exosomes containing KL-6 protein; 3—A detection antibody labeled with a luminescent substance.
  • 1 magnetic beads labeled with capture antibody
  • 2 exosomes containing KL-6 protein
  • 3 A detection antibody labeled with a luminescent substance.
  • a magnetic bead-exosome-detection antibody complex is constructed. Since the detection antibody is labeled with a luminescent substance, Therefore, the information of KL-6 protein content on exosomes can be indirectly reflected by detecting the luminescence intensity of the reaction system.
  • the specific value of the KL-6 protein content can be calculated.
  • the detection method provided by this application has good stability, and the detection results obtained based on the detection method provided by this application are significantly correlated with the results obtained by traditional serum detection, which proves that the detection method of this application has high accuracy and feasibility , which has important application value in the fields of exosome-related basic research and other fields.
  • the present application found for the first time that the KL-6 protein content on exosomes has a greater correlation with lung diseases. This discovery is of great significance for the mechanism research and clinical diagnosis of lung diseases.
  • the reaction temperature in step (1) is 33-40°C, such as 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, etc.
  • the reaction time is 5-15 min, such as 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, 11 min, 12 min, 13 min, 14 min, 15 min, etc.
  • the reaction temperature in step (2) is 33-40°C, such as 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, etc.
  • the reaction time is 5-15 min, such as 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, 11 min, 12 min, 13 min, 14 min, 15 min, etc.
  • the reaction temperature in step (3) is 33-40°C, such as 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, etc.
  • the reaction time is 5-10 min, such as 5 min, 6 min, 7 min, 8 min, 9 min, 10 min and so on.
  • the exosome sample to be tested is prepared from any sample to be tested in whole blood, plasma, serum, bronchoalveolar lavage fluid, cerebrospinal fluid or urine.
  • the preparation method of the exosome sample to be tested includes any one of centrifugation, ultrafiltration, magnetic bead immunization, polyethylene glycol precipitation or kit extraction, preferably centrifugation.
  • the rotational speed of the centrifuge is 10000-20000g, such as 10000g, 11000g, 12000g, 13000g, 14000g, 15000g, 16000g, 17000g, 18000g, 19000g, 20000g, etc.
  • the centrifugation time is 1-2h, such as 1h, 1.1h, 1.2h, 1.3h, 1.4h, 1.5h, 1.6h, 1.7h, 1.8h, 1.9h, 2h and so on.
  • the method for detecting the KL-6 protein content on the exosomes includes the following steps:
  • step (3) Take the KL-6 standard substance of the same volume as the exosome sample to be tested, repeat the operation of step (2), and fit the standard curve according to the luminous intensity detected by the standard substance, and calculate the standard curve method to calculate the KL-6 content in the sample.
  • 0.5-3 times can be 0.5 times, 0.6 times, 0.7 times, 0.8 times, 0.9 times, 1 times, 1.2 times, 1.5 times, 1.7 times, 2 times, 2.2 times, 2.5 times, 2.7 times, 3 times etc.
  • the specific numerical value in the above 0.8-1.2mg can be 0.8mg, 0.85mg, 0.9mg, 0.95mg, 1mg, 1.05mg, 1.1mg, 1.15mg, 1.2mg.
  • the specific values in the above 400-600 ⁇ L can be 400 ⁇ L, 420 ⁇ L, 440 ⁇ L, 450 ⁇ L, 460 ⁇ L, 480 ⁇ L, 500 ⁇ L, 520 ⁇ L, 540 ⁇ L, 550 ⁇ L, 560 ⁇ L, 580 ⁇ L, 600 ⁇ L, etc.
  • the specific values in the above 150-250 ⁇ L can be 150 ⁇ L, 160 ⁇ L, 170 ⁇ L, 180 ⁇ L, 190 ⁇ L, 200 ⁇ L, 210 ⁇ L, 220 ⁇ L, 230 ⁇ L, 240 ⁇ L, 250 ⁇ L, etc.
  • 80-120 ⁇ g may be 80 ⁇ g, 85 ⁇ g, 90 ⁇ g, 95 ⁇ g, 100 ⁇ g, 105 ⁇ g, 110 ⁇ g, 115 ⁇ g, 120 ⁇ g, etc.
  • the specific values in the above 2-3h can be 2h, 2.1h, 2.2h, 2.3h, 2.4h, 2.5h, 2.6h, 2.7h, 2.8h, 2.9h, 3h, etc.
  • the specific values in the above 8-12 ⁇ L can be 8 ⁇ L, 8.5 ⁇ L, 9.5 ⁇ L, 10 ⁇ L, 10.5 ⁇ L, 11 ⁇ L, 11.5 ⁇ L, 12 ⁇ L, etc.
  • the specific values in the above 8-12 times can be 8 times, 8.5 times, 9.5 times, 10 times, 10.5 times, 11 times, 11.5 times, 12 times and so on.
  • the specific values in the above 10-30 ⁇ L can be 10 ⁇ L, 12 ⁇ L, 15 ⁇ L, 18 ⁇ L, 20 ⁇ L, 22 ⁇ L, 25 ⁇ L, 28 ⁇ L, 30 ⁇ L, etc.
  • 80-120 ⁇ g may be 80 ⁇ g, 85 ⁇ g, 90 ⁇ g, 95 ⁇ g, 100 ⁇ g, 105 ⁇ g, 110 ⁇ g, 115 ⁇ g, 120 ⁇ g, etc.
  • the specific values in the above 200-300 ⁇ L can be 200 ⁇ L, 210 ⁇ L, 220 ⁇ L, 230 ⁇ L, 240 ⁇ L, 250 ⁇ L, 260 ⁇ L, 270 ⁇ L, 280 ⁇ L, 290 ⁇ L, 300 ⁇ L, etc.
  • the specific values in the above 180-220 ⁇ L can be 180 ⁇ L, 185 ⁇ L, 190 ⁇ L, 195 ⁇ L, 200 ⁇ L, 205 ⁇ L, 210 ⁇ L, 215 ⁇ L, 220 ⁇ L, etc.
  • the present application provides the application of the method for detecting KL-6 protein content on exosomes as described in the first aspect in the preparation of a kit for diagnosing lung diseases.
  • the present application provides a kit for diagnosing lung diseases
  • the kit for diagnosing lung diseases includes capture antibody-labeled magnetic beads, luminescent-labeled detection antibodies, luminescent substrate and KL-6 standard .
  • the present application creatively provides a kit for diagnosing lung diseases. Based on the test results, the cut-off value (CUTOFF value) is set according to 2.1 times the concentration of exosome KL-6 in healthy people, that is, 50U/mL, and the sample to be tested is determined. Exosome KL-6 test results are negative and positive, and this is used to confirm the patient's lung disease status.
  • the kit provided by the present application has better sensitivity in the diagnosis of lung diseases, reduces false negatives of diagnostic results, and has important application value.
  • the capture antibody comprises 8MKL-61.
  • the detection antibody includes 8MKL-62.
  • the kit is used to detect the KL-6 protein content level on exosomes.
  • the incubation temperature is 15-40°C, such as 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 2.5-4h, such as 2.5h, 2.6h, 2.7h, 2.8h, 2.9h, 3h, 3.1h, 3.2h, 3.3h, 3.4h, 3.5h, 3.6h, 3.7h, 3.8 h, 3.9h, 4h, etc.
  • the mass ratio of the capture antibody to the magnetic beads is 1:(8-12), such as 1:8, 1:8.5, 1:9, 1:9.5, 1:10, 1:10.5, 1:11 , 1:11.5, 1:12, etc.
  • the detection antibody labeled with a luminescent substance is prepared by a preparation method comprising the following steps:
  • the incubation temperature is 15-40°C, such as 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 20-60 min, such as 20 min, 25 min, 30 min, 35 min, 40 min, 45 min, 50 min, 55 min, 60 min and so on.
  • the type of lung disease includes pneumonia or interstitial lung disease.
  • the present application provides a method of using the kit for diagnosing lung diseases as described in the third aspect, the method of using includes:
  • step (2) Mix the magnetic bead-exosome complex obtained in step (1) with the detection antibody labeled with a luminescent substance, and react to obtain the magnetic bead-exosome-detection antibody complex;
  • step (3) Mix the magnetic bead-exosome-detection antibody complex obtained in step (2) with a luminescent substrate, react, and use chemiluminescence to detect the luminous intensity of the reaction system;
  • the KL-6 protein content of the exosome sample to be tested is calculated according to the luminescence intensity detected in step (3) by the standard curve method.
  • the reaction temperature in step (1) is 33-40°C, and the reaction time is 5-15min;
  • the reaction temperature in step (2) is 33-40°C, and the reaction time is 5-15min;
  • the reaction temperature in step (3) is 33-40° C.
  • the reaction time is 5-10 min.
  • the exosome sample to be tested is prepared from any sample to be tested in whole blood, plasma, serum, bronchoalveolar lavage fluid, cerebrospinal fluid or urine.
  • the preparation method of the exosome sample to be tested includes any one of centrifugation, ultrafiltration, magnetic bead immunization, polyethylene glycol precipitation or kit extraction, preferably centrifugation.
  • the rotational speed of the centrifugation is 10000-20000g, and the centrifugation time is 1-2h.
  • the method for using the kit for diagnosing lung diseases includes the following steps:
  • step (3) Take the KL-6 standard substance of the same volume as the exosome sample to be tested, repeat the operation of step (2), fit the standard curve according to the luminous intensity detected by the standard substance, and use the standard curve method to calculate the KL-6 content in the sample.
  • This application proposes a detection method for KL-6 protein content on exosomes for the first time, and it is applied to the detection of serum samples, and the detection method has high stability.
  • the detection results obtained based on the detection method provided by the application are significantly correlated with the results obtained by traditional serum detection, which proves that the detection method of the application has high accuracy and feasibility.
  • the detection method of KL-6 protein content on exosomes provided by this application has important application value in the fields of exosome-related basic research and other fields.
  • This application also found for the first time that the KL-6 protein content on exosomes has a greater correlation with lung diseases, and this discovery is of great significance for the mechanism research and clinical diagnosis of lung diseases.
  • the application also provides a kit for diagnosing lung diseases.
  • the cut-off value (CUTOFF value) is set according to 2.1 times the concentration of exosome KL-6 in healthy people, that is, 50 U/mL, and the exosomes to be tested are determined.
  • the negative and positive results of the secretory KL-6 test can be used to confirm the patient's lung disease status.
  • the kit provided by the present application has better sensitivity in the diagnosis of lung diseases, reduces false negatives of diagnostic results, and has important application value.
  • Figure 1 is a schematic diagram of the detection method for the detection of KL-6 protein content on exosomes.
  • 1 magnetic beads labeled with capture antibody
  • 2 exosomes containing KL-6 protein
  • 3 detection antibody labeled with luminescent substance.
  • Fig. 2 is a morphological characterization diagram of exosomes prepared in step (1) in Example 1.
  • Figure 3 is a comparison chart of the detection results of samples from healthy people and samples from patients with ILDs.
  • Fig. 4 is a correlation analysis diagram between the test results of Example 1 and the traditional serum test results.
  • Fig. 5 is a comparative analysis chart of the clinical reference value of serum KL-6 and the cut-off value of exosomal KL-6 of the present application.
  • the serum samples of healthy people involved in the following examples come from healthy volunteers, and the serum samples of ILDs patients come from the First affiliated Hospital of Guangzhou Medical University. All personnel were informed and agreed to this experiment before sampling, and the collection of serum samples followed hospital standards .
  • This embodiment provides a method for detecting the KL-6 protein content on exosomes, comprising the following steps:
  • Sample pretreatment Centrifuge the serum sample (1 mL) at 10,000 g for 10 min at 4°C to remove impurities in the sample, and transfer the centrifuged supernatant to a new centrifuge tube.
  • Supernatant pretreatment add 4 times the sample volume of pre-cooled 0.01M PBS to the impurity-removed supernatant, and place the mixture at 4°C for 2 hours.
  • Magnetic bead pretreatment Add 500 ⁇ L of equilibrium solution (0.01M PBS) to 1 mg of magnetic beads, vortex and shake to mix, and remove the liquid on the magnetic stand. Add 200 ⁇ L of magnetic bead activation buffer (10 mg/mL EDC, 10 mg/mL NHS), vortex and mix well, and incubate at 25°C for 30 min with a vortex shaker.
  • Antibody coupling remove the magnetic bead activation buffer on the magnetic stand, add capture antibody (8MKL-61 100 ⁇ g), vortex and mix, and incubate at 25°C for 3h with a vortex shaker.
  • Magnetic bead blocking remove the unconjugated antibody on the magnetic stand, add 500 ⁇ L of blocking solution (5% BSA prepared in 0.01M PBS), vortex and mix well, and use a vertical mixer to block upside down at 25°C for 2 hours. After washing, add 1 mL of magnetic bead preservation solution (3% BSA prepared in 0.01M PBS), and store at 4°C for later use.
  • Antibody blocking Take 20 ⁇ L of blocking solution (5% lysine) into the incubated AP-labeled detection antibody, mix well, and use a shaker to shake and block for 30 minutes at 25°C in the dark. Dialyze, add 1mL AP preservation solution (0.01M PBS) to resuspend, and store at 4°C for later use.
  • blocking solution 5% lysine
  • step (2) Take 250 ⁇ L of the capture antibody-labeled magnetic beads prepared in step (2), add 10 ⁇ L of test solution, mix thoroughly and incubate at 37°C for 10 min. The reaction solution was washed repeatedly to obtain magnetic beads for capturing exosomes.
  • the standard curve was fitted according to the luminescence intensity detected by the standard, and the KL-6 content in the sample to be tested was calculated through the standard curve.
  • This embodiment provides a method for detecting the KL-6 protein content on exosomes, comprising the following steps:
  • Sample pretreatment Centrifuge the serum sample (1 mL) at 10,000 g for 10 min at 4°C to remove impurities in the sample, and transfer the centrifuged supernatant to a new centrifuge tube.
  • Supernatant pretreatment add 1 times the sample volume of pre-cooled PBS (concentration: 0.01M) to the impurity-removed supernatant to obtain a mixed solution.
  • Magnetic bead pretreatment Add 400 ⁇ L of equilibrium solution (0.01M PBS) to 0.9 mg of magnetic beads, vortex to mix, and remove the liquid on the magnetic stand. Add 160 ⁇ L of magnetic bead activation buffer (10 mg/mL EDC, 10 mg/mL NHS), vortex and mix well, and incubate at 25°C for 50 min with a vortex shaker.
  • Antibody coupling Remove the magnetic bead activation buffer on the magnetic stand, add capture antibody (8MKL-61 100 ⁇ g), vortex and mix, and incubate at 25°C for 2.5h with a vortex shaker.
  • Magnetic bead blocking Remove the unconjugated antibody on the magnetic stand, add 450 ⁇ L of blocking solution (5% BSA prepared in 0.01M PBS), vortex and mix, and use a vertical mixer to block upside down at 25°C for 3 hours. After washing, add 1 mL of magnetic bead preservation solution (3% BSA prepared in 0.01M PBS), and store at 4°C for later use.
  • Antibody blocking Take 20 ⁇ L of blocking solution (5% lysine) into the incubated AE-labeled detection antibody, mix well, and use a shaker to shake and block for 40 minutes at 25°C in the dark. Dialyze, add 1mL AE preservation solution (0.01M PBS, pH7.4) to resuspend, and store at 4°C for later use.
  • blocking solution 5% lysine
  • test sample test solution 10 ⁇ L calibrator (1040U/ml of KL-6) with 180 ⁇ L sample diluent, and mix thoroughly to obtain a high-value calibrator test solution.
  • step (2) Take 200 ⁇ L of the capture antibody-labeled magnetic beads prepared in step (2), add 10 ⁇ L of test solution, mix well and incubate at 37°C for 15 min. The reaction solution was washed repeatedly to obtain magnetic beads for capturing exosomes.
  • This embodiment provides a method for detecting the KL-6 protein content on exosomes, comprising the following steps:
  • Sample pretreatment Centrifuge the serum sample (1 mL) at 10,000 g for 10 min at 4°C to remove impurities in the sample, and transfer the centrifuged supernatant to a new centrifuge tube.
  • Supernatant pretreatment add 2 times the sample volume of pre-cooled PBS (concentration: 0.01M) to the impurity-removed supernatant to obtain a mixed solution.
  • Magnetic bead pretreatment Add 600 ⁇ L of equilibrium solution (0.01M PBS) to 1.1 mg of magnetic beads, vortex and shake to mix, and remove the liquid on the magnetic stand. Add 300 ⁇ L of magnetic bead activation buffer (10 mg/mL EDC, 10 mg/mL NHS), vortex and mix well, and incubate at 25°C for 20 min with a vortex shaker.
  • Antibody coupling remove the magnetic bead activation buffer on the magnetic stand, add capture antibody (8MKL-61 100 ⁇ g), vortex and mix, and incubate at 25°C for 3h with a vortex shaker.
  • Magnetic bead blocking remove the unconjugated antibody on the magnetic stand, add 600 ⁇ L of blocking solution (5% BSA prepared in 0.01M PBS), vortex and mix well, and use a vertical mixer to block upside down at 25°C for 2.5h. After washing, add 1 mL of magnetic bead preservation solution (3% BSA prepared in 0.01M PBS), and store at 4°C for later use.
  • Antibody blocking Take 25 ⁇ L of blocking solution (5% lysine) into the incubated AP-labeled detection antibody, mix well, and use a shaker to shake and block for 20 minutes at 25°C in the dark. Resuspend the dialyzed AP-labeled antibody in AP preservation solution, and store at 4°C for later use.
  • blocking solution 5% lysine
  • test sample test solution 10 ⁇ L calibrator (1040U/ml of KL-6) with 180 ⁇ L sample diluent, and mix thoroughly to obtain a high-value calibrator test solution.
  • step (2) Take 280 ⁇ L of the capture antibody-labeled magnetic beads prepared in step (2), add 10 ⁇ L of test solution, mix well and incubate at 37°C for 10 min. The reaction solution was washed repeatedly to obtain magnetic beads for capturing exosomes.
  • the standard curve was fitted according to the luminescence intensity detected by the standard, and the KL-6 content in the sample to be tested was calculated through the standard curve.
  • the shape of the exosome sample to be tested prepared in step (1) of Example 1 was characterized by transmission electron microscopy, and the results are shown in Figure 2. As shown in the figure, the extracted exosomes have a typical vesicle-like structure, and the particle size distribution is 40-100nm, which is consistent with the results reported in relevant literature, which proves the successful extraction of exosomes.
  • test results are shown in Table 1.
  • Example 1 Take 12 cases of serum samples from ILDs patients, and use the detection method provided in Example 1 to detect the KL-6 protein content on exosomes (sample size is 1mL). The detection results of 12 healthy human samples obtained by the detection method provided in Example 1 are also listed in Table 2 for comparison.
  • the cut-off value (CUTOFF value) is set according to 2.1 times the concentration of exosome KL-6 in healthy people, that is, 50U/mL.
  • the negative or positive test result of exosome KL-6 in the sample to be tested can be determined according to this CUTOFF value, and the This is to confirm the patient's lung disease status.
  • the serum samples of 12 ILDs patients in Test Example 2 were detected by traditional serum KL-6 content detection method, the detection results were compared with the detection results obtained by the method of the present application, and correlation analysis was carried out, see the attached Figure 4.
  • the results showed that the test results obtained by the two methods were significantly correlated, the correlation coefficient was 0.9349, P ⁇ 0.0001.
  • the accuracy and feasibility of this detection method are illustrated.
  • the clinical reference value is 500 U/mL.
  • the vertical dotted line in the figure indicates that the serum KL-6 detection value is 500 U/mL
  • the horizontal dotted line on the figure indicates the exosome KL-6 detection cut-off value of 50 U/mL provided by this application.
  • the serum KL-6 values of the four patients marked by the arrows in the figure were lower than 500U/mL, so they should be judged to be negative according to the traditional detection method of serum KL-6 content, and the exosomal KL-6 values of these four patients
  • the -6 value is higher than the cut-off value 50U/mL provided by this application, so they should be judged as positive according to the test kit provided by this application.
  • This result shows that the kit for detecting lung diseases provided by the present application has better sensitivity in the diagnosis of lung diseases compared with traditional serum detection, reduces false negatives of diagnostic results, and has important application value.
  • this application proposes a detection method for KL-6 protein content on exosomes for the first time, and it is applied to the detection of serum samples, and the detection method has high stability.
  • the detection results obtained based on the detection method provided by the application are significantly correlated with the results obtained by traditional serum detection, which proves that the detection method of the application has high accuracy and feasibility.
  • the detection method of KL-6 protein content on exosomes provided by this application has important application value in the fields of exosome-related basic research and other fields.
  • This application also found for the first time that the KL-6 protein content on exosomes has a greater correlation with lung diseases, and this discovery is of great significance for the mechanism research and clinical diagnosis of lung diseases.
  • the application also provides a kit for diagnosing lung diseases.
  • the cut-off value (CUTOFF value) is set according to 2.1 times the concentration of exosome KL-6 in healthy people, that is, 50 U/mL, and the exosomes to be tested are determined.
  • the negative and positive results of the secretory KL-6 test can be used to confirm the patient's lung disease status.
  • the kit provided by the present application has better sensitivity in the diagnosis of lung diseases, reduces false negatives of diagnostic results, and has important application value.

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Abstract

一种外泌体上KL-6蛋白含量的检测方法及其应用，检测方法包括：利用免疫磁珠捕获法将磁珠与外泌体上的KL-6蛋白结合，利用化学发光免疫法检测体系的发光强度，最后通过标准曲线法计算得到外泌体上KL-6的蛋白含量。外泌体上KL-6蛋白含量的检测方法是首次提出，具有较好的稳定性和可行性，在外泌体相关基础研究等领域具有重要的应用价值。外泌体上KL-6蛋白含量与肺部患病情况有较大相关性是首次发现，提供了外泌体上KL-6蛋白含量的参考值作为肺部患病的评价标准，并提供了一种诊断肺部疾病的试剂盒，试剂盒相比于传统血清检测在肺部疾病的诊断上具有更好的灵敏度，减少了诊断结果的假阴性，具有重要的应用价值。

Description

一种外泌体上KL-6蛋白含量的检测方法及其应用 技术领域
本申请属于蛋白质检测技术领域，涉及一种外泌体上KL-6蛋白含量的检测方法及其应用，具体涉及一种外泌体上KL-6蛋白含量的检测方法以及一种诊断肺部疾病的试剂盒。
背景技术
KL-6(涎液化糖链抗原，Krebs Von den Lungen-6)，是Kohno于1985年发现的，利用人肺腺癌细胞系(VMRC-LCR)免疫小鼠制备了多株单克隆抗体，将其中的第6号抗体识别的涎液化糖链抗原命名为KL-6。KL-6主要由增殖、再生的或受损的肺泡II型上皮细胞分泌，在正常肺组织中，KL-6在II型肺泡上皮细胞、呼吸细支气管上皮细胞和支气管腺浆液细胞上表达。当肺部发生炎症尤其是与II型肺泡相关的肺炎时，II型肺泡的大量死亡和再生以及肺间质上皮细胞屏障的破损会导致KL-6大量释放到血液中。多项针对KL-6的研究表明，其作为间质性肺疾病(Interstitial Lung Diseases，ILDs)的血清学指标显示出积极的临床意义。因此测量外周血KL-6的水平可用于间质性肺炎的诊断和治疗策略的确定。其中，血清被认为是理想的测试样品，因为它可以无创地重复收集，然而，其中包含的99％以上的蛋白质是无关蛋白(如白蛋白等)，从检测角度考虑，这些蛋白质是杂质，会影响检测的准确度。
外泌体是指包含了复杂RNA和蛋白质的小膜泡(30-150nm)，现今，其特指直径在40-100nm的盘状囊泡。1983年，外泌体首次于绵羊网织红细胞中被发现，1987年Johnstone将其命名为“exosome”。多种细胞在正常及病理状态下均可分泌外泌体，且外泌体天然存在于体液中，如血液、唾液、尿液、脑脊液和乳汁中。外泌体被视为特异性分泌的膜泡，能够参与细胞间通讯，近年来，人们对外泌体的研究兴趣日益增长。研究表明，外泌体中含有多种与各种细胞生理活动相关的蛋白质，然而外泌体上的KL-6蛋白的含量情况及其功能尚不清楚，目前有关外泌体上KL-6蛋白的检测方法也未见报道。
因此，开发一种外泌体上KL-6蛋白含量的检测方法将对于外泌体相关研究以及KL-6蛋白的功能研究等领域具有重要的应用价值。
发明内容
本申请提供了一种外泌体上KL-6蛋白含量的检测方法及其应用，具体提供一种外泌体中KL-6蛋白含量的检测方法以及一种诊断肺部疾病的试剂盒。
第一方面，本申请提供一种外泌体上KL-6蛋白含量的检测方法，所述检测方法包括如下步骤：
(1)将捕获抗体标记的磁珠与待测外泌体样本混合，反应，得磁珠-外泌体复合物；
(2)将步骤(1)得到的磁珠-外泌体复合物与发光物标记的检测抗体混合，反应，得磁珠-外泌体-检测抗体复合物；
(3)将步骤(2)得到的磁珠-外泌体-检测抗体复合物与发光底物混合，反应，利用化学发光法检测反应体系的发光强度；以及
(4)利用标准曲线法，根据步骤(3)检测得到的发光强度计算得到待测外泌体样本的KL-6蛋白含量。
本申请首次提出外泌体上KL-6蛋白含量的检测方法，检测的原理如附图1所示，图中1—捕获抗体标记的磁珠；2—含有KL-6蛋白的外泌体；3—发光物标记的检测抗体。通过捕获抗体与KL-6蛋白的特异性结合作用以及检测抗体与KL-6蛋白的特异性结合作用，构建得到磁珠-外泌体-检测抗体复合物，由于检测抗体上标记有发光物，因此通过检测反应体系的发光强度可以间接反映外泌体上KL-6蛋白含量信息。通过标准曲线法，即可计算出KL-6蛋白含量的具体数值。本申请提供的检测方法具有较好的稳定性，基于本申请提供的检测方法得到的检测结果与传统的血清检测得到的结果显著相关，证明了本申请检测方法具有较高的准确性和可行性，在外泌体相关基础研究等领域具有重要的应用价值。基于此检测方法，本申请首次发现外泌体上KL-6蛋白含量与肺部患病情况有较大的相关性，此发现对于肺部疾病的机理研究以及临床诊断等方面具有重要意义。
优选地，步骤(1)所述反应的温度为33-40℃，例如33℃、34℃、35℃、36℃、37℃、38℃、39℃、40℃等。所述反应的时间为5-15min，例如5min、6min、7min、8min、9min、10min、11min、12min、13min、14min、15min等。
优选地，步骤(2)所述反应的温度为33-40℃，例如33℃、34℃、35℃、 36℃、37℃、38℃、39℃、40℃等。所述反应的时间为5-15min，例如5min、6min、7min、8min、9min、10min、11min、12min、13min、14min、15min等。
优选地，步骤(3)所述反应的温度为33-40℃，例如33℃、34℃、35℃、36℃、37℃、38℃、39℃、40℃等。所述反应的时间为5-10min，例如5min、6min、7min、8min、9min、10min等。
优选地，所述待测外泌体样本由全血、血浆、血清、支气管肺泡灌洗液、脑脊液或尿液中的任意一种待测样本制备得到。
优选地，所述待测外泌体样本的制备方法包括离心法、超滤法、磁珠免疫法、聚乙二醇沉淀法或试剂盒提取法中的任意一种，优选为离心法。
优选地，所述离心的转速为10000-20000g，例如10000g、11000g、12000g、13000g、14000g、15000g、16000g、17000g、18000g、19000g、20000g等。所述离心的时间为1-2h，例如1h、1.1h、1.2h、1.3h、1.4h、1.5h、1.6h、1.7h、1.8h、1.9h、2h等。
作为本申请的一个优选方案，所述外泌体上KL-6蛋白含量的检测方法包括如下步骤：
(1)向待测样本中加入0.5-3倍样本体积的磷酸盐缓冲液进行稀释，在0-4℃下以10000-20000g的转速离心1-2h，收集沉淀，加入磷酸盐缓冲液进行重悬，得待测外泌体样本；
向0.8-1.2mg磁珠中加入400-600μL平衡液，混合，移去液体，加入150-250μL磁珠活化液，混合，15-40℃下孵育20-60min，移去液体，加入80-120μg捕获抗体，15-40℃下孵育2.5-4h，移去液体，加入400-600μL封闭液，15-40℃下封闭2-3h，洗涤，加入磁珠保存液，得捕获抗体标记的磁珠；
取发光物8-12μL，用无水二甲基甲酰胺稀释8-12倍后，取10-30μL与80-120μg待标记检测抗体混合，15-40℃下避光孵育20-60min，加入10-30μL封闭液，15-40℃下避光封闭20-60min，加入保存液，得发光物标记的检测抗体；
(2)取8-12μL待测外泌体样本与200-300μL捕获抗体标记的磁珠混合，33-40℃下反应5-15min，洗涤，除去液体，加入200-300μL发光物标记的检测抗体，33-40℃下反应5-15min，洗涤，除去液体，加入180-220μL发光底物，33-40℃下孵育5-10min，在477nm波长下检测反应体系的发光强度；以及
(3)取与待测外泌体样本等体积的KL-6标准品，重复步骤(2)的操作，依据标准品检测得的发光强度拟合成标准曲线，利用标准曲线法计算出待测样本中KL-6含量。
上述0.5-3倍中的具体数值可以为0.5倍、0.6倍、0.7倍、0.8倍、0.9倍、1倍、1.2倍、1.5倍、1.7倍、2倍、2.2倍、2.5倍、2.7倍、3倍等。
上述0.8-1.2mg中的具体数值可以为0.8mg、0.85mg、0.9mg、0.95mg、1mg、1.05mg、1.1mg、1.15mg、1.2mg。
上述400-600μL中的具体数值可以为400μL、420μL、440μL、450μL、460μL、480μL、500μL、520μL、540μL、550μL、560μL、580μL、600μL等。
上述150-250μL中的具体数值可以为150μL、160μL、170μL、180μL、190μL、200μL、210μL、220μL、230μL、240μL、250μL等。
上述80-120μg中的具体数值可以为80μg、85μg、90μg、95μg、100μg、105μg、110μg、115μg、120μg等。
上述2-3h中的具体数值可以为2h、2.1h、2.2h、2.3h、2.4h、2.5h、2.6h、2.7h、2.8h、2.9h、3h等。
上述8-12μL中的具体数值可以为8μL、8.5μL、9.5μL、10μL、10.5μL、11μL、11.5μL、12μL等。
上述8-12倍中的具体数值可以为8倍、8.5倍、9.5倍、10倍、10.5倍、11倍、11.5倍、12倍等。
上述10-30μL中的具体数值可以为10μL、12μL、15μL、18μL、20μL、22μL、25μL、28μL、30μL等。
上述80-120μg中的具体数值可以为80μg、85μg、90μg、95μg、100μg、105μg、110μg、115μg、120μg等。
上述200-300μL中的具体数值可以为200μL、210μL、220μL、230μL、240μL、250μL、260μL、270μL、280μL、290μL、300μL等。
上述180-220μL中的具体数值可以为180μL、185μL、190μL、195μL、200μL、205μL、210μL、215μL、220μL等。
第二方面，本申请提供如第一方面所述的外泌体上KL-6蛋白含量的检测方法在制备诊断肺部疾病的试剂盒中的应用。
第三方面，本申请提供一种诊断肺部疾病的试剂盒，所述诊断肺部疾病的试剂盒包括捕获抗体标记的磁珠、发光物标记的检测抗体、发光底物和KL-6标准品。
本申请创造性地提供了一种诊断肺部疾病的试剂盒，基于检测结果，依照健康人外泌体KL-6浓度的2.1倍设置截断值(CUTOFF值)，即50U/mL，确定待测样本外泌体KL-6检测结果的阴阳性，并以此来确认患者肺部疾病状态。本申请提供的试剂盒相比于传统血清检测在肺部疾病的诊断上具有更好的灵敏度，减少了诊断结果的假阴性，具有重要的应用价值。
优选地，所述捕获抗体包括8MKL-61。
优选地，所述检测抗体包括8MKL-62。
优选地，所述试剂盒用于检测外泌体上KL-6蛋白含量水平。
所述捕获抗体标记的磁珠由包括如下步骤的制备方法制备得到：
将捕获抗体与活化后的磁珠混合，孵育，封闭磁珠，即得。
优选地，所述孵育的温度为15-40℃，例如15℃、17℃、20℃、22℃、25℃、27℃、30℃、32℃、35℃、37℃、40℃等。所述孵育的时间为2.5-4h，例如2.5h、2.6h、2.7h、2.8h、2.9h、3h、3.1h、3.2h、3.3h、3.4h、3.5h、3.6h、3.7h、3.8h、3.9h、4h等。
优选地，所述捕获抗体与磁珠的质量比为1:(8-12)，例如1:8、1:8.5、1:9、1:9.5、1:10、1:10.5、1:11、1:11.5、1:12等。
优选地，所述发光物标记的检测抗体由包括如下步骤的制备方法制备得到：
将发光物与待标记的检测抗体混合，避光孵育，封闭抗体，即得。
优选地，所述孵育的温度为15-40℃，例如15℃、17℃、20℃、22℃、25℃、27℃、30℃、32℃、35℃、37℃、40℃等。所述孵育的时间为20-60min，例如20min、25min、30min、35min、40min、45min、50min、55min、60min等。
优选地，所述肺部疾病的类型包括肺炎或间质性肺部疾病。
第四方面，本申请提供一种如第三方面所述的诊断肺部疾病的试剂盒的使用方法，所述使用方法包括：
(1)将捕获抗体标记的磁珠与待测外泌体样本混合，反应，得磁珠-外泌体复合物；
(2)将步骤(1)得到的磁珠-外泌体复合物与发光物标记的检测抗体混合， 反应，得磁珠-外泌体-检测抗体复合物；
(3)将步骤(2)得到的磁珠-外泌体-检测抗体复合物与发光底物混合，反应，利用化学发光法检测反应体系的发光强度；以及
(4)利用KL-6标准品，通过标准曲线法，根据步骤(3)检测得到的发光强度计算得到待测外泌体样本的KL-6蛋白含量。
优选地，步骤(1)所述反应的温度为33-40℃，所述反应的时间为5-15min；
优选地，步骤(2)所述反应的温度为33-40℃，所述反应的时间为5-15min；
优选地，步骤(3)所述反应的温度为33-40℃，所述反应的时间为5-10min。
优选地，所述待测外泌体样本由全血、血浆、血清、支气管肺泡灌洗液、脑脊液或尿液中的任意一种待测样本制备得到。
优选地，所述待测外泌体样本的制备方法包括离心法、超滤法、磁珠免疫法、聚乙二醇沉淀法或试剂盒提取法中的任意一种，优选为离心法。
优选地，所述离心的转速为10000-20000g，所述离心的时间为1-2h。
作为本申请的一个优选方案，所述诊断肺部疾病的试剂盒的使用方法包括如下步骤：
(1)向待测样本中加入0.5-3倍样本体积的磷酸盐缓冲液进行稀释，在0-4℃下以10000-20000g的转速离心1-2h，收集沉淀，加入磷酸盐缓冲液进行重悬，得待测外泌体样本；
向0.8-1.2mg磁珠中加入400-600μL平衡液，混合，移去液体，加入150-250μL磁珠活化液，混合，15-40℃下孵育20-60min，移去液体，加入80-120μg捕获抗体，15-40℃下孵育2.5-4h，移去液体，加入400-600μL封闭液，15-40℃下封闭2-3h，洗涤，加入磁珠保存液，得捕获抗体标记的磁珠；
取发光物8-12μL，用无水二甲基甲酰胺稀释8-12倍后，取10-30μL与80-120μg待标记检测抗体混合，15-40℃下避光孵育20-60min，加入10-30μL封闭液，15-40℃下避光封闭20-60min，加入保存液，得发光物标记的检测抗体；
(2)取8-12μL待测外泌体样本与200-300μL捕获抗体标记的磁珠混合，33-40℃下反应5-15min，洗涤，除去液体，加入200-300μL发光物标记的检测抗体，33-40℃下反应5-15min，洗涤，除去液体，加入180-220μL发光底物，33-40℃下孵育5-10min，在477nm波长下检测反应体系的发光强度；以及
(3)取与待测外泌体样本等体积的KL-6标准品，重复步骤(2)的操作， 依据标准品检测得的发光强度拟合成标准曲线，利用标准曲线法计算出待测样本中KL-6含量。
相对于现有技术，本申请具有以下有益效果：
本申请首次提出外泌体上KL-6蛋白含量的检测方法，并应用于血清样本的检测，检测方法具有较高的稳定性。基于本申请提供的检测方法得到的检测结果与传统的血清检测得到的结果显著相关，证明了本申请检测方法具有较高的准确性和可行性。本申请提供的外泌体上KL-6蛋白含量的检测方法在外泌体相关基础研究等领域具有重要的应用价值。本申请还首次发现外泌体上KL-6蛋白含量与肺部患病情况有较大相关性，此发现对于肺部疾病的机理研究以及临床诊断等方面具有重要意义。本申请还提供了一种诊断肺部疾病的试剂盒，基于检测结果，依照健康人外泌体KL-6浓度的2.1倍设置截断值(CUTOFF值)，即50U/mL，确定待测样本外泌体KL-6检测结果的阴阳性，并以此来确认患者肺部疾病状态。本申请提供的试剂盒相比于传统血清检测在肺部疾病的诊断上具有更好的灵敏度，减少了诊断结果的假阴性，具有重要的应用价值。
附图说明
图1是外泌体上KL-6蛋白含量的检测方法的检测原理图。其中1—捕获抗体标记的磁珠；2—含有KL-6蛋白的外泌体；3—发光物标记的检测抗体。
图2是实施例1中步骤(1)制备得到的外泌体的形态表征图。
图3是健康人样本与ILDs患者样本的检测结果对比图。
图4是实施例1的检测结果与传统血清检测结果的相关性分析图。
图5是血清KL-6临床参考值与本申请的外泌体KL-6截断值对比分析图。
具体实施方式
下面通过具体实施方式来进一步说明本申请的技术方案。本领域技术人员应该明了，所述实施例仅仅是帮助理解本申请，不应视为对本申请的具体限制。
下述实施例所涉及的健康人血清样本来源于健康志愿者，ILDs患者的血清样本来源于广州医科大学附属第一医院，所有人员采样前均对本实验知情并同意，血清样本的采集遵循医院标准。
实施例1
本实施例提供一种外泌体上KL-6蛋白含量的检测方法，包括如下步骤：
(1)分离提取外泌体
样本预处理：将血清样本(1mL)于4℃以10000g的转速离心10min，去除样本中杂质，将离心后上清液转移至新的离心管中。
上清液预处理：在去除杂质的上清液中加入4倍样本体积的预冷的0.01M PBS，将混合液放置于4℃静置2h。
分离外泌体：将混合液于4℃以10000g的转速离心1h，弃上清，收集沉淀，加入1/2样本体积的预冷的0.01M PBS，均匀吹打混匀，即得待测外泌体样本。
(2)制备捕获抗体标记的磁珠
磁珠预处理：向1mg磁珠中加入500μL平衡液(0.01M PBS)，涡旋震荡混匀，于磁力架上移去液体。加入200μL磁珠活化Buffer(10mg/mL EDC，10mg/mL NHS)，涡旋混匀，利用涡旋振荡器在25℃下震荡孵育30min。
抗体偶联：磁力架上移去磁珠活化Buffer，加入捕获抗体(8MKL-61 100μg)，涡旋混匀，利用涡旋振荡器在25℃下震荡孵育3h。
磁珠封闭：在磁力架上移去未偶联的抗体，加入500μL封闭液(0.01M PBS配制的5％BSA)，涡旋混匀，利用垂直混匀器在25℃下颠倒封闭2h。洗涤后，加入1mL磁珠保存液(0.01M PBS配制的3％BSA)，4℃保存备用。
(3)制备发光物标记的检测抗体
取AP(碱性磷酸酶)10μL，加入90μL无水DMF(无水二甲基甲酰胺)，混合均匀后取20μL至100μL加入到待标记的检测抗体(8MKL-62)中，混匀后利用振荡器在25℃避光下孵育30min。
抗体封闭：取20μL封闭液(5％赖氨酸)至孵育完成的AP标记的检测抗体中，混匀后利用振荡器在25℃避光下震荡封闭30min。透析，加入1mL AP保存液(0.01M PBS)重悬，4℃保存备用。
(4)待测外泌体样本中KL-6含量的检测与计算
将10μL步骤(1)制得的待测外泌体样本与180μL样本稀释液(0.01M PBS，0.05％吐温-20)混合，充分混匀，得待测样本测试液。将10μL标准品(已知浓度的KL-6重组蛋白)与180μL样本稀释液混合，充分混匀，得标准品测试液。将10μL校准品(440U/ml的KL-6)与180μL样本稀释液混合，充分混匀，得低值校准品测试液。将10μL校准品(1040U/ml的KL-6)与180μL样本稀 释液混合，充分混匀，得高值校准品测试液。分别对待测样本测试液、标准品测试液(6种浓度的KL-6重组蛋白分别进行操作)、低值校准品测试液和高值校准品测试液进行如下操作：
取250μL步骤(2)制得的捕获抗体标记的磁珠，加入10μL测试液，充分混匀后37℃孵育10min。反复清洗去除反应液，得捕获外泌体的磁珠。
将250μL步骤(3)制得的AP标记的抗体与捕获外泌体的磁珠混合，充分混匀后37℃孵育10min。反复清洗去除反应液，得抗体结合的磁珠复合物。向其中加入AMPPD底物液200μL，充分混匀后37℃反应5min。在477nm波长下检测反应体系的发光强度。
依据标准品检测得的发光强度拟合成标准曲线，通过标准曲线计算出待测样本中KL-6含量。
实施例2
本实施例提供一种外泌体上KL-6蛋白含量的检测方法，包括如下步骤：
(1)分离提取外泌体
样本预处理：将血清样本(1mL)于4℃以10000g的转速离心10min，去除样本中杂质，将离心后上清液转移至新的离心管中。
上清液预处理：在去除杂质的上清液中加入1倍样本体积的预冷的PBS(浓度为0.01M)，得混合液。
分离外泌体：将混合液于4℃以20000g的转速离心1h，弃上清，收集沉淀，加入1倍样本体积的预冷的PBS，均匀吹打混匀，即得待测外泌体样本。
(2)制备捕获抗体标记的磁珠
磁珠预处理：向0.9mg磁珠中加入400μL平衡液(0.01M PBS)，涡旋震荡混匀，于磁力架上移去液体。加入160μL磁珠活化Buffer(10mg/mL EDC，10mg/mL NHS)，涡旋混匀，利用涡旋振荡器在25℃下震荡孵育50min。
抗体偶联：磁力架上移去磁珠活化Buffer，加入捕获抗体(8MKL-61 100μg)，涡旋混匀，利用涡旋振荡器在25℃下震荡孵育2.5h。
磁珠封闭：在磁力架上移去未偶联的抗体，加入450μL封闭液(0.01M PBS配制的5％BSA)，涡旋混匀，利用垂直混匀器在25℃下颠倒封闭3h。洗涤后，加入1mL磁珠保存液(0.01M PBS配制的3％BSA)，4℃保存备用。
(3)制备发光物标记的检测抗体
取AE(吖啶酯)10μL，加入90μL无水DMF(无水二甲基甲酰胺)，混合均匀后取20μL至100μL加入到待标记的检测抗体(8MKL-62)中，混匀后利用振荡器在25℃避光下孵育40min。
抗体封闭：取20μL封闭液(5％赖氨酸)至孵育完成的AE标记的检测抗体中，混匀后利用振荡器在25℃避光下震荡封闭40min。透析，加入1mL AE保存液(0.01M PBS，pH7.4)重悬，4℃保存备用。
(4)待测外泌体样本中KL-6含量的检测与计算
将10μL步骤(1)制得的待测外泌体样本与180μL样本稀释液(0.01M PBS，0.05％吐温-20)混合，充分混匀，得待测样本测试液。将10μL标准品(已知浓度的KL-6重组蛋白)与180μL样本稀释液混合，充分混匀，得标准品测试液。将10μL校准品(440U/ml的KL-6)与180μL样本稀释液混合，充分混匀，得低值校准品测试液。将10μL校准品(1040U/ml的KL-6)与180μL样本稀释液混合，充分混匀，得高值校准品测试液。分别对待测样本测试液、标准品测试液(6种浓度的KL-6重组蛋白分别进行操作)、低值校准品测试液和高值校准品测试液进行如下操作：
取200μL步骤(2)制得的捕获抗体标记的磁珠，加入10μL测试液，充分混匀后37℃孵育15min。反复清洗去除反应液，得捕获外泌体的磁珠。
将200μL步骤(3)制得的AE标记的抗体与捕获外泌体的磁珠混合，充分混匀后35℃孵育8min。反复清洗去除反应液，得抗体结合的磁珠复合物。向其中加入AE预激发液100μL，立即加入100μL激发液，充分混匀后测定最大发光强度。依据标准品检测得的发光强度拟合成标准曲线，通过标准曲线计算出待测样本中KL-6含量。
实施例3
本实施例提供一种外泌体上KL-6蛋白含量的检测方法，包括如下步骤：
(1)分离提取外泌体
样本预处理：将血清样本(1mL)于4℃以10000g的转速离心10min，去除样本中杂质，将离心后上清液转移至新的离心管中。
上清液预处理：在去除杂质的上清液中加入2倍样本体积的预冷的PBS(浓度为0.01M)，得混合液。
分离外泌体：将混合液于4℃以12000g的转速离心2h，弃上清，收集沉 淀，加入1/2样本体积的预冷的PBS，均匀吹打混匀，即得待测外泌体样本。
(2)制备捕获抗体标记的磁珠
磁珠预处理：向1.1mg磁珠中加入600μL平衡液(0.01M PBS)，涡旋震荡混匀，于磁力架上移去液体。加入300μL磁珠活化Buffer(10mg/mL EDC，10mg/mL NHS)，涡旋混匀，利用涡旋振荡器在25℃下震荡孵育20min。
抗体偶联：磁力架上移去磁珠活化Buffer，加入捕获抗体(8MKL-61 100μg)，涡旋混匀，利用涡旋振荡器在25℃下震荡孵育3h。
磁珠封闭：在磁力架上移去未偶联的抗体，加入600μL封闭液(0.01M PBS配制的5％BSA)，涡旋混匀，利用垂直混匀器在25℃下颠倒封闭2.5h。洗涤后，加入1mL磁珠保存液(0.01M PBS配制的3％BSA)，4℃保存备用。
(3)制备发光物标记的检测抗体
取AP(碱性磷酸酶)10μL，加入100μL无水DMF(无水二甲基甲酰胺)，混合均匀后取20μL至100μL加入到待标记的检测抗体(8MKL-62)中，混匀后利用振荡器在25℃避光下孵育20min。
抗体封闭：取25μL封闭液(5％赖氨酸)至孵育完成的AP标记的检测抗体中，混匀后利用振荡器在25℃避光下震荡封闭20min。AP保存液重悬透析过的AP标记抗体，4℃保存备用。
(4)待测外泌体样本中KL-6含量的检测与计算
将10μL步骤(1)制得的待测外泌体样本与180μL样本稀释液(0.01M PBS，0.05％吐温-20)混合，充分混匀，得待测样本测试液。将10μL标准品(已知浓度的KL-6重组蛋白)与180μL样本稀释液混合，充分混匀，得标准品测试液。将10μL校准品(440U/ml的KL-6)与180μL样本稀释液混合，充分混匀，得低值校准品测试液。将10μL校准品(1040U/ml的KL-6)与180μL样本稀释液混合，充分混匀，得高值校准品测试液。分别对待测样本测试液、标准品测试液(6种浓度的KL-6重组蛋白分别进行操作)、低值校准品测试液和高值校准品测试液进行如下操作：
取280μL步骤(2)制得的捕获抗体标记的磁珠，加入10μL测试液，充分混匀后37℃孵育10min。反复清洗去除反应液，得捕获外泌体的磁珠。
将280μL步骤(3)制得的AP标记的抗体与捕获外泌体的磁珠混合，充分混匀后38℃孵育6min。反复清洗去除反应液，得抗体结合的磁珠复合物。向其 中加入AMPPD底物液200μL，充分混匀后36℃反应5min。在477nm波长下检测反应体系的发光强度。
依据标准品检测得的发光强度拟合成标准曲线，通过标准曲线计算出待测样本中KL-6含量。
测试例1
外泌体形态表征
利用透射电镜对实施例1的步骤(1)中制得的待测外泌体样本进行形态表征，结果见附图2。如图所示，提取到的外泌体具有典型的囊泡状结构，且粒径分布在40-100nm，与相关文献报道的结果一致，证明了外泌体的成功提取。
测试例2
方法的稳定性
取健康志愿者血清样本12例，每例3mL，分别采用实施例1-3提供的检测方法进行外泌体上KL-6蛋白含量的检测(样本量均为1mL)，3种检测方法得到的检测结果见表1。
表1
Figure PCTCN2021143493-appb-000001
Figure PCTCN2021143493-appb-000002
结果显示：实施例1-3的检测方法对同一样本的检测结果较为一致，标准误差较小，说明本申请提供的检测方法稳定性高，具有一定的应用价值。
测试例3
健康人样本与ILDs患者样本检测结果对比
取ILDs患者血清样本12例，采用实施例1提供的检测方法进行外泌体上KL-6蛋白含量的检测(样本量均为1mL)，检测结果见表2，并将测试例2中采用实施例1提供的检测方法得到的12例健康人样本的检测结果也列于表2进行对比。
表2
Figure PCTCN2021143493-appb-000003
结果显示：整体来看，ILDs患者的外泌体上的KL-6蛋白含量显著高于健康志愿者样本的外泌体上的KL-6蛋白含量，将表中数据绘制成箱线图(见附图3)更是可以明显看出此现象。各样本的外泌体KL-6浓度存在个体差异，其中健康人样本的外泌体KL-6浓度范围为9-51U/mL，均值为24。ILDs患者的外泌体KL-6浓度范围为51-10000U/mL，均值为1147。依照健康人外泌体KL-6浓度的2.1倍设置截断值(CUTOFF值)，即50U/mL，可依据此CUTOFF值来 确定待测样本外泌体KL-6检测结果的阴阳性，并以此来确认患者肺部疾病状态。
测试例4
本申请的检测方法(即试剂盒检测)与传统血清检测方法的结果对比
采用传统的血清KL-6含量检测方法对测试例2中的12例ILDs患者的血清样本进行检测，将其检测结果与本申请的方法得到的检测结果进行对比，并进行相关性分析，见附图4。结果显示：两种方法得到的检测结果显著相关，相关系数0.9349，P<0.0001。说明了本检测方法的准确性和可行性。
目前，通过传统的血清KL-6含量检测方法进行肺部患病情况的阴阳性判定时，临床上给出的参考值是500U/mL。如附图5所示，图中竖虚线表示血清KL-6检测值为500U/mL，图上横虚线表示本申请提供的外泌体KL-6检测截断值50U/mL。图中箭头标出的四例患者的血清KL-6值低于500U/mL，因此若依照传统的血清KL-6含量检测方法则应判定他们为阴性，而这四例患者的外泌体KL-6值高于本申请提供的截断值50U/mL，因此依照本申请提供的试剂盒检测则应判定他们为阳性。这一结果表明，本申请提供的检测肺部疾病的试剂盒相较于传统的血清检测在肺部疾病的诊断上具有更好的灵敏度，减少了诊断结果的假阴性，具有重要的应用价值。
综上，本申请首次提出外泌体上KL-6蛋白含量的检测方法，并应用于血清样本的检测，检测方法具有较高的稳定性。基于本申请提供的检测方法得到的检测结果与传统的血清检测得到的结果显著相关，证明了本申请检测方法具有较高的准确性和可行性。本申请提供的外泌体上KL-6蛋白含量的检测方法在外泌体相关基础研究等领域具有重要的应用价值。本申请还首次发现外泌体上KL-6蛋白含量与肺部患病情况有较大相关性，此发现对于肺部疾病的机理研究以及临床诊断等方面具有重要意义。本申请还提供了一种诊断肺部疾病的试剂盒，基于检测结果，依照健康人外泌体KL-6浓度的2.1倍设置截断值(CUTOFF值)，即50U/mL，确定待测样本外泌体KL-6检测结果的阴阳性，并以此来确认患者肺部疾病状态。本申请提供的试剂盒相比于传统血清检测在肺部疾病的诊断上具有更好的灵敏度，减少了诊断结果的假阴性，具有重要的应用价值。
申请人声明，本申请通过上述实施例来说明本申请的外泌体上KL-6蛋白含量的检测方法及其应用，但本申请并不局限于上述实施例，即不意味着本申请必须依赖上述实施例才能实施。所属技术领域的技术人员应该明了，对本申请 的任何改进，对本申请产品各原料的等效替换及辅助成分的添加、具体方式的选择等，均落在本申请的保护范围和公开范围之内。
以上详细描述了本申请的优选实施方式，但是，本申请并不限于上述实施方式中的具体细节，在本申请的技术构思范围内，可以对本申请的技术方案进行多种简单变型，这些简单变型均属于本申请的保护范围。
另外需要说明的是，在上述具体实施方式中所描述的各个具体技术特征，在不矛盾的情况下，可以通过任何合适的方式进行组合，为了避免不必要的重复，本申请对各种可能的组合方式不再另行说明。

Claims (13)

  1. 一种外泌体上KL-6蛋白含量的检测方法，其包括如下步骤：
    (1)将捕获抗体标记的磁珠与待测外泌体样本混合，反应，得磁珠-外泌体复合物；
    (2)将步骤(1)得到的磁珠-外泌体复合物与发光物标记的检测抗体混合，反应，得磁珠-外泌体-检测抗体复合物；
    (3)将步骤(2)得到的磁珠-外泌体-检测抗体复合物与发光底物混合，反应，利用化学发光法检测反应体系的发光强度；以及
    (4)利用标准曲线法，根据步骤(3)检测得到的发光强度计算得到待测外泌体样本的KL-6蛋白含量。
  2. 如权利要求1所述的外泌体上KL-6蛋白含量的检测方法，其中，步骤(1)所述反应的温度为33-40℃，所述反应的时间为5-15min。
  3. 如权利要求1所述的外泌体上KL-6蛋白含量的检测方法，其中，步骤(2)所述反应的温度为33-40℃，所述反应的时间为5-15min。
  4. 如权利要求1所述的外泌体上KL-6蛋白含量的检测方法，其中，步骤(3)所述反应的温度为33-40℃，所述反应的时间为5-10min。
  5. 如权利要求1-4中任一项所述的外泌体上KL-6蛋白含量的检测方法，其中，所述待测外泌体样本由全血、血浆、血清、支气管肺泡灌洗液、脑脊液或尿液中的任意一种待测样本制备得到。
  6. 如权利要求1-5中任一项所述的外泌体上KL-6蛋白含量的检测方法，其中，所述待测外泌体样本的制备方法包括离心法、超滤法、磁珠免疫法、聚乙二醇沉淀法或试剂盒提取法中的任意一种，优选为离心法；
    优选地，所述离心的转速为10000-20000g，所述离心的时间为1-2h。
  7. 如权利要求1-6中任一项所述的外泌体上KL-6蛋白含量的检测方法在制备诊断肺部疾病的试剂盒中的应用。
  8. 一种诊断肺部疾病的试剂盒，其包括捕获抗体标记的磁珠、发光物标记的检测抗体、发光底物和KL-6标准品。
  9. 如权利要求8所述的诊断肺部疾病的试剂盒，其中，所述捕获抗体包括8MKL-61；
    优选地，所述检测抗体包括8MKL-62。
  10. 如权利要求8或9所述的诊断肺部疾病的试剂盒，其中，所述试剂盒 用于检测外泌体上KL-6蛋白含量水平。
  11. 如权利要求8-10任一项所述的诊断肺部疾病的试剂盒，其中，所述捕获抗体标记的磁珠由包括如下步骤的制备方法制备得到：
    将捕获抗体与活化后的磁珠混合，孵育，封闭磁珠，即得；
    优选地，所述孵育的温度为15-40℃，所述孵育的时间为2.5-4h；
    优选地，所述捕获抗体与磁珠的质量比为1:(8-12)。
  12. 如权利要求8-11中任一项所述的诊断肺部疾病的试剂盒，其中，所述发光物标记的检测抗体由包括如下步骤的制备方法制备得到：
    将发光物与待标记的检测抗体混合，避光孵育，封闭抗体，即得；
    优选地，所述孵育的温度为15-40℃，所述孵育的时间为20-60min。
  13. 如权利要求8-12中任一项所述的诊断肺部疾病的试剂盒，其中，所述肺部疾病的类型包括肺炎或间质性肺部疾病。
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