CN116626296A - Application of serum exosomes as markers in preparation of tumor diagnosis kit - Google Patents
Application of serum exosomes as markers in preparation of tumor diagnosis kit Download PDFInfo
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Abstract
The invention discloses application of serum exosomes as markers in preparation of tumor diagnosis kits. The kit takes the quantitative result of the serum exosomes as an index of tumor diagnosis, and the quantitative result of the serum exosomes is quantified through CD9 proteins on the exosomes. The quantitative method of the CD9 protein comprises the following steps: the exosome CD9 protein is marked by adopting a luminophore, and the CD9 protein is quantitatively detected by a magnetic immunochemiluminescence detection method. The invention takes the exosome of serum source as a basic unit and is used as a novel biomarker for tumor diagnosis; quantitative results of serum exosomes are used for early diagnosis of tumors. The number of exosomes is quantified by a chemiluminescence method to perform tumor correlation diagnosis, detection can be completed within 1 hour, and automation can be realized.
Description
Technical Field
The invention belongs to the technical field of biomedical diagnosis, and particularly relates to application of serum exosomes as markers in preparation of tumor diagnosis kits.
Background
Exosomes (exosomes) are 30-150 nm-sized, extremely low density extracellular nanoscale vesicular structures formed by cells undergoing a series of regulatory processes such as "endocytosis-fusion-efflux". Exosomes have not attracted the attention of researchers for a long time after being discovered until the introduction of nanoanalytical techniques and the publication of the nobel biomedical prize in 2013, where particles with diameters of only tens of nanometers have received unprecedented attention.
Exosomes are reported to be present in most body fluids in the human body, including blood, urine, milk, etc. There are studies that find that for differences in the number of exosomes between healthy and tumor patients, the prediction of tumors can be made by measuring the number of exosomes in body fluids.
Currently, early screening of tumors on the market is mainly focused on ctDNA: ctDNA is a new tumor marker, plays an important role in early diagnosis, treatment, prognosis detection and the like of tumors, and in clinical work, the occurrence probability and the tumor type of tumors can be judged by detecting whether ctDNA carries tumor-specific mutation or other related genome change information.
ctDNA detection has certain limitations because ctDNA is not detected in the blood of all cancer patients; and ctDNA is mainly used for diagnosing whether cancer is caused by comparing sequence differences among the DNA, cfDNA is possibly generated in vivo if a patient is infected with other viruses or microorganisms, so that the cost of ctDNA detection is increased to a certain extent, and the accuracy of detection is possibly reduced.
Disclosure of Invention
The invention aims to provide application of serum exosomes as markers in preparation of tumor diagnosis kits and the tumor diagnosis kits. Mainly solves the technical problems of high cost and low accuracy of tumor diagnosis in the prior art.
The technical scheme adopted by the invention for solving the technical problems is as follows:
the application of the serum exosomes as markers in the preparation of tumor diagnosis kits takes the quantitative result of the serum exosomes as an index of tumor diagnosis.
As a preferred embodiment, the quantification of the serum exosomes is quantified by CD9 proteins on the exosomes.
As a preferred embodiment, the method for quantifying the CD9 protein is: the exosome CD9 protein is marked by adopting a luminophore, and the CD9 protein is quantitatively detected by a magnetic immunochemiluminescence detection method.
The invention also provides a tumor diagnosis kit, which comprises an immunomagnetic bead suspension, a washing liquid, an antibody detection liquid, an excitation liquid A and an excitation liquid B; the immunomagnetic bead suspension is used for separating and purifying exosomes; the antibody detection solution is used for detecting CD9 protein of exosomes.
As a preferred embodiment, the magnetic beads in the immune magnetic bead suspension are carboxyl magnetic beads with the particle diameter of 150 nm-4 um, the concentration of the magnetic beads is 1-30 mg/mL, the surface of the magnetic beads is coated with exosome specific antibodies, the concentration of the antibodies is 0.01-1 mg/mL, and the solvent is water.
As a preferred embodiment, the magnetic beads are preferably carboxyl magnetic beads with the particle size of 1um, and the exosome specific antibody coated on the surface of the magnetic beads is one or two of CD63 and CD81, and the concentration is 0.1mg/mL.
As a preferred embodiment, the composition of the washing liquid is: na at a concentration of 1 to 20mM 2 HPO 4 NaH at a concentration of 0.5 to 10mM 2 PO 4 NaCl with concentration of 0.1-2M and water as solvent. The composition of the washing liquid is further preferably: na at a concentration of 8mM 2 HPO 4 NaH at a concentration of 2mM 2 PO 4 And NaCl at a concentration of 0.15M.
As a preferred embodiment, the antibody detection solution comprises 0.01-1 μg/mL of CD9 antibody, and the solvent is water.
As a preferred embodiment, the CD9 antibody surface is labeled with the luminophore acridinium ester.
Further preferably, the CD9 antibody concentration is 0.015. Mu.g/mL, and the amount of acridine ester is 10 as antibody/acridine ester: 1.
As a preferred embodiment, the composition of the excitation liquid a is: HNO with concentration of 0.1-0.2M 3 H with concentration of 0.3-0.6% 2 O 2 The solvent is water; the composition of the excitation liquid B is as follows: naOH with concentration of 0.25-0.5M and water as solvent.
The invention uses the quantitative result of serum exosomes as the index of tumor diagnosis, which comprises the following steps:
1) Serum samples for the experimental study were collected, requiring at least 20 healthy human sera and at least 20 tumor patient sera.
2) According to the experimental design, samples were grouped according to healthy and tumor patients and luminescence values were determined using the present method.
3) The method comprises the following steps of sample luminescence value detection:
a. adding 1mL of serum sample into a centrifuge tube, adding 50 mu L of immunomagnetic beads (0.1 mg/mL), fully shaking and uniformly mixing, and shaking and incubating for 15min at room temperature;
b. centrifuging the centrifuge tube instantaneously for about 5s, placing the centrifuge tube on a magnetic rack for 1min, and sucking and discarding liquid;
c. 1mL of the washing solution (8 mM Na was added 2 HPO 4 NaH at 2mM 2 PO 4 And 0.15M NaCl), fully vibrating and uniformly mixing, instantly centrifuging for about 5s, placing on a magnetic rack for 1min, and absorbing and discarding liquid;
d. repeating the washing 3 times;
e. adding 50 mu L of antibody detection solution (the concentration of the antibody detection solution CD9 antibody is 0.015 mu g/mL), shaking and mixing uniformly, and then shaking and incubating for 5min at 37 ℃;
f. centrifuging the centrifuge tube instantaneously for about 5s, placing the centrifuge tube on a magnetic rack for 1min, and sucking and discarding liquid;
g. 100uL of excitation solution A (0.1M HNO was added 3 、0.4％H 2 O 2 ) After gentle mixing, 100uL of challenge solution B was added and mixed well (0.45 m noh);
detecting a luminescence value at a wavelength of 430 nm;
4) Performing quantitative calculation on exosomes of each sample according to a known relative quantitative standard curve;
5) Analyzing the luminous value of the test sample, and determining the number cut-off value of exosomes between healthy people and tumor patients through statistical analysis, wherein the number cut-off value is used for screening the tumor sample;
6) And detecting the luminescence value of the unknown sample to be detected to calculate a quantitative value, and comparing the quantitative value with a cut-off value to discriminate tumor.
The English abbreviations related to the invention are noted as follows:
ctDNA: circulating tumor genes
cfDNA: circulating DNA
NTA: nanoparticle tracking analysis (Nanoparticle Tracking Analysis, NTA)
Compared with the prior art, the invention has the following beneficial effects:
1, the serum-derived exosomes are taken as basic units and are used as novel biomarkers for tumor diagnosis; quantitative results of serum exosomes are used for early diagnosis of tumors.
2, the invention quantifies the number of exosomes by a chemiluminescence method to diagnose the relevance of the tumor, and can complete detection within 1 hour, and can realize automation.
3, the invention uses CD9 protein to quantify during exosome quantitative detection, and exosome quantitative detection can be carried out without exosome cracking, thus diagnosing tumor.
Drawings
FIG. 1 is a graph showing the establishment of a standard curve of the correspondence between the concentration of exosomes and the luminescence values in example 1 of the present invention.
Fig. 2 is a data graph of the detected luminescence values of each sample in embodiment 2 of the present invention.
Fig. 3 is a ROC graph of a sample detection luminescence value in example 2 of the present invention.
Detailed Description
The following describes the technical scheme of the present invention in detail by referring to examples. The reagents and biological materials used hereinafter are commercial products unless otherwise specified.
Example 1: serum-derived exosome quantitative standard curve drawing
1) Serum samples of healthy people were collected in 10mL and the serum was pre-treated as follows:
centrifuge at 16,000g,40 min, then pass through a 0.2 μm filter.
2) Subjecting the pretreated serum to ultracentrifugation to separate exosomes:
centrifuge at 150,000g,45 for 3h. The suspension was resuspended in PBS (Thermo Fisher cat# 10010023). NTA measures the concentration of resuspended exosomes. (mother liquor concentration was 3.0E+11 parts/mL).
3) The exosomes after concentration measurement were subjected to gradient dilution with PBS to obtain samples drawing quantitative standard curves of the exosomes, and the specific sample concentrations are shown in table 1.
TABLE 1
4) The sample luminescence value detection steps are as follows:
a. adding 100 mu L of a sample to be detected into a centrifuge tube, then adding 50 mu L of immunomagnetic beads (0.1 mg/mL), fully vibrating and uniformly mixing, and vibrating and incubating for 15min at room temperature;
b. centrifuging the centrifuge tube instantaneously for about 5s, placing the centrifuge tube on a magnetic rack for 1min, and sucking and discarding liquid;
c. 400. Mu.L of the washing solution (8 mM Na 2 HPO 4 NaH at 2mM 2 PO 4 And 0.15M NaCl), fully vibrating and uniformly mixing, instantly centrifuging for about 5s, placing on a magnetic rack for 1min, and absorbing and discarding liquid;
d. repeating the washing step c 3 times;
e. adding 50 mu L of antibody detection solution (the concentration of CD9 antibody in the antibody detection solution is 0.015 mu g/mL), shaking and mixing uniformly, and then shaking and incubating for 5min at 37 ℃;
f. centrifuging the centrifuge tube instantaneously for about 5s, placing the centrifuge tube on a magnetic rack for 1min, and sucking and discarding liquid;
g. 100uL of excitation solution A (0.1M HNO was added 3 、0.4％H 2 O 2 ) After gentle mixing, 100uL of excitation solution B was added and mixed well (0.45M NaOH);
detecting a luminescence value at a wavelength of 430 nm;
5) The luminescence values detected for each sample in table 1 are shown in table 2. A standard curve is established by corresponding the exosome concentration of the sample to the luminescence detection value, and referring to fig. 1, the standard curve is drawn.
TABLE 2
Example 2: quantitative results of serum-derived exosomes and tumor correlation
1) Serum samples for the experimental study were collected, requiring at least 20 healthy human serum and at least 20 tumor patient serum (healthy human samples labeled M1-M20, tumor patient samples labeled M21-M40).
2) According to the experimental design, samples were grouped according to healthy and tumor patients and luminescence values were determined using the present method.
3) The specific steps of the method for detecting the sample luminescence value are as follows:
a. adding 100 mu L of serum sample into a centrifuge tube, adding 50 mu L of immunomagnetic beads (0.1 mg/mL), fully shaking and uniformly mixing, and shaking and incubating for 15min at room temperature;
b. centrifuging the centrifuge tube instantaneously for about 5s, placing the centrifuge tube on a magnetic rack for 1min, and sucking and discarding liquid;
c. 400. Mu.L of the washing solution (8 mM Na 2 HPO 4 NaH at 2mM 2 PO 4 And 0.15M NaCl), fully vibrating and uniformly mixing, instantly centrifuging for about 5s, placing on a magnetic rack for 1min, and absorbing and discarding liquid;
d. repeating the washing step c 3 times;
e. adding 50 mu L of antibody detection solution (the concentration of CD9 antibody in the antibody detection solution is 0.015 mu g/mL), shaking and mixing uniformly, and then shaking and incubating for 5min at 37 ℃;
f. centrifuging the centrifuge tube instantaneously for about 5s, placing the centrifuge tube on a magnetic rack for 1min, and sucking and discarding liquid;
g. 100uL of excitation solution A (0.1M HNO was added 3 、0.4％H 2 O 2 ) After gentle mixing, 100. Mu.L of excitation solution B (0.45M NaOH) was added;
detecting a luminescence value at a wavelength of 430 nm;
4) Calculating the exosome concentration in each sample;
5) And analyzing the exosome concentration of the test sample, and determining the cut-off value between the healthy person and the tumor patient through statistical analysis, so as to be used for screening the tumor sample. The test results for each sample are shown in Table 3 and the analysis for the test sample is shown in Table 4. Referring to fig. 2, a data graph of the detected luminescence value of each sample is shown. Referring to fig. 3, a ROC graph of luminescence values is detected for a sample.
From the data results of tables 3 and 4, it can be obtained: the invention is studied between healthy and tumor patients with a method specificity of 0.835. The cut-off value between healthy and tumor patients was analyzed to be around 3.56E+09particles/mL.
TABLE 3 Table 3
TABLE 4 Table 4
Example 3: detection of clinical samples by the method
1) Adding 100 mu L of a sample to be detected into a centrifuge tube, then adding 50 mu L of immunomagnetic beads (0.1 mg/mL), fully vibrating and uniformly mixing, and vibrating and incubating for 15min at room temperature;
2) Centrifuging the centrifuge tube instantaneously for about 5s, placing the centrifuge tube on a magnetic rack for 1min, and sucking and discarding liquid;
3) 400. Mu.L of the washing solution (8 mM Na 2 HPO 4 NaH at 2mM 2 PO 4 And 0.15M NaCl), fully vibrating and uniformly mixing, instantly centrifuging for about 5s, placing on a magnetic rack for 1min, and absorbing and discarding liquid;
4) Repeating the washing step c 3 times;
5) Adding 50 mu L of antibody detection solution (the concentration of CD9 antibody in the antibody detection solution is 0.015 mu g/mL), shaking and mixing uniformly, and then shaking and incubating for 5min at 37 ℃;
6) Centrifuging the centrifuge tube instantaneously for about 5s, placing the centrifuge tube on a magnetic rack for 1min, and sucking and discarding liquid;
7) 100uL of excitation solution A (0.1M HNO was added 3 、0.4％H 2 O 2 ) After gentle mixing, 100uL of excitation solution B was added and mixed well (0.45M NaOH);
8) Detecting a luminescence value at a wavelength of 430 nm;
9) Sample exosome concentrations were calculated from the standard curve. See table 5 for test results for clinical samples.
TABLE 5
The data results in Table 5 can be seen: by using the method for clinical sample detection, the quantitative concentrations of the exosomes in the tumor positive samples are smaller than the cut-off value, and healthy people and tumor patients can be well screened through the quantitative results of the exosomes in serum. Provides great help for painless diagnosis and early diagnosis of tumors.
Example 4: the method of the invention is compared with the prior art
The invention takes 4 clinical serum samples as comparison test samples, and the comparison experiment is a plasma sample with the same source. Serum and plasma samples were all centrifuged at 10000g for 30min at 4℃before the experiment.
The method of the invention is as follows:
1) 100 mu L of serum is added into a centrifuge tube, 50 mu L of immunomagnetic beads (0.1 mg/mL) are added, and the mixture is fully and uniformly mixed by shaking, and the mixture is incubated for 15min by shaking at room temperature;
2) Centrifuging the centrifuge tube instantaneously for about 5s, placing the centrifuge tube on a magnetic rack for 1min, and sucking and discarding liquid;
3) 400. Mu.L of the washing solution (8 mM Na 2 HPO 4 NaH at 2mM 2 PO 4 And 0.15M NaCl), fully vibrating and uniformly mixing, instantly centrifuging for about 5s, placing on a magnetic rack for 1min, and absorbing and discarding liquid;
4) Repeating the washing step c 3 times;
5) Adding 50 mu L of antibody detection solution (the concentration of CD9 antibody in the antibody detection solution is 0.015 mu g/mL), shaking and mixing uniformly, and then shaking and incubating for 5min at 37 ℃;
6) Centrifuging the centrifuge tube instantaneously for about 5s, placing the centrifuge tube on a magnetic rack for 1min, and sucking and discarding liquid;
7) 100uL of excitation solution A (0.1M HNO was added 3 、0.4％H 2 O 2 ) After gentle mixing, 100uL of excitation solution B was added and mixed well (0.45M NaOH);
8) And detecting the luminescence value at the wavelength of 430nm, and calculating the concentration of the exosomes according to a standard curve.
The comparison method comprises the following steps:
exosome extraction and purification
1) 100 mu L of plasma is added into a centrifuge tube, 200 mu L of exosome extract (TotalExosome Isolation, invitrogen) is added into the centrifuge tube for mixing, after shaking is uniform, the mixture is incubated overnight at 4 ℃ for 6 to 16 hours, and then the incubated mixture is centrifuged at 4 ℃ for 1 hour at 10000g, and after supernatant is removed, the total exosomes (exosomes) of the plasma deposited at the bottom of the centrifuge tube are obtained;
2) Re-suspending the total exosomes of the plasma obtained in step 1) with 0.5mL of 1 x pbs to obtain a re-suspension of the total exosomes;
3) After 20 μl of antibody beads (EpCAM beads, invitrogen) and 1mL of 1 x pbs were mixed in another new centrifuge tube, the tube was magnetically rack-adsorbed for 2 minutes, and the liquid was removed, so that EpCAM antibody-labeled beads were adsorbed in the centrifuge tube.
4) 0.5mL of the total exosomes obtained in the step 2) above was added to the centrifuge tube with the EpCAM antibody-labeled magnetic beads adsorbed thereon obtained in the step 3) above, and incubated overnight at 4 ℃ (all of which may be used for 6 to 16 hours).
5) Placing the centrifuge tube in the step 4) in a magnetic rack, adsorbing for 2 minutes, and removing liquid in the tube;
6) Adding 1ml of 1 x PBS into the centrifuge tube in the step 5), placing the centrifuge tube in a magnetic rack, adsorbing for 2 minutes, and removing liquid in the tube; repeating the process once; the exosomes bound by the magnetic beads are obtained.
Elisa detection of exosomes
1) The obtained exosomes were resuspended in 200 μl of 1 x pbs and coated with 100 μl/Kong Jiazhi of high binding capacity elisa plate. Post-incubation liquid in wells was discarded overnight (6-16 hours) at 4℃and 200. Mu.L of 1 XPBS (0.05% Tween) was used to wash the plates 3 times, 1-2min each time, 200. Mu.L/well. Removing the supernatant liquid;
2) 200. Mu.L of PBST containing 5% skimmed milk was added to the wells, incubated at 37℃for 2h for blocking, and the supernatant was removed. Washing the plate with 200 μL of 1 x PBS for 3 times, soaking for 1-2min each time, and removing supernatant;
3) CD9 primary antibody (proteontech) was added to the wells at a dilution of 1:5000 of 100. Mu.L, incubated at 37℃for 1h and the supernatant removed. 200 μL of 1 x PBS was washed 3 times, each time soaked for 1-2min, and the supernatant fluid was removed;
4) HRP-enzyme-labeled secondary antibody (sigma) was added to the wells at 1:10000 dilution of 100. Mu.L, incubated at 37℃for 1h and the supernatant removed. 200 μL of 1 x PBS was washed 3 times, each time soaked for 1-2min, and the supernatant fluid was removed;
5) Adding 100 μL TMB color development solution into the hole, developing for 15-20min in dark, and if the color is light, developing at 37deg.C for no more than 30min;
6) 100. Mu.L of the stop solution was added to the wells and the liquid color changed from blue to yellow. The microplate was immediately placed in an ELISA reader and the Optical Density (OD) of each well was measured sequentially at a wavelength of 450 nm.
The detection results of the method of the invention and the Elisa method are shown in Table 6.
TABLE 6
Table 6 data results indicate: the detection result of the invention on the clinical sample is consistent with the existing method, and the invention has the advantages of quick and complete detection within 1 h. The comparison method is complex in operation and can be completed in 3 days.
The foregoing is only a part of the preferred embodiments of the present invention, and the present invention is not limited to the contents of the embodiments. It will be apparent to those skilled in the art that various changes and modifications can be made within the scope of the technical solution of the present invention, and any changes and modifications are within the scope of the present invention.
Claims (10)
1. The application of serum exosomes as markers in preparing tumor diagnosis kit is characterized in that: the quantitative result of serum exosomes was used as an index for tumor diagnosis.
2. Use of the serum exosome of claim 1 as a marker in the preparation of a tumor diagnostic kit, characterized in that: the quantification of the serum exosomes was performed by CD9 proteins on the exosomes.
3. The use of the serum exosome of claim 1 as a marker in the preparation of a tumor diagnostic kit, wherein the method for quantifying the CD9 protein comprises: the exosome CD9 protein is marked by adopting a luminophore, and the CD9 protein is quantitatively detected by a magnetic immunochemiluminescence detection method.
4. A tumor diagnostic kit, characterized in that: the kit comprises an immunomagnetic bead suspension, a washing liquid, an antibody detection liquid, an excitation liquid A and an excitation liquid B; the immunomagnetic bead suspension is used for separating and purifying exosomes; the antibody detection solution is used for detecting CD9 protein of exosomes.
5. The tumor diagnostic kit according to claim 4, wherein: the magnetic beads in the immune magnetic bead suspension are carboxyl magnetic beads with the particle size of 150 nm-4 um, the concentration of the magnetic beads is 1-30 mg/mL, the surface of the magnetic beads is coated with exosome specific antibodies, the concentration of the antibodies is 0.01-1 mg/mL, and the solvent is water.
6. The tumor diagnostic kit according to claim 5, wherein: the exosome specific antibody coated on the surface of the magnetic bead is one or two of CD63 and CD81, and the concentration is 0.1mg/mL.
7. The tumor diagnostic kit according to claim 4, wherein the composition of the washing solution is: na at a concentration of 1 to 20mM 2 HPO 4 NaH at a concentration of 0.5 to 10mM 2 PO 4 NaCl with concentration of 0.1-2M and water as solvent.
8. The tumor diagnostic kit according to claim 4, wherein: the antibody detection solution comprises 0.01-1 mug/mL of CD9 antibody, and the solvent is water.
9. The tumor diagnostic kit according to claim 8, wherein: the CD9 antibody surface is marked with a luminous substance acridinium ester.
10. The tumor diagnostic kit according to claim 4, wherein the excitation liquid a has a composition of: HNO with concentration of 0.1-0.2M 3 H with concentration of 0.3-0.6% 2 O 2 The solvent is water; the composition of the excitation liquid B is as follows: naOH with concentration of 0.25-0.5M and water as solvent.
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