CN116609523A - Kit for diagnosing interstitial lung diseases by using CA15-3 protein on exosome and application - Google Patents

Kit for diagnosing interstitial lung diseases by using CA15-3 protein on exosome and application Download PDF

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CN116609523A
CN116609523A CN202310543518.9A CN202310543518A CN116609523A CN 116609523 A CN116609523 A CN 116609523A CN 202310543518 A CN202310543518 A CN 202310543518A CN 116609523 A CN116609523 A CN 116609523A
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刘晨旭
洪锐槟
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Guangzhou Zhaorui Medical Biotechnology Co ltd
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Guangzhou Zhaorui Medical Biotechnology Co ltd
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Abstract

The application relates to a kit for auxiliary diagnosis of interstitial lung diseases by taking CA15-3 protein detection on exosomes as a marker and a detection method thereof; the application belongs to the technical field of protein detection, and the detection method comprises the following steps: after an exosome sample is treated by using a sample treatment liquid, exosomes containing CA15-3 proteins in the sample react with a magnetic bead coating working liquid and a luminescent substance labeling antibody working liquid to form an immune complex, and a chemiluminescent method is used for detecting the luminous intensity and calculating the concentration value; the application provides a method for assisting in diagnosis of interstitial lung diseases by using CA15-3 on exosomes, which has higher specificity and sensitivity compared with the existing serum detection kit on the market, and has important clinical value and popularization prospect in assisting in diagnosis of interstitial lung diseases.

Description

Kit for diagnosing interstitial lung diseases by using CA15-3 protein on exosome and application
Technical Field
The application belongs to the technical field of protein detection, and relates to a kit for auxiliary diagnosis of interstitial lung diseases by using CA15-3 protein on exosomes, a detection method and application.
Background
Saccharide antigen 15-3 (CA 15-3) is derived from mammary epithelial cells, is mutated from its surface saccharide antigen, and is released into the blood circulation. CA15-3 is a soluble form of Mucin1 (Mucin 1 or MUC 1) after hydrolysis. CA15-3 was initially found to be a tumor antigen recognized by both monoclonal antibodies DF3 and 115D8, where DF3 recognizes the MUC1 core protein and 115D8 recognizes a portion of the glycan chains on MUC 1. MUC1 is a highly glycosylated membrane-bound protein expressed on the apical surface of tissue epithelial cells such as the gastrointestinal tract, esophagus, etc., belongs to the mucin family, and has a molecular weight of about 400-450kDa. MUC1 is composed of alpha and beta subunits, the N terminal alpha subunit has cell adhesiveness, can be used as adhesion protein or anti-adhesion protein to provide protective mucus barrier for epithelial cells, prevents bacterial and enzymatic attack, and the C terminal beta subunit of MUC1 can participate in regulating ERK, SRC and NF-kappa-B pathway cell signaling through phosphorylation and protein interaction, and can influence Ras/MAPK pathway in activating T cells to promote tumor development; CA15-3 is considered a relatively promising serological indicator, but CA15-3 has less research on the occurrence, progression, efficacy and prognosis of ILD lesions.
Exosomes refer to extracellular vesicles with diameters of 40-160 nm and average diameters of 100nm, and are now becoming hot spots for diagnosis and treatment of various diseases. CA15-3 is a glycoprotein located on the cell membrane, so in theory, the membrane protein can be secreted into the circulatory system through exosomes, and detection of exosomes CA15-3 has important potential clinical value in serodiagnosis. A plurality of cells can secrete exosomes under normal and pathological conditions, but the relation between CA15-3 on exosomes and interstitial pneumonia is not reported at present.
The existing detection reagent based on the target spot in serum has low sensitivity and insignificant clinical benefit for auxiliary diagnosis of interstitial lung diseases, so that the establishment of a method for accurately detecting the lung disease related protein on exosomes is of great clinical value and research value for judging the disease type and progress state of interstitial pneumonia.
Disclosure of Invention
The application aims to provide a kit for auxiliary diagnosis of interstitial lung diseases by taking CA15-3 protein on exosomes as a marker, and a detection method and application thereof.
In a first aspect, the application provides an application of CA15-3 protein on exosomes in preparing a kit for diagnosing lung diseases, which adopts the following technical scheme:
an application of CA15-3 protein on exosomes in preparing a kit for diagnosing lung diseases.
The kit can be used for auxiliary diagnosis of interstitial pneumonia and other lung diseases.
In a second aspect, the application provides a kit for diagnosing pulmonary diseases, which adopts the following technical scheme:
a kit for diagnosing lung diseases comprises a sample treatment solution, a magnetic bead coated by a CA15-3 capture antibody, a luminescent substance-marked CA15-3 detection antibody, a luminescent substrate and a CA15-3 calibrator.
The application discloses a kit for auxiliary diagnosis of interstitial lung diseases by taking CA15-3 protein detection on exosomes as a marker, and further, the kit is used for auxiliary diagnosis of interstitial pneumonia or other lung diseases.
In a specific embodiment, the sample processing fluid comprises the following raw materials in weight percent: 0.5 to 1.5 percent of TRIS, 0.3 to 3 percent of NaCl, 0.5 to 3 percent of BSA, 0.1 to 1 percent of Triton X-100, 0.1 to 0.5 percent of Tween-80, 0.01 to 0.1 percent of EDTA-2Na, 0.1 to 1.0 percent of polyvinylpyrrolidone-K30 (PVP-K30), 0.2 to 2 percent of bicine (N, N-dihydroxyethylglycine), 0.1 to 0.3 percent of reducing agent, 0.05 to 0.5 percent of ProClin and the balance of water.
Preferably, the reducing agent is at least one of DTT, DEAE, beta-mercaptoethanol and TCEP.
The pretreatment mode is added to reduce the influence of components in the exosome extraction eluent on exosome protein detection, effectively shorten the detection time and facilitate the realization of exosome protein full-automatic detection; and the detection accuracy can be further improved after pretreatment, and the detection time can be greatly shortened without additional incubation.
Through researches, the detection luminescence value of the CA15-3 protein on the exosomes of the ILD patients is obviously higher than that of the CA15-3 protein on the exosomes of healthy people, and the ILD patients show very obvious differences. Therefore, the detection of the exosome CA15-3 has higher sensitivity and specificity, and can effectively identify samples which cannot be diagnosed by the conventional serum KL-6 detection reagent, so that the exosome CA15-3 detection can have important potential value in the auxiliary diagnosis and treatment process of the interstitial lung diseases.
In a third aspect, the application provides a chemiluminescent detection method for detecting CA15-3 protein on exosomes, which adopts the following technical scheme:
the kit is used for detecting the protein content on exosomes, and comprises the following steps: before the exosome sample to be detected is detected, preprocessing is carried out on the exosome sample to be detected, and the exosome sample to be detected is mixed with a sample processing liquid to obtain the preprocessed exosome sample to be detected.
After the exosome sample is pretreated by using a sample treatment liquid, exosomes in the sample are cracked, CA15-3 protein on the exosomes is released into the solution, and immune complexes are formed by reacting with a magnetic bead coating working liquid and a illuminant labeling antibody working liquid, and the luminous intensity is detected by using a chemiluminescence method, and the concentration value is calculated.
In a specific embodiment, the kit is used in a method for detecting the content of CA15-3 protein on exosomes, comprising the following steps:
a chemiluminescent detection method for detecting CA15-3 protein on exosomes comprises the following steps:
(1) Pretreatment is carried out on an exosome sample to be tested: mixing the exosome sample to be tested with a sample treatment liquid for reaction to obtain a pretreated exosome sample to be tested;
(2) Mixing the pretreated exosome sample to be tested obtained in the step (1) with a magnetic bead coating working solution, and reacting to obtain a magnetic bead-antigen complex;
(3) Mixing the magnetic bead-antigen complex obtained in the step (2) with a illuminant labeled antibody working solution, and reacting to obtain a magnetic bead-antigen-detection antibody complex;
(4) Mixing the magnetic bead-antigen-detection antibody complex obtained in the step (3) with a luminescent substrate, reacting, and detecting the luminous intensity;
(5) And (3) calculating the protein content in the exosome sample to be detected according to the luminous intensity obtained by the detection in the step (4) by using a standard curve method.
In a specific embodiment, the volume ratio of the exosome sample to be tested in step (1) to the sample processing fluid is 1: (2-30).
Preferably, in the step (1), the volume ratio of the exosome sample to be tested to the sample treatment fluid is 1:2, 1:2.5, 1: 5. 1:10, 1:12, 1:15, 1:18, 1:20, 1:22, 1:25, 1:28, or 1:30.
In a specific embodiment, the exosome sample in step (1) is reacted with the sample treatment fluid for a period of time ranging from 1 to 120 minutes; preferably, the reaction time is 1 to 10 minutes.
Preferably, the reaction time is 1min, 2min, 3min, 4min, 5min, 6min, 7min, 8min, 9min or 10min.
In a specific embodiment, the exosome sample in step (1) is reacted with the sample treatment fluid at a temperature of 25-42 ℃; preferably, the reaction temperature is 35-40 ℃.
Preferably, the reaction temperature is 35 ℃, 36 ℃, 37 ℃, 38 ℃, 39 ℃, 40 ℃.
The preparation method of the illuminant-labeled antibody working solution in the step (3) comprises the following steps: diluting the detection antibody marked by the luminescent substance by using a marker diluent to obtain a luminescent marker working solution;
wherein the marker diluent comprises the following raw materials in percentage by weight: 0.5-3% of MES, 0.9-3.0% of NaCl, 1-3% of BSA, 0.1-0.5% of Tween-20, 0.1-1% of sarcosyl (NLS), 0.1-5% of zwitterionic surfactant, 0.5-5% of water-soluble polymer, 0.005-0.05% of bovine gamma-globulin, 0.05-0.5% of Proclin and the balance of water.
Preferably, the water-soluble polymer is at least one of dextran, aminodextran, chitosan, and chitin.
Preferably, the zwitterionic surfactant is at least one of SB3-14, SB3-12, SB3-10, SB3-8, CHAPS, CHAPSO.
In a specific embodiment, the reaction time in step (2) and step (3) is from 1 to 120 minutes; preferably, the reaction time is 5-15min.
Preferably, the reaction time is 5min, 6min, 7min, 8min, 9min, 10min, 11min, 12min, 13min, 14min, 15min.
In a specific embodiment, the reaction temperature in step (2) and step (3) is 25-42 ℃; preferably, the reaction temperature is 35-40 ℃.
Preferably, the reaction temperature is 35 ℃, 36 ℃, 37 ℃, 38 ℃, 39 ℃, 40 ℃.
The exosome sample to be detected used in the application can be any one of whole blood, serum, plasma, bronchoalveolar perfusate or urine to be detected.
The preparation method of the exosome sample to be detected in the application is any one of a full-automatic exosome extraction instrument or a manual extraction method (such as a centrifugation method, a kit method, an ultrafiltration method and a magnetic bead immunization method), and preferably, the full-automatic exosome extraction instrument and the kit method are used.
In a specific embodiment, the capture antibody coated magnetic beads (also referred to as bead coatings) are prepared as follows:
1) Placing the magnetic beads on a blood mixing instrument until the magnetic beads are completely mixed, and then using the magnetic beads;
2) Preparing 1 centrifuge tube, removing 5-10mg of magnetic beads by a pipette, removing the supernatant by magnetic separation, and adding 1mL of magnetic bead activation buffer (aqueous solution containing MES with mass concentration of 1%) into the centrifuge tube;
3) Sequentially adding 25 mu L of EDC solution (final concentration of 10-30 mg/mL) and 25 mu LNHS solution (final concentration of 30-50 mg/mL) into the magnetic bead solution, reacting for 15-120min at room temperature, and removing the supernatant by magnetic separation;
4) Using 1mL of magnetic bead coupling buffer solution (aqueous solution containing MES with mass concentration of 1%) to resuspend the magnetic beads, then adding a capture antibody to be coated, and incubating for 1-4h at room temperature to obtain a magnetic bead-antibody conjugate solution; the mass ratio of the capture antibody to the magnetic beads is 0.01-0.1:1, particularly preferably in a ratio of 0.01 to 0.04:1.
5) 100. Mu.L of blocking agent was added to the above 1mL solution of the magnetic bead-antibody conjugate and incubated for 1-4h at room temperature.
6) The above solution was placed on a magnetic separator, and the supernatant was removed by magnetic separation, and this was repeated 3 times.
7) And placing the magnetic bead solution on a touch type ultrasonic cell crusher to break up the aggregated magnetic beads.
8) And placing the magnetic bead solution after the ultrasonic treatment on a magnetic separator, and removing the supernatant through magnetic separation to obtain the capture antibody coated magnetic beads.
Preparing a magnetic bead coating working solution: the magnetic bead coating working solution is obtained by diluting the capture antibody coated magnetic beads with the magnetic bead coating diluent, and the concentration range of the magnetic bead coating working solution obtained after dilution is 0.2-1.5mg/mL, preferably the dilution concentration is 0.3-0.8mg/mL.
The magnetic bead coating diluent mainly comprises the following components: TRIS 0.05-0.1%, naCl 0.9-3.0%, BSA 1-3%, tween-20.1-0.5%, sodium azide 0.1% and water in balance.
The preparation method of the luminescent substance labeled detection antibody (also called a marker) comprises the following steps:
1) 100-300. Mu.g of the detection antibody was added to 300-600. Mu.L of the labeling buffer (0.2M NaHCO3,pH9.0) prepared in advance, and 10-100. Mu.L of acridine ester was added thereto so that the final concentration of the luminescent material was 0.25-2.5mg/mL.
2) Placing the mixed liquid obtained in the step 1) on a constant temperature mixing instrument for reaction for 30-120min;
3) After the completion, adding 100 mu L of quenching agent into the reaction solution in the step 2), reacting for 30-120min at room temperature, and removing unreacted substances by a dialysis or desalting column method to obtain a luminescent substance marker; and diluting the obtained luminescent substance marker to the concentration of 100-200 mug/mL by using a marker preservation solution, and preserving at the temperature of 2-8 ℃ to obtain the luminescent substance-marked detection antibody.
In the method for preparing the detection antibody marked by the luminescent substance, the luminescent substance can be any one of acridine ester, alkaline phosphatase and horseradish peroxidase.
Preparation of a luminescent substance marking working solution (also referred to as a marking working solution): the luminescent substance labeled detection antibody is diluted to a solution with a target concentration by using a label diluent, and the concentration after dilution is in the range of 0.05-1.00 mug/mL, preferably, the concentration is in the range of 0.1-0.5 mug/mL.
The marker diluent comprises the following raw materials in percentage by weight: 0.5-3% of MES, 0.9-3.0% of NaCl, 1-3% of BSA, 0.1-0.5% of Tween-20, 0.1-1% of sarcosyl (NLS), 0.1-5% of zwitterionic surfactant, 0.5-5% of water-soluble polymer, 0.005-0.05% of bovine gamma-globulin, 0.05-0.5% of Proclin and the balance of water.
Preferably, the water-soluble polymer is at least one of dextran, aminodextran, chitosan, and chitin.
Preferably, the zwitterionic surfactant is at least one of SB3-14, SB3-12, SB3-10, SB3-8, CHAPS, CHAPSO.
Compared with the prior art, the application has the following advantages:
1. the application provides the detection of the CA15-3 protein on the exosome for auxiliary diagnosis of the interstitial lung diseases for the first time, and compared with the traditional serum detection kit, the diagnosis method has higher specificity and sensitivity, and has important clinical value and popularization prospect in auxiliary diagnosis of the interstitial lung diseases;
2. the application realizes the stable detection of the protein on the exosome by establishing the detection method of the CA15-3 protein on the exosome, and has important value for the research of the exosome protein.
Drawings
FIG. 1 is a bar graph comparing the results of testing healthy human samples with samples of ILD+serum KL-6-and ILD+serum KL-6+ patients.
FIG. 2 is a ROC graph of serum CA15-3 versus exosome CA 15-3.
Detailed Description
In order that the application may be more readily understood, the application will be further described in detail with reference to the following examples, which are given by way of illustration only and are not limiting in scope of application. Within the scope of the technical idea of the application, a number of simple variants can be made to the technical solution of the application, all of which fall within the scope of protection of the application. The starting materials or components used in the present application may be prepared by commercial or conventional methods unless specifically indicated.
Abbreviations and key term definitions in this embodiment:
TRIS trimethylolaminomethane
BSA Bovine serum albumin
Tween-20 Tween 20
Tween-80 Tween 80
DTT Dithiothreitol
bicine N, N-dihydroxyethyl glycine
EDTA-2Na Ethylene diamine tetraacetic acid disodium salt
MES Morpholinoethanesulfonic acid
EDC 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride
NHS N-hydroxysuccinimide
DMSO Dimethyl sulfoxide
PVP Polyvinylpyrrolidone
CHAPS 3- [3- (choledoxypropyl) dimethylammonium]-1-propanesulfonic acid inner salt
SB3-10 Xin Xianliu substituted betaines
Proclin 300 Liquid biological preservative (Proclin 300)
Example 1:
the embodiment provides a detection method of CA15-3 protein on exosomes, comprising the following steps:
1. the preparation method of the magnetic bead coating comprises the following steps:
1) Placing the magnetic beads on a blood mixing instrument for more than 10min until the magnetic beads are completely mixed;
2) Preparing 1 centrifuge tube, transferring 10mg of magnetic beads by a pipette, removing the supernatant by magnetic separation, and adding 1mL of magnetic bead activation buffer (aqueous solution containing MES with mass concentration of 1%) into the centrifuge tube to make the concentration of the magnetic beads be 10mg/mL;
3) To the above magnetic bead solution, 25. Mu.L of EDC solution (final concentration 20 mg/mL) and 25. Mu.L of LNHS (final concentration 40 mg/mL) were sequentially added, and reacted at room temperature for 60 minutes, after which the supernatant was removed by magnetic separation.
4) The beads were resuspended using 1mL of a bead coupling buffer (aqueous solution containing MES at a mass concentration of 1%), then 200 μg of capture antibody to be coated was added and incubated at room temperature for 2h to give a solution of the bead-antibody conjugate.
5) 100. Mu.L of blocking agent (aqueous solution containing bovine serum albumin at a mass concentration of 1%) was added to the above 1mL buffer of the magnetic bead-antibody conjugate and incubated at room temperature for 1h.
6) The above solution was placed on a magnetic separator, and the supernatant was removed by magnetic separation, and this was repeated 3 times.
7) And placing the magnetic bead solution on a touch type ultrasonic cell crusher to break up the aggregated magnetic beads.
8) And placing the magnetic bead solution after the ultrasonic treatment on a magnetic separator, and removing the supernatant through magnetic separation to obtain the capture antibody coated magnetic beads.
Preparing a magnetic bead coating working solution: diluting the capture antibody coated magnetic beads by using a magnetic bead coating diluent to obtain a magnetic bead coating working solution, wherein the concentration of the magnetic bead coating working solution obtained after dilution is 0.8mg/mL, and the magnetic bead coating diluent mainly comprises the following components: TRIS 0.1%, naCl 0.9%, BSA3%, tween-20.1%, sodium azide 0.1% and the balance of water.
(2) Label-detection antibody complex preparation steps:
1) 200. Mu.g of detection antibody was added to 500. Mu.L of labeling buffer (0.2M NaHCO) 3 pH 9.0), 25. Mu.L of acridinium ester (final concentration 10 mg/mL) was added thereto to give a final concentration of 0.5mg/mL of the luminescent material.
2) Placing the mixed liquid obtained in the step 1) on a constant temperature mixing instrument for reacting for 60min;
3) After the completion, 100. Mu.L of a quenching agent (0.2M NaHCO3 solution containing 10% lysine by mass, pH 9.0) was added to the reaction solution of the step 2), the reaction was carried out at room temperature for 30 minutes, and unreacted substances were removed by a desalting column method to obtain a luminescent substance marker; and diluting the obtained luminescent substance marker to the concentration of 200 mug/mL by using a marker preservation solution, and preserving at the temperature of 2-8 ℃ to obtain a luminescent substance-marked detection antibody (CA 15-3 detection antibody acridine lipid marker).
Preparing an acridine lipid marker working solution: the detection antibody labeled with the luminescent material (CA 15-3 detection antibody acridine lipid marker) was diluted with acridine lipid marker dilution to obtain an acridine lipid marker working solution with a concentration of 0.4. Mu.g/mL.
Wherein the acridine lipid marker diluent comprises the following raw materials in percentage by weight: MES 1%, naCl 0.9%, BSA 2%, tween-20.1%, sarcosyl (NLS) 0.2%, CHAPS 0.2%, dextran 750K 1%, bovine gamma-globulin 0.05%, proclin 300.1%, and the balance water, pH6.0.
(3) Pretreatment of exosome samples:
pretreatment of exosome samples: mu.L of the exosome sample was mixed with 180. Mu.L of the sample treatment solution, and then reacted at 37℃for 5 minutes.
The sample treatment fluid comprises the following raw materials in percentage by weight: TRIS 0.5%, naCl 0.9%, BSA 2%, triton X-100.1%, tween-80.2%, EDTA-2Na 0.05%, polyvinylpyrrolidone-K30.5%, bicine0.5%, DTT 0.1%, proClin 300.1%, and water in balance, pH7.5.
(4) Detecting and calculating the CA15-3 content in an exosome sample to be detected:
taking 20 mu L of the pretreated sample, adding 50 mu L of the magnetic bead coating working solution, uniformly mixing, and reacting for 5min at 37 ℃; then, unbound material is removed by magnetic separation, washing, and the supernatant is aspirated to obtain the magnetic bead-antigen complex.
Adding 50 mu L of a luminescent substance labeled antibody working solution (acridine lipid label working solution) into the magnetic bead-antigen complex, uniformly mixing, and reacting for 5min at 37 ℃; then, the unbound material is removed by magnetic separation and washing, and the supernatant is removed by suction, thereby obtaining the magnetic bead-antigen-detection antibody complex.
Adding 100 mu L of AE pre-excitation liquid and 100 mu L of excitation liquid into the magnetic bead-antigen-detection antibody complex, fully and uniformly mixing, and then measuring the maximum luminous intensity; fitting the luminous intensity detected by the standard substance into a standard curve, and calculating the CA15-3 content in the sample to be detected through the standard curve.
Example 2:
the main difference from example 1 is that: the sample treatment fluid used was different.
The sample treatment fluid comprises the following raw materials in percentage by weight: TRIS 1%, naCl 0.9%, BSA 2%, triton X-100.2%, tween-80.1%, EDTA-2Na 0.1%, polyvinylpyrrolidone-K30% 1%, bicine 0.2%, DTT 0.1%, proClin 300.1%, and water in balance, pH7.5.
Example 3:
the main difference from example 1 is that: the sample treatment fluid used was different.
The sample treatment fluid comprises the following raw materials in percentage by weight: TRIS 1%, naCl 0.9%, BSA 2%, triton X-100.5%, tween-80.1%, EDTA-2Na 0.05%, polyvinylpyrrolidone-K30.2%, bicine0.5%, DEAE 0.1%, proClin 300.1%, and water in balance, pH7.5.
Example 4:
the main difference from example 1 is that: the sample treatment fluid used was different.
The sample treatment fluid comprises the following raw materials in percentage by weight: TRIS 0.5%, naCl 0.5%, BSA 2%, triton X-100.5%, tween-80.3%, EDTA-2Na 0.1%, polyvinylpyrrolidone-K30% 1%, bicine0.5%, DTT 0.1%, proClin 300.1%, and water in balance, pH7.5. Example 5:
the main difference from example 1 is that: the acridine lipid marker dilutions used were different.
The marker diluent comprises the following raw materials in percentage by weight: MES 1%, naCl 0.9%, BSA 2%, tween-20.1%, sarcosyl (NLS) 0.2%, CHAPS 0.5%, dextran 2000K 1%, bovine gamma-globulin 0.02%, proclin 300.1%, and the balance water, pH6.5.
Example 6:
the main difference from example 1 is that: the acridine lipid marker dilutions used were different.
The marker diluent comprises the following raw materials in percentage by weight: MES 0.5%, naCl 0.9%, BSA 2%, tween-20.2%, sarcosyl (NLS) 0.3%, SB 3-10.3%, aminodextran 1%, bovine gamma-globulin 0.02%, proclin 300.1%, and water in balance, pH6.0.
Comparative example
Comparative example 1:
this comparative example differs from example 1 in that the exosome sample was directly tested without pretreatment.
Comparative example 2:
this comparative example differs from example 1 in that the samples were pre-treated with commercially available conventional sample dilutions.
The manufacturer of conventional sample dilutions was Eimer technology Co., ltd, cat# ICT-647.
Comparative example 3:
this comparative example differs from example 1 in that the samples were pre-treated with PBS buffer containing 1% BSA by mass.
Wherein, the PBS buffer solution comprises the following raw materials in percentage by weight: naCl 0.8%, KCl 0.02%, na 2 HPO 4 0.144％、KH 2 PO 4 0.024%, the balance being water, pH7.4.
Comparative example 4:
the main difference between this comparative example and example 1 is that: the acridine lipid marker dilutions used were different and did not contain water-soluble polymers.
The marker diluent comprises the following raw materials in percentage by weight: MES 1%, naCl 0.9%, BSA 2%, tween-20.1%, sarcosyl (NLS) 0.2%, CHAPS 0.2%, bovine gamma-globulin 0.05%, proclin 3000.1%, the balance water, pH6.0.
Comparative example 5:
the main difference between this comparative example and example 1 is that: the acridine lipid marker dilutions used were different and did not contain zwitterionic surfactants.
The marker diluent comprises the following raw materials in percentage by weight: MES 1%, naCl 0.9%, BSA 2%, tween-20.1%, sarcosyl (NLS) 0.2%, dextran 750K 1%, bovine gamma-globulin 0.05%, proclin 300.1%, and the balance water, pH6.0.
Test example:
test example 1:
effect of different methods on CA15-3 detection results on exosomes signal-to-noise ratio:
CA15-3 test kits were prepared as described in examples 1-6 and comparative examples 1-5, and sample dilutions (manufacturer: eimer technologies Co., ltd., cat# ICT-647) and 2 ILD positive samples were tested and the effect of different methods on the signal to noise ratio was compared by calculating the signal to noise ratio based on the luminescence values. The test results are shown in Table 1.
TABLE 1 influence of different methods on the signal-to-noise ratio
Note that: the above experimental results were all achieved using a full-automatic chemiluminescence immunoassay (shine i 2910).
Analysis of results: as can be seen from the results in Table 1, the pretreatment method of the application is adopted to detect the exosome CA15-3 protein in cooperation, and the signal to noise ratio of the detection result of the reagent is obviously improved compared with the method which does not carry out pretreatment, uses PBS buffer solution and conventional diluent solution treatment, so that the addition of the pretreatment mode in the application reduces the influence of components in the exosome extraction eluent on the exosome protein detection, effectively shortens the detection time, and is beneficial to the realization of the exosome protein full-automatic detection. In addition, the combination of the zwitterionic surfactant and the water-soluble polymer in the acridine lipid marker diluent can improve the signal intensity and keep the reagent detection background at a lower level, thereby obviously improving the detection signal-to-noise ratio.
Test example 2:
comparing the detection result of the healthy human sample with the detection result of the ILD patient sample:
serum samples of 20 cases of healthy people and 40 cases of ILD patients (20 cases in which commercial serum KL-6 is positive and 20 cases in which commercial serum KL-6 is negative) were taken. After extracting exosomes, the detection method provided in example 1 was used to detect the content of CA15-3 protein on exosomes, and ROC curves of exosome CA15-3 detection results were prepared, and the results are shown in Table 2.
TABLE 2 detection of CA15-3 protein content on exosomes
Analysis of results: as can be seen from table 2 and fig. 1, the luminescence value of the CA15-3 protein detection on the exosomes of ILD patients was significantly higher than that of the CA15-3 protein detection on the exosomes of the sample of healthy people, and showed very significant differences (P < 0.0001). Based on the definition of yin and yang of a sample in clinical diagnosis of interstitial lung diseases, a ROC curve (shown in figure 2) is manufactured, and cutoff is preliminarily determined to be 4500, it can be seen that for a sample positive for serum KL-6 positive ILD, exosome CA15-3 can be detected; for a serum KL-6 negative ILD positive sample, 90% (18/20) of the exosome CA15-3 can be detected, and the result shows that the exosome CA15-3 has higher detection rate for interstitial pneumonia, and the sensitivity of the exosome CA15-3 is obviously superior to that of the serum diagnostic kit in the existing market. The exosome CA15-3 has higher sensitivity and specificity in detecting interstitial pneumonia, and can have important potential value in the auxiliary diagnosis and treatment process of interstitial lung diseases.
Test example 3:
comparing the detection result of the healthy human sample with the detection result of the ILD patient sample:
serum samples of 20 cases of healthy people and 40 cases of ILD patients (20 cases in which commercial serum KL-6 is positive and 20 cases in which commercial serum KL-6 is negative) were taken. The detection method provided in example 1 was used to detect the CA15-3 protein content in serum. Then, based on clinical diagnosis of interstitial lung diseases, yin and yang of the sample are defined, and an ROC curve of a serum CA15-3 detection result is produced and compared. The results are shown in Table 3 and FIG. 2.
TABLE 3 detection of serum CA15-3 protein content
Analysis of results: based on clinical diagnosis of interstitial lung diseases, yin and yang of a sample are defined, ROC curves (see figure 2) are respectively manufactured, and serum CA15-3 detection cutoff is preliminarily determined to be 50000. For ILD samples whose serum KL-6 detection result is negative, 65% (13/20) of the samples were able to detect positive by serum CA15-3, which is weaker than the detection capacity of exosome CA 15-3. The auc= 0.9875 of ROC curve of CA15-3 (serum-exo) on exosomes, auc=0.85 of ROC curve of serum CA15-3 (serum), can give a preliminary conclusion: the sensitivity of the CA15-3 assay on the exosomes is higher than that of the serum CA15-3 assay. The above results indicate that exosome CA15-3 detection is more advantageous than serum CA15-3 detection in the diagnosis of interstitial pneumonia.
It should be noted that the above-described embodiments are only for explaining the present application and do not constitute any limitation of the present application. The application has been described with reference to exemplary embodiments, but it is understood that the words which have been used are words of description and illustration, rather than words of limitation. Modifications may be made to the application as defined in the appended claims, and the application may be modified without departing from the scope and spirit of the application. Although the application is described herein with reference to particular means, materials and embodiments, the application is not intended to be limited to the particulars disclosed herein, as the application extends to all other means and applications which perform the same function.

Claims (10)

1. An application of CA15-3 protein on exosomes in preparing a kit for diagnosing lung diseases.
2. A kit for diagnosing a pulmonary disorder, comprising: comprises sample treatment fluid, a magnetic bead coated by a CA15-3 capture antibody and a CA15-3 detection antibody marked by a luminescent substance.
3. A kit for diagnosing a pulmonary disease according to claim 2, wherein: the kit also includes a luminescent substrate and a protein calibrator.
4. The kit for diagnosing a pulmonary disease according to claim 2, wherein the sample processing liquid comprises the following raw materials in weight percent: 0.5 to 1.5 percent of TRIS, 0.3 to 3 percent of NaCl, 0.5 to 3 percent of BSA, 0.1 to 1 percent of Triton X-100, 0.1 to 0.5 percent of Tween-80, 0.01 to 0.1 percent of EDTA-2Na, 0.1 to 1.0 percent of polyvinylpyrrolidone-K, 0.2 to 2 percent of bicine, 0.1 to 0.3 percent of reducing agent, 0.05 to 0.5 percent of ProClin and the balance of water.
5. The kit for diagnosing a pulmonary disease according to claim 4, wherein the reducing agent is at least one of DTT, DEAET, β -mercaptoethanol, TCEP.
6. A method for detecting the protein content of exosomes using the kit of any one of claims 1-5, characterized by the steps of:
(1) Mixing an exosome sample to be tested with a sample treatment liquid to obtain a pretreated exosome sample to be tested;
(2) Mixing the pretreated exosome sample to be tested obtained in the step (1) with a magnetic bead coating working solution, and reacting to obtain a magnetic bead-antigen complex;
(3) Mixing the magnetic bead-antigen complex obtained in the step (2) with a illuminant labeled antibody working solution to obtain a magnetic bead-antigen-detection antibody complex;
(4) Mixing the magnetic bead-antigen-detection antibody complex obtained in the step (3) with a luminescent substrate, and detecting the luminous intensity;
(5) And (3) calculating the protein content in the exosome sample to be detected according to the luminous intensity obtained by the detection in the step (4) by using a standard curve method.
7. The method of detecting according to claim 6, wherein: in the step (1), the volume ratio of the exosome sample to be measured to the sample treatment fluid is 1: (2-30).
8. The method of detecting according to claim 6, wherein: the reaction time in the step (1) is 1-120min; the reaction temperature is 25-42 ℃.
9. The method of detecting according to claim 6, wherein: the preparation method of the illuminant-labeled antibody working solution in the step (3) comprises the following steps: diluting the detection antibody marked by the luminescent substance by using a marker diluent to obtain a luminescent marker working solution;
wherein the marker diluent comprises the following raw materials in percentage by weight: 0.5-3% of MES, 0.9-3.0% of NaCl, 1-3% of BSA, 0.1-0.5% of Tween-20, 0.1-1% of sarcosyl, 0.1-5% of zwitterionic surfactant, 0.5-5% of water-soluble polymer, 0.005-0.05% of bovine gamma-globulin, 0.05-0.5% of Proclin and the balance of water.
10. The method of claim 9, wherein: the water-soluble polymer is at least one of dextran, aminodextran, chitosan and chitin; the zwitterionic surfactant is at least one of SB3-14, SB3-12, SB3-10, SB3-8, CHAPS, CHAPSO.
CN202310543518.9A 2023-05-15 2023-05-15 Kit for diagnosing interstitial lung diseases by using CA15-3 protein on exosome and application Pending CN116609523A (en)

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