A kit and method for the combined detection of multiple immune markers on the surface of peripheral blood extracellular vesicles

Abstract

The present invention belongs to the field of medical testing technology and discloses a kit for the combined detection of multiple immune markers on the surface of peripheral blood extracellular vesicles. The kit comprises: immunomagnetic beads modified with CD63 antibodies; three different oligonucleotide chains coupled to three antibodies, respectively, to form three antibody-oligonucleotide conjugates: CD9, CD33, and CD34; three pairs of primers for amplifying the three different oligonucleotide chains or their complementary paired sequences; and three fluorescent probes, each containing a different fluorescent marker. The nucleotide sequences of the oligonucleotide chains coupled to CD9, CD33, and CD34 antibodies are shown in SEQ ID NO: 1, SEQ ID NO: 5, and SEQ ID NO: 9, respectively. The kit can achieve non-invasive screening for AML patients whose symptoms are not obvious in the early stages of the disease and has excellent detection sensitivity and stability.

Description

Technical Field

[0001] The present invention belongs to the field of medical detection technology, and specifically relates to a kit and method for the combined detection of multiple immune markers on the surface of peripheral blood extracellular vesicles. Background Art

[0002] Leukemia is a rare but highly malignant tumor that causes a significant number of cancer-related deaths. Acute myeloid leukemia (AML) is the most common type of leukemia in adults. It is characterized by the arrest of proliferation and differentiation of myeloid blasts, leading to dysregulation of normal hematopoiesis and the lowest survival rate of all leukemias. The distribution of AML cases shows that approximately 42.8% of patients are over 65 years old, indicating that the incidence of AML gradually increases with age.

[0003] Peripheral blood and bone marrow cell samples play a key role in the diagnosis and treatment of AML. Peripheral blood drawn through a vein in the arm is the first test in the diagnosis of AML. Because leukemia originates in the bone marrow, examining the bone marrow for leukemia cells is a core component of diagnosis. Bone marrow samples are primarily obtained through bone marrow aspiration and biopsy, typically from the hipbone. In cases where AML may have spread to the brain and spinal cord, a doctor may perform a lumbar puncture to remove a sample of cerebrospinal fluid (CSF) for testing. This test is typically performed when a patient presents with symptoms to determine whether leukemia cells have spread to the central nervous system. During a lumbar puncture, the patient lies on their side. After administering local anesthesia, the doctor inserts a thin needle through the space between the vertebrae and draws a sample of CSF. Lumbar punctures are used not only for testing but also sometimes to administer chemotherapy drugs directly into the CSF to prevent or treat the spread of leukemia to the central nervous system. These diagnostic procedures are crucial for confirming the diagnosis of AML, developing a treatment plan, and monitoring treatment effectiveness. AML develops rapidly and has a high mortality rate, making early detection crucial for patient outcomes. However, because AML's early clinical symptoms are subtle, often manifested only by abnormal peripheral blood test results, clinicians can easily miss the diagnosis. Furthermore, bone marrow aspiration is an invasive procedure, making it difficult for most patients to accept.

[0004] Therefore, there is an urgent need for a non-invasive and simple detection method that can detect potential AML patients in seemingly healthy people, so as to identify them early and intervene in treatment. Summary of the Invention

[0005] The purpose of the present invention is to provide a kit for the combined detection of multiple immune markers on the surface of extracellular vesicles in peripheral blood, which can achieve non-invasive screening of AML patients whose symptoms are not obvious in the early stage of the disease.

[0006] The above-mentioned objectives are achieved by the following technical solutions.

[0007] The first aspect of the present invention provides a kit for the combined detection of multiple immune markers on the surface of extracellular vesicles in peripheral blood, the kit comprising:

[0008] Immunomagnetic beads with CD63 antibodies modified on the surface;

[0009] Three different oligonucleotide chains are coupled to three antibodies respectively, forming three antibody oligonucleotide conjugates, wherein the three antibodies are CD9 antibody, CD33 antibody and CD34 antibody;

[0010] and three pairs of primers and three fluorescent probes for amplifying three different oligonucleotide chains or their complementary paired sequences, wherein the fluorescent label in each fluorescent probe is different.

[0011] The nucleotide sequence of the oligonucleotide chain coupled to the CD9 antibody is shown in SEQ ID NO: 1, the nucleotide sequence of the oligonucleotide chain coupled to the CD33 antibody is shown in SEQ ID NO: 5, and the nucleotide sequence of the oligonucleotide chain coupled to the CD34 antibody is shown in SEQ ID NO: 9.

[0012] In some embodiments, the primer pair sequences for amplifying the oligonucleotide chain conjugated to CD9 antibody are shown in SEQ ID NO: 2 and SEQ ID NO: 3; and / or,

[0013] The primer pair sequences for amplifying the oligonucleotide chain conjugated to CD33 antibody are shown in SEQ ID NO: 6 and SEQ ID NO: 7; and / or,

[0014] The sequences of the primer pair used to amplify the oligonucleotide chain conjugated to CD34 antibody are shown in SEQ ID NO: 10 and SEQ ID NO: 11.

[0015] In some embodiments, the fluorescent probe sequence used to amplify the oligonucleotide chain coupled to the CD9 antibody is shown in SEQ ID NO: 4; and / or,

[0016] The fluorescent probe sequence for amplifying the oligonucleotide chain coupled to the CD33 antibody is shown in SEQ ID NO: 8; and / or,

[0017] The fluorescent probe sequence used to amplify the oligonucleotide chain coupled to the CD34 antibody is shown in SEQ ID NO:12.

[0018] In some embodiments, when the oligonucleotide chain conjugate is prepared, the ratio of the antibody to the oligonucleotide chain is 1:3-15; preferably, the ratio of the antibody to the oligonucleotide chain is 1:3-5.

[0019] In some embodiments, the amount of CD63 antibody labeled in the immunomagnetic beads is 800ug / ml to 1600ug / ml.

[0020] A second aspect of the present invention provides a use of the above-mentioned kit for the combined detection of multiple immune markers on the surface of peripheral blood extracellular vesicles in the preparation of a product for detecting acute myeloid leukemia.

[0021] A third aspect of the present invention provides a method for jointly detecting multiple immune markers on the surface of extracellular vesicles of peripheral blood, using the above-mentioned kit for jointly detecting multiple immune markers on the surface of extracellular vesicles of peripheral blood, comprising the following steps:

[0022] Immunomagnetic beads modified with CD63 antibodies were added to the collected peripheral blood samples and mixed, and then the immunomagnetic beads-extracellular vesicle immune complexes were obtained by magnetic adsorption;

[0023] mixing the three antibody-oligonucleotide conjugates with the immunomagnetic bead-extracellular vesicle immune complex to obtain an immunomagnetic bead-extracellular vesicle-antibody oligonucleotide conjugate immune complex;

[0024] The immunomagnetic beads-extracellular vesicles-antibody oligonucleotide conjugate immune complex is amplified by qPCR to obtain an amplified product.

[0025] In some embodiments, the number of cycles of the qPCR amplification is 43 to 47 times; preferably, the number of cycles of the qPCR amplification is 45 times.

[0026] In some embodiments, the concentration of extracellular vesicles in the peripheral blood sample is 0.274×10 7 Particles / mL~8.78×10 7 particles / mL.

[0027] In some embodiments, the volume ratio of the added peripheral blood sample to the immunomagnetic beads is 5 μL:15 μL-17 μL, and the concentration of the immunomagnetic beads is 8 mg / ml-12 mg / ml.

[0028] In the present invention, by constructing immunomagnetic beads with CD63 antibodies modified on the surface, the immunomagnetic beads can specifically capture extracellular vesicles in peripheral blood, thereby separating the extracellular vesicles from the peripheral blood. Then, by constructing three antibody-oligonucleotide chain conjugates, they specifically identify CD9, CD33 and CD34 molecules on the extracellular vesicles, respectively. By amplifying and analyzing the oligonucleotide chains on the antibody-oligonucleotide chain conjugates, the expression levels of each CD9, CD33 and CD34 molecule on the extracellular vesicles are analyzed. The expression levels of CD9, CD33 and CD34 molecules can be used to achieve non-invasive screening of AML patients with subtle symptoms in the early stage of the disease. The method has excellent detection sensitivity and stability and can provide a new clinical reference for the screening of AML. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a schematic diagram of the principle of the combined detection method of multiple immune markers on the surface of extracellular vesicles in the peripheral blood of patients with acute myeloid leukemia.

[0030] Figure 2 This is a diagram of the clinical application scenario of the combined detection method of multiple immune markers on the surface of extracellular vesicles in the peripheral blood of patients with acute myeloid leukemia.

[0031] Figure 3 Identification of extracellular vesicles in the peripheral blood of patients with acute myeloid leukemia: (A) Transmission electron microscopy image of extracellular vesicles; (B) NTA particle size analysis of extracellular vesicle samples; (C) Western blot detection of the expression of CD81, CD63, TSG101, and HSP-70 in extracellular vesicle samples.

[0032] Figure 4 This is the difference verification of extracellular vesicle surface immune markers at different concentrations: (A) difference verification of CD9 molecules on the extracellular vesicle surface; (B) difference verification of CD33 molecules on the extracellular vesicle surface; (C) difference verification of CD34 molecules on the extracellular vesicle surface; (D) difference verification of CD63 molecules on the extracellular vesicle surface.

[0033] Figure 5 NTA is used to measure the concentration of extracellular vesicles in peripheral blood samples.

[0034] Figure 6 The difference verification of extracellular vesicle surface immune markers at the same concentration: (A) difference verification of CD9 molecules on the extracellular vesicle surface; (B) difference verification of CD33 molecules on the extracellular vesicle surface; (C) difference verification of CD34 molecules on the extracellular vesicle surface; (D) difference verification of CD63 molecules on the extracellular vesicle surface

[0035] Figure 7Preparation of immunomagnetic beads (IMB): (A) Variation of antibody levels on immunomagnetic beads; (B) Characterization of immunomagnetic beads after labeling.

[0036] Figure 8 The saturation amount of antibody labeled with immunomagnetic beads (IMB): (A) antibody concentration standard curve; (B) saturation amount of antibody labeled with immunomagnetic beads.

[0037] Figure 9 The maximum amount of extracellular vesicles captured by immunomagnetic beads (IMB): (A) Western Blot verification of the maximum amount of extracellular vesicles captured by immunomagnetic beads (IMB); (B) Image J verification of the maximum amount of extracellular vesicles captured by immunomagnetic beads (IMB).

[0038] Figure 10 It is Ab-Oligo preparation-Oligo screening.

[0039] Figure 11 This is Ab-Oligo characterization and verification: (A) SDS-PAGE verification of CD9 Ab-Oligo conjugate; (B) SDS-PAGE verification of CD34 Ab-Oligo conjugate; (C) SDS-PAGE verification of CD33 Ab-Oligo conjugate.

[0040] Figure 12 The Ab-Oligo conditions were optimized: (A) the Ab-Oligo conjugate dosage ratio was 1:15; (B) the Ab-Oligo conjugate dosage ratio was 1:15; (C) the Ab-Oligo conjugate dosage ratio was 1:15.

[0041] Figure 13 This is the feasibility verification of the detection method: (A) IMB-EVs-Ab-oligo amplification reaction; (B) clinical sample verification of the detection method.

[0042] Figure 14 The results are as follows: (A) qPCR reaction conditions for detecting extracellular vesicles in normal human peripheral blood; (B) qPCR reaction conditions for detecting extracellular vesicles in acute myeloid leukemia cells.

[0043] Figure 15 The sensitivity of the detection method is as follows: (A) standard curve of the detection method for detecting CD9 molecules; (B) standard curve of the detection method for detecting CD33 molecules; (C) standard curve of the detection method for detecting CD34 molecules.

[0044] Figure 16 The stability of the detection method is: (A) the stability of the detection method for detecting CD9 molecules; (B) the stability of the detection method for detecting CD33 molecules; (C) the stability of the detection method for detecting CD34 molecules.

[0045] Figure 17 Clinical sample validation of the detection method: (A) CD9 molecule CT value results of peripheral blood samples from patients with acute myeloid leukemia repeated three times; (B) Scatter plot of CD9 molecule detection in normal subjects and patients with acute myeloid leukemia.

[0046] Figure 18 Clinical sample validation of the detection method: (A) CD33 molecule CT value results of peripheral blood samples from patients with acute myeloid leukemia repeated three times; (B) Scatter plot of CD33 molecule detection in normal subjects and patients with acute myeloid leukemia.

[0047] Figure 19 Clinical sample validation of the detection method: (A) CD34 molecule CT value results of peripheral blood samples from patients with acute myeloid leukemia repeated three times; (B) Scatter plot of CD34 molecule detection in normal subjects and patients with acute myeloid leukemia

[0048] Figure 20 This is the clinical sample validation of the detection method: ROC curve of CD9 / CD33 / CD34. DETAILED DESCRIPTION

[0049] To facilitate understanding of the present invention, the present invention will be described more fully below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the present disclosure more thorough and comprehensive.

[0050] In the following examples, the experimental methods without specific conditions are generally carried out under conventional conditions or conditions recommended by the manufacturers. The various commonly used chemical reagents used in the examples are all commercially available products.

[0051] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art to which this invention pertains. The terms used in this specification are for the purpose of describing specific embodiments only and are not intended to limit the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0052] Example 1

[0053] 1) Preparation of main solution

[0054] (1) SDS-PAGE destaining buffer: Weigh 10 mL of glacial acetic acid, 40 mL of ultrapure water, and 50 mL of methanol, then add 1000 mL of ultrapure water and mix thoroughly. Store at room temperature until ready to use. Prepare the desired amount in proportion.

[0055] (2) 0.015 M pH 7.4 PBS buffer: weigh 8 g of NaCl, 0.2 g of KCl, 1.14 g of Na2HPO4, and 0.27 g of KH2PO4, add 900 mL of ultrapure water to fully dissolve, add ultrapure water to make up to 1 L, mix well, and store at 4°C until use. Prepare the solution in proportion according to the amount required for the experiment.

[0056] (3) 0.1 M pH 8.0 Tris-HCl buffer: Weigh 12.11 g of Tris, add 900 mL of ultrapure water, and dissolve completely. Adjust the pH to 8.0 with NaOH solution, then add ultrapure water to 1 L. Store at 4°C until ready to use. Prepare the buffer according to the required amount for the experiment.

[0057] (4) Electrophoresis buffer: Weigh 3.03 g of Trix base, 14.4 g of glycine, and 1 g of SDS, add ultrapure water to a volume of 1 L, and store at room temperature until use (can be recycled). Prepare the solution in proportion to the amount required for the experiment.

[0058] (5) Transfer solution: Weigh 3.03 g of Trix base, 14.4 g of glycine, and 200 mL of methanol, and dilute to 1 L with ultrapure water. Once prepared, store at room temperature until ready for use (can be recycled). Prepare the solution in proportion to the amount required for the experiment.

[0059] (6) 2% milk blocking solution: According to the experimental requirements, weigh 2g of skim milk powder and add ultrapure water to make up to 1L. After preparation, store at room temperature until use.

[0060] (7) Protein Blotting Developing Solution: Weigh 0.50 mL of A and 0.50 mL of B according to the experimental requirements. After preparation, store at room temperature until ready for use.

[0061] (8) Clinical sample collection

[0062] 8.1 The inclusion criteria for clinical samples are shown in Table 1.

[0063] Table 1

[0064]

[0065] 8.2 Sample Source and Ethical Approval

[0066] This study used clinical samples from patients with acute myeloid leukemia and healthy individuals. The samples were collected by Guangdong Second People's Hospital and approved by the hospital's Research Ethics Review Committee. All clinical samples were handled in strict accordance with World Health Organization standard operating procedures, and informed consent was obtained from all participants or their legal representatives.

[0067] (9) The main reagent materials are as follows Table 2

[0068] Table 2

[0069]

[0070]

[0071] 2) Experimental methods

[0072] (1) Characterization and identification of extracellular vesicles

[0073] Transmission electron microscopy characterization of extracellular vesicles: Place 10 μL of extracellular vesicle resuspension on a dedicated copper grid and let it stand at room temperature for 2 minutes, then use filter paper to absorb the excess liquid; add 20 μL of electron microscopy fixative (2% phosphotungstic acid staining solution) dropwise to the copper grid and let it stand at room temperature for 10 minutes, then use filter paper to absorb the excess liquid; after negative staining, wash the copper grid twice with deionized water, absorb the excess liquid with filter paper, and air-dry; place the dry copper grid in the mounting slot and fix the gasket; after aligning the sample tube with the microscope inlet, you can start observation.

[0074] Nanoparticle tracking analyzer for particle size analysis of extracellular vesicles: Install the sealing ring in the sample tank and clean the glass area with lens paper; then clean the pipeline with sterile PBS or ultrapure water (filtered through a 0.22μm membrane to remove excess particles), remove all bubbles from the pool during the cleaning process, and turn on the camera when the liquid flow is stable to eliminate interference from bright spots without particles; slowly inject the diluted sample, and shoot and analyze after the flow rate stabilizes.

[0075] Western blot analysis of extracellular vesicle surface immunomarkers: Prepare the protein standards in the kit at various concentrations. Then, add 25 μL of each standard to 200 μL of Coomassie Brilliant Blue reagent, shake thoroughly, and measure the absorbance at 462 nm. Construct a standard curve based on the absorbance and the corresponding protein concentration (μg / mL) of the standard. Similarly, add 10 μL of the test sample to 200 μL of Coomassie Brilliant Blue reagent, shake thoroughly, and measure the absorbance at 462 nm. The absorbance values ​​are converted to the corresponding concentration using the standard curve. The extracellular vesicle sample is mixed with loading buffer (10× SDS) in a 9:1 ratio and heated in a water bath at 100°C for 10–15 minutes. After protein denaturation, store in a refrigerator at -20°C. Assemble the gel mold and prepare the lower layer gel (1.50 mm thick) first; take 4 mL of equal volume of lower layer gel solution (2×) and lower layer gel buffer (2×), put them into the gel cup and mix them evenly; add 80 μL of improved coagulant to the mixed solution and mix well to avoid bubbles; add the mixed lower layer gel solution to the gel mold so that the liquid level is 1.5 cm away from the upper edge of the glass plate, then cover with a layer of distilled water to keep the gel surface flat and avoid bubbles; let it stand at room temperature (25°C) for 6 -10 minutes, when a boundary appears between the lower gel and the overlay aqueous phase, it indicates that the gel has solidified; then prepare the upper gel, slowly pour off the overlay phase, take equal volumes of upper gel solution (2×) and color layer gel buffer (2×), 1.0 mL each, and place them in a new gel preparation cup to mix thoroughly; add 20 μL of the improved coagulant to the mixture and stir to avoid bubbles; add the upper gel solution to the upper layer of the lower gel until the gel solution reaches the top of the glass plate, and slowly insert the comb into the gel to avoid bubbles. Let it stand for 10-15 minutes, wait for the upper gel to solidify, carefully remove the comb, use the tip of the pipette to draw up the electrophoresis buffer, rinse the sample wells, and then proceed to load the sample for electrophoresis.After cleaning the glass plate, align the glass plate and place it on the glue pouring rack; then add deionized water to the upper edge of the glass plate; after standing for 20 minutes, if the liquid level drops less than 5mm, it means that the glue can be poured; then add separation glue to 2 / 3 of the upper edge of the glass plate and add deionized water / isopropanol to the upper edge of the glass plate, let it stand for 30 minutes, wait for the separation glue to solidify (that is, a clear boundary with deionized water); absorb the upper deionized water and dry it with filter paper, add concentrated glue to the upper edge of the glass plate and immediately insert the sample comb vertically (no bubbles can be generated), let it stand for 30 minutes. min, wait for the stacking gel to solidify; then remove the loading comb, fix the two glass plates on the electrophoresis tank, fill the space between the two glass plates with electrophoresis solution, and add the electrophoresis solution from the inside out to 5mm from the upper edge of the glass plate; then add a certain volume of sample and the corresponding marker indicator to the loading port, and start electrophoresis; at the beginning of electrophoresis, use low voltage to concentrate the sample (80V, 30min) until bromophenol blue enters the separation gel; then switch to high voltage (120V, 90min) until bromophenol blue reaches the bottom of the glass plate (can be adjusted according to the required bands). Remove the gel and cut the gel according to the location of the target protein; cover the surface of the gel with a PVDF membrane soaked in methanol (about 15 seconds) (no bubbles) and cover it with a sponge pad; then place it on a transfer clip and transfer it to a transfer tank filled with transfer buffer for transfer (100mA, 90min); incubate the membrane in blocking buffer (37℃, 1h); then wash the blocking buffer clean (3 times, 5min / time, 80r / min); after washing, add the corresponding primary antibody dilution and incubate overnight (4℃, shaker 80r / min); then wash the primary antibody dilution with 0.05% PBST (6 times, 5min / time) and incubate in secondary antibody dilution (room temperature, 60min, shaker 80r / min); then wash the secondary antibody dilution clean (6 times, 5min / time) and soak in PBST; finally, after cleaning off the excess PBST, soak the membrane in exposure solution (A:B = 1:1 mixture, 1mL) and perform imaging analysis.

[0076] (2) Preparation of immunomagnetic beads (IMB) modified with CD63 antibodies

[0077] Thoroughly mix the streptavidin-modified magnetic beads by vortexing for 30 seconds. Transfer 100 μl of the beads to a 1.5 ml EP tube or eight-well strip, place on a magnetic rack, and let stand for 1 minute to allow the beads to separate. Discard the supernatant. Remove the EP tube from the magnetic rack and resuspend the beads in 100 μl of PBST buffer (for binding protein). Mix thoroughly by pipetting 10 times or by vortexing for approximately 10 seconds. After a brief centrifugation, place the EP tube on a magnetic rack and let stand for 1 minute to allow the beads to separate. Discard the supernatant. Repeat this step once. Resuspend the beads in 50 μl of PBST buffer (for binding protein). Add 50 μl of biotin-modified CD63 antibody and mix thoroughly by pipetting 10 times or by vortexing for approximately 10 seconds. Incubate at room temperature in a vertical rotating mixer for 15-30 minutes. After a brief centrifugation, place the EP tube on a magnetic stand and let it stand for 1 minute to allow the magnetic beads to separate. Transfer the supernatant to a new EP tube for subsequent use. Resuspend the magnetic beads in 100 μl of PBST buffer (binding protein). Gently pipette 10 times or place on a vortex shaker for about 10 seconds to mix thoroughly. After a brief centrifugation, place the EP tube on a magnetic stand and let it stand for 1 minute to allow the magnetic beads to separate. Discard the supernatant. Repeat this step once. Add the appropriate PBS to resuspend the magnetic beads for use. This is the immunomagnetic beads (IMB) modified with CD63 antibodies.

[0078] (3) Characterization and verification of immunomagnetic beads (IMB)

[0079] The prepared immunomagnetic bead resuspension, the supernatant from the immunomagnetic bead separation process, and the precipitate were collected. Protein standards from the BCA kit were prepared at different concentrations. Subsequently, 25 μL of each standard concentration was added to 200 μL of Coomassie Brilliant Blue reagent, shaken thoroughly, and the corresponding absorbance (462 nm) was measured. A standard curve was constructed based on the absorbance and the protein concentration (μg / mL) corresponding to the standard. Similarly, 10 μL of each of the three test samples (prepared immunomagnetic bead resuspension, the supernatant from the immunomagnetic bead separation process, and the precipitate) were added to 200 μL of Coomassie Brilliant Blue reagent, shaken thoroughly, and the corresponding absorbance (462 nm) was measured. The absorbance values ​​were converted to the corresponding concentrations using the standard curve. Western blot analysis was performed on the immunomagnetic beads labeled with CD63 antibodies.

[0080] (4) Optimization of immunomagnetic beads (IMB) conditions

[0081] Thoroughly mix the streptavidin-modified magnetic beads by vortexing for 30 seconds. Transfer 100 μl of the beads to a 1.5 ml EP tube or an eight-well strip, place on a magnetic rack, and let stand for 1 minute to allow the beads to separate. Discard the supernatant. Remove the EP tube from the magnetic rack and resuspend the beads in 100 μl of PBST buffer (for binding protein). Mix thoroughly by pipetting 10 times or by vortexing for approximately 10 seconds. After a brief centrifugation, place the EP tube on a magnetic rack and let stand for 1 minute to allow the beads to separate. Discard the supernatant. Repeat this step once. Resuspend the beads in 50 μl of PBST buffer (for binding protein). Add 0, 0.5, 1, or 1.5 μg (1 mg / ml) of biotin-modified CD63 antibody, respectively. Mix thoroughly by pipetting 10 times or by vortexing for approximately 10 seconds. At room temperature, place in a vertical rotating mixer and incubate for 15-30 minutes. After a brief centrifugation, place the EP tube on a magnetic stand and let it stand for 1 minute to separate the magnetic beads. Move the supernatant to a new EP tube for subsequent use. Resuspend the magnetic beads in 100μl PBSTBuffer (binding protein), gently pipette 10 times or place it on a vortex shaker for about 10 seconds to mix thoroughly. After a brief centrifugation, place the EP tube on a magnetic stand and let it stand for 1 minute to separate the magnetic beads. Discard the supernatant. Repeat this step once. Add appropriate PBS to resuspend the magnetic beads for use. Then use the BCA protein concentration kit to measure the concentration of immunomagnetic beads modified with different amounts of antibodies.

[0082] (5) Immunomagnetic Bead (IMB) Performance Verification

[0083] 0, 1, 2, 4, 8, 16, and 32 μL of immunomagnetic beads labeled with CD63 antibodies were added to 5 μL of peripheral blood, mixed, and incubated for 15 minutes. After magnetic separation, these samples were analyzed by Western blot and grayscale analysis using Image J software.

[0084] (6) Preparation of antibody oligonucleotide chain conjugates

[0085] Oligonucleotide screening: Synthesize three single-stranded DNAs and modify their 5' ends with amino groups. Design complementary primers and fluorescent probes based on these three DNA strands. See Table 2.1 for specific sequences. All probes were reconstituted in DEPC water and stored at -20°C.

[0086] Table 2.1

[0087]

[0088] Verify single-strand DNA amplification by agarose gel electrophoresis: Rinse the gel casting tank with deionized water, air dry, place on a level work surface, and insert a sample comb. Weigh 300 mg of agarose and dissolve it in 30 ml of 1× electrophoresis buffer. Heat in a microwave oven until completely dissolved. Remove and shake well. Add 3 μl of 10,000× nucleic acid dye to prepare the agarose gel. Gently pour the agarose solution, cooled to 60°C, onto the gel casting tank. After the agarose gel solidifies, transfer the gel to an electrophoresis tank, add electrophoresis buffer, and remove the comb. Mix the samples (PCR amplification products of T1, T2, and T3) with 6× loading buffer in a ratio of 5:1. Use a pipette to add the mixture to the sample tank, adding 10-20 μl to each tank. Record the order and volume of the samples loaded. Install the electrode wires, connect one end of the sample well to the negative electrode and the other end to the positive electrode. Turn on the power supply and adjust the voltage to 120V. Run the electrophoresis for about 1.5 hours. Stop the electrophoresis when the bromophenol blue moves to 1-2 cm from the front of the gel. Remove the gel and observe the bands on a gel imaging system.

[0089] To conjugate the antibody to the oligonucleotide chain: Add 100 μL of oligonucleotide to the Oligonucleotide Activation Reagent vial. Mix gently and incubate at room temperature for 30 minutes. During this incubation, the antibody is activated. Add 100 μL of antibody (at a concentration of 1 mg / mL) to the Antibody Activation Reagent vial. Mix gently and incubate at room temperature for 30 minutes. During this incubation, the activation reagent is desalted. Mount each column vertically. First, open the top cap. Then, open the bottom cap and allow the storage liquid to pass through the column. Discard the liquid that flows out of the column. Add 3 mL of Wash Buffer to the top of the column and allow the liquid to drain by gravity to equilibrate each column. Discard the liquid that flows out. Repeat this step four times. After another 30-minute incubation, add 100 μL of the activated oligonucleotide or antibody to the top of the column and allow the liquid to be completely absorbed by the column. Collect the liquid that flows out and allow it to settle. Add 550 μL of Wash Buffer to the top of the column again. Push the activated reagent to the bottom of the column. Wait until the liquid is completely absorbed before proceeding to the next step. Collect the outflowing liquid and let it stand. Place a clean microcentrifuge tube under the separation column. Add 300 μL of wash buffer from the top of the separation column. Collect the eluate from the bottom of the separation column. The eluate (300 μL) contains the activated oligonucleotide or antibody and is ready for coupling. Add 300 μL of activated antibody to 300 μL of activated oligonucleotide and wash buffer. Mix well and incubate at room temperature for 1 hour.

[0090] (7) Optimization of antibody oligonucleotide conjugate conditions

[0091] Reconstitute the activated oligonucleotide and antibody, which have been stored at -20°C, at room temperature. Add 300 μL of activated antibody to 300 μL, 200 μL, and 60 μL of activated oligonucleotide and wash buffer, respectively. Mix thoroughly and incubate at room temperature for 1 hour. Analyze the three samples using SDS-PAGE to determine the optimal ratio of antibody to oligonucleotide.

[0092] (8) SDS-PAGE verification of antibody-oligonucleotide conjugates

[0093] Place the sealing silicone frame on the flat glass, then overlap the concave glass with the flat glass, stand up the two pieces of glass so that the bottom ends touch the table, clamp the two pieces of glass with your hands and put them into the electrophoresis tank, then insert the inclined plate to an appropriate degree, and then you can pour the glue. Prepare the lower layer of glue (1.50mm thick) first; take equal volumes of lower layer glue solution (2×) and lower layer glue buffer solution (2×) 4mL each, put them into the glue cup and mix them; add 80μL of improved coagulant to the mixed solution, mix and stir to avoid bubbles; add the mixed lower layer glue solution to the glue mold so that the liquid surface is 1.5cm away from the upper edge of the glass plate, then cover with a layer of distilled water to keep the gel surface flat and do not generate bubbles; let it stand at room temperature (25℃) for 6-10m in. When a boundary appears between the lower gel and the overlay aqueous phase, it indicates that the gel has solidified. Then prepare the upper gel, slowly pour off the overlay phase, take equal volumes of upper gel solution (2×) and color layer gel buffer (2×), 1.0 mL each, and place them in a new gel preparation cup to mix thoroughly. Add 20 μL of the improved coagulant to the mixture and stir to avoid bubbles. Add the upper gel solution to the upper layer of the lower gel until the gel solution reaches the top of the glass plate. Slowly insert the comb into the gel to avoid bubbles. Let it stand for 10-15 minutes, wait for the upper gel to solidify, carefully remove the comb, use the tip of the pipette to draw up the electrophoresis buffer to rinse the sample wells, and then proceed to load the sample for electrophoresis. After cleaning the glass plate, align the glass plate and place it on the glue pouring rack; then add deionized water to the upper edge of the glass plate; after standing for 20 minutes, if the liquid level drops less than 5mm, it means that the glue can be poured; then add separation glue to 2 / 3 of the upper edge of the glass plate and add deionized water / isopropanol to the upper edge of the glass plate, let it stand for 30 minutes, wait for the separation glue to solidify (that is, a clear boundary with deionized water); absorb the upper deionized water and dry it with filter paper, add concentrated glue to the upper edge of the glass plate and immediately insert the sample comb vertically (no bubbles can be generated), let it stand for 30 minutes. min, wait for the stacking gel to solidify; then remove the sample comb, fix the two glass plates on the electrophoresis tank, fill the space between the two glass plates with electrophoresis solution, and add the electrophoresis solution from the inside out to 5mm above the upper edge of the glass plate; then add a certain volume of sample and the corresponding marker indicator to the sample loading port, and start electrophoresis; at the beginning of electrophoresis, use low voltage to concentrate the sample (80V, 30min) until the bromophenol blue enters the separating gel; then switch to high voltage (120V, 90min) until the bromophenol blue reaches the bottom of the glass plate (this can be adjusted according to the desired banding). After the electrophoresis is completed, turn off the power, remove the glass plates, and gently pry with a knife in the gap between the long and short glass plates to separate the gel surface from one glass plate. Then gently lift the film, insert a copper wire into the center of the indicator zone as a mark, and place it in a large culture dish for staining with 0.25% Coomassie Brilliant Blue solution for 2-4 hours. Discard the staining solution, rinse the gel surface several times with distilled water, then add destaining solution for diffusion destaining, and change the destaining solution frequently until the protein band is clear.Observe the antibody-oligonucleotide chain conjugate for any lag reaction.

[0094] (9) Feasibility verification of a combined detection method for multiple immune markers on the surface of extracellular vesicles in the peripheral blood of patients with acute myeloid leukemia

[0095] (1) Prepare the mixture shown in Table 12 in a qPCR tube.

[0096] Table 12

[0097]

[0098]

[0099] (2) Perform qPCR reaction according to the following conditions in Table 13

[0100] Table 13

[0101]

[0102] (10) Optimization of the combined detection method of multiple immune markers on the surface of extracellular vesicles in peripheral blood of patients with acute myeloid leukemia

[0103] After preparing the samples in the eight-tube strips, place them on a vortex mixer, mix thoroughly, centrifuge, and place them in the qPCR instrument, keeping the temperature and other conditions unchanged. Set different cycle numbers (30, 35, 40, 45, 50, and 55) and record the Ct values.

[0104] (11) Detection process of combined detection method of multiple immune markers on the surface of extracellular vesicles in peripheral blood of patients with acute myeloid leukemia

[0105] Sample collection: Use EDTA-K2 vacuum blood collection tubes to collect venous blood. Avoid hemolysis during the collection process. Centrifuge at 3500-4000rpm for 15 minutes twice to remove platelets, and aspirate the supernatant. Sample storage: The collected samples should be tested within 24 hours. If the test cannot be completed, they should be stored at -20℃ for no more than 7 days. If it exceeds 7 days, they should be stored at -80℃. Sample testing: Add the collected and processed plasma to the EP tube, add the immunomagnetic beads prepared in advance, and mix well. Use a magnet to adsorb to the bottom of the EP tube and pour out the supernatant; at this time, add the antibody oligonucleotide chain conjugate prepared in advance and mix well. Take the sample for real-time fluorescence quantitative PCR reaction. Read the Ct value to determine the result.

[0106] (12) Performance verification of a combined detection method for multiple immune markers on the surface of extracellular vesicles in the peripheral blood of patients with acute myeloid leukemia

[0107] Plasma with known extracellular vesicle concentrations was diluted into a series of concentration gradients (0.274, 0.611, 1.098, 2.195, 4.39, 8.78×10 7 Particles / mL), add the immunomagnetic beads prepared in advance, mix and separate, then add the antibody oligonucleotide chain conjugate prepared in advance and mix. Take 1 μL of each concentration solution and add it to the sample well, and repeat 3 times. Draw a standard curve with Ct value as the vertical axis and concentration as the horizontal axis. Then take low, medium and high (0.274, 1.647, 8.78×10 7 1 μL of each of the three concentrations (particles / mL) was added to the sample wells and repeated 10 times. Coefficient of variation (CV) = (standard deviation SD / mean) × 100%.

[0108] 3) Results

[0109] 3.1 Principles of the Construction of a Combined Detection Method for Multiple Immune Markers on the Surface of Extracellular Vesicles in the Peripheral Blood of Patients with Acute Myeloid Leukemia

[0110] like Figure 1 As shown in Figure 2, the development of a combined detection method for multiple immune markers on the surface of extracellular vesicles (EVs) in the peripheral blood of patients with acute myeloid leukemia consists of three main components. The first component involves the isolation of EVs from peripheral blood using immunomagnetic beads. First, the magnetic beads are streptavidin-labeled, followed by the biotinylation of CD63 antibodies. The streptavidin-labeled magnetic beads and the biotinylated CD63 antibodies are then linked to form immunomagnetic beads. Because CD63 is a universal membrane protein of EVs, immunomagnetic beads labeled with CD63 antibodies can specifically capture EVs from peripheral blood, allowing for their isolation. The immunomagnetic beads specifically recognize EVs, and the immunomagnetic bead-EV complex is attracted to a magnet, allowing the EVs to be isolated from the peripheral blood.

[0111] The second part involves detecting extracellular vesicles. Antibodies against CD33, CD34, and CD9 are selected, and three different oligonucleotide single chains are screened. Using an Ab-Oligo conjugate kit, the antibodies and oligonucleotide chains are linked to form three Ab-Oligo conjugates: CD9Ab-Oligo, CD33Ab-Oligo, and CD34Ab-Oligo. The prepared Ab-Oligo conjugates are added to isolated extracellular vesicles. Because extracellular vesicles contain CD33, CD34, and CD9 membrane proteins, the three Ab-Oligo conjugates specifically recognize extracellular vesicles, ultimately forming an immunomagnetic bead-extracellular vesicle-Ab-Oligo conjugate immune complex. A single extracellular vesicle can simultaneously bind to multiple different Ab-Oligo conjugates.

[0112] The third part is the results of the methodological analysis to distinguish healthy people from patients with acute myeloid leukemia. Figure 2 As shown in Figure 2, the oligonucleotide chains on the Ab-Oligo conjugates complement each other with different primers and fluorescent probes with different fluorophores, and amplification reactions are performed via qPCR. The concentration of membrane proteins on extracellular vesicles is revealed by the conversion of the oligonucleotide chains on the Ab-Oligo conjugates, and the concentration of membrane proteins is inversely proportional to the Ct value measured by the qPCR reaction.

[0113] 3.2 Characterization and Identification of Extracellular Vesicles in Peripheral Blood of Patients with Acute Myeloid Leukemia

[0114] In order to verify that the samples used in this study contained extracellular vesicles, conventional extracellular vesicle identification methods were used for identification, and transmission electron microscopy showed the ultrastructure of extracellular vesicles: e.g. Figure 3 As shown, extracellular vesicles are double-membrane vesicles approximately 200 nm in size. NTA revealed the concentration and size distribution of extracellular vesicles. Western blotting was used to identify extracellular vesicle membrane proteins, revealing the presence of CD81, TSG101, HSP-70, and CD63 proteins on the extracellular vesicle membrane. Surface extracellular vesicles were successfully isolated.

[0115] 3.3 Verification of differences in extracellular vesicle surface immune markers

[0116] 3.3.1 Verification of the difference of surface immune markers of extracellular vesicles at different concentrations

[0117] Studies have shown that the levels of three CD molecules, CD9, CD33, and CD34, on cells derived from acute myeloid leukemia are significantly more expressed than those in normal people, and there are also differences in the three CD molecules CD9, CD33, and CD34 on extracellular vesicles in the peripheral blood of normal people and patients with acute myeloid leukemia. We used Western Blot to verify the differences in the levels of the three CD molecules, and used ImageJ grayscale analysis software to perform semi-quantitative experiments on the data. Because CD63 is a universal membrane protein on extracellular vesicles, it was also verified. A total of four samples were prepared, namely, a single normal peripheral blood and peripheral blood of patients with acute myeloid leukemia, and a mixed multi-person normal peripheral blood and peripheral blood of patients with acute myeloid leukemia. Figure 4As shown in Figure 2, the results showed that the levels of CD9, CD33, and CD34 on extracellular vesicles in the peripheral blood of patients with acute myeloid leukemia were significantly higher than those in the peripheral blood of healthy controls (P < 0.001). CD63 is a universal membrane protein on extracellular vesicles in peripheral blood. Theoretically, the level of CD63 on extracellular vesicles in the peripheral blood of patients with acute myeloid leukemia should be equal to that in the peripheral blood of healthy controls. Considering that the number of extracellular vesicles contained in the peripheral blood samples was different despite the same volume, the extracellular vesicle concentration should be standardized for comparison.

[0118] 3.3.2 Uniform extracellular vesicle concentration

[0119] In order to more accurately verify the content of CD molecules on extracellular vesicles in peripheral blood, the concentration of extracellular vesicles in peripheral blood samples was measured using NTA and diluted to the same concentration before subsequent experiments. Figure 5 As shown, the results show that the extracellular vesicle size distribution in the three samples is highly similar and very concentrated, with a small size span and very close size values, with average particle sizes of 69 nm, 70 nm, and 67 nm, respectively. The extracellular vesicle concentrations in the three samples were 8.78E+12, 4.82E+12, and 2.72E+12 (particles / mL), respectively. All three samples were diluted to 1.0E+12 (particles / mL) for subsequent downstream experiments to verify the differences in the content of extracellular vesicle immunomarkers by Western Blot.

[0120] 3.3.3 Verification of differences in surface immune markers of EVs at the same concentration

[0121] In order to more accurately analyze the content of CD molecules on extracellular vesicles in peripheral blood, the extracellular vesicle concentration in plasma was unified to 1.0E+12 (particles / mL) using NTA, and then the differences in the content of the three CD molecules were verified by Western Blot. The data were semi-quantitatively analyzed using ImageJ grayscale analysis software. Figure 6 The results showed that the levels of CD9, CD33, and CD34 on extracellular vesicles in the peripheral blood of patients with acute myeloid leukemia were higher than those in the peripheral blood of normal controls, and the expression of these proteins was statistically significant (P < 0.001). The level of CD63 on extracellular vesicles in the peripheral blood of patients with acute myeloid leukemia was lower than that in the peripheral blood of normal controls, and the expression of these proteins was statistically significant (P < 0.001).

[0122] Because CD63 is a universal membrane protein, it is present on extracellular vesicles secreted by all cells. This indirectly shows that CD9, CD33, and CD34 on extracellular vesicles are of practical significance for distinguishing normal people from acute myeloid leukemia.

[0123] 3.4 Preparation of Immunomagnetic Beads (IMB)

[0124] 3.4.1 IMB Characterization and Verification

[0125] To verify that the CD63 antibody was successfully labeled on the streptavidin-labeled magnetic beads, we used the BCA protein concentration method to measure the antibody-magnetic bead mixture after antibody labeling and the supernatant separated after three times of magnet adsorption of the magnet-antibody complex. Figure 7 As shown, the results show that the concentration of the antibody-magnetic bead mixture is higher than the supernatant separated after three magnetic adsorptions of the magnet-antibody complex, indicating that the antibody has been consumed and the streptavidin-labeled magnetic beads have been labeled with the biotinylated antibody. We also prepared samples of the immunomagnetic beads and verified the CD63 antibody labeling by Western Blot.

[0126] 3.4.2IMB Condition Optimization

[0127] There are a certain number of epitopes on the immunomagnetic beads that will bind to the antibody. In order to verify the maximum saturation amount that the immunomagnetic beads can label, we use the BCA method to determine the protein concentration to verify the antibody saturation amount. When preparing the immunomagnetic beads, we added 0, 0.5, 1.0, and 1.5 μg of CD63 antibody and tested the protein concentration of the prepared immunomagnetic beads. Figure 8 As shown in the figure, when the amount of antibody added is 0.5ug (antibody concentration 1000ug / ml), the antibody labeling amount on the magnetic beads (antibody labeling amount is 1400ug / ml) tends to saturate, which is the maximum antibody labeling amount of the immunomagnetic beads.

[0128] 3.4.3 IMB Performance Verification

[0129] In order to determine the maximum amount of extracellular vesicles that can be captured by immunomagnetic beads in a certain volume of peripheral blood, Western Blot was used for verification, such as Figure 9 As shown in the figure, the results show that when 16 μL of magnetic beads (10 mg / ml) are added, the amount of extracellular vesicles that can be captured by the immunomagnetic beads tends to be saturated. This is the maximum amount of extracellular vesicles that can be captured by the immunomagnetic beads.

[0130] 3.5 Preparation of Antibody-Oligonucleotide Conjugates (Ab-Oligo)

[0131] 3.5.1Oligo screening

[0132] Antibody oligonucleotide chains are linked based on chemical functional groups. In order to successfully prepare antibody oligonucleotide chain conjugates and perform subsequent real-time fluorescence quantitative PCR, we first used agarose gel electrophoresis to verify that the oligonucleotide chains can be successfully amplified with primers. The concentrations of the oligonucleotide chains added during the PCR reaction were 0.01μM, 0.1μM, 1μM, 10μM, and 100μM, respectively. Figure 10 As shown, there are bands at each concentration, indicating that the oligonucleotide chain can react with the primer. We finally chose the oligonucleotide chain at a concentration of 100 μM for subsequent experiments.

[0133] 3.5.2 Ab-Oligo Characterization and Validation

[0134] In order to verify the success of Ab-Oligo conjugate, we used SDS-PAGE experiment to verify. When the antibody is connected to the oligonucleotide chain, the surface charge will change, and a hysteresis reaction will occur. The results show that Ab-Oligo conjugate has a hysteresis reaction compared to a single antibody. Figure 11 As shown, it shows that the antibody and oligonucleotide chain are successfully coupled.

[0135] 3.5.3 Ab-Oligo Condition Optimization

[0136] After the Ab-Oligo conjugate is successfully prepared, we change the ratio of antibody to oligonucleotide chain to select the optimal ratio of antibody to oligonucleotide chain. The ratio of antibody to oligonucleotide chain is 1:3, 1:10, and 1:15 respectively. Figure 12 As shown in the figure, the results showed that the bands were clearest when the ratio of antibody to oligonucleotide was 1:3, so the ratio of 1:3 was selected for subsequent experiments.

[0137] 3.6 Feasibility Verification of a Combined Detection Method for Multiple Immune Markers on the Surface of Extracellular Vesicles in Peripheral Blood of Patients with Acute Myeloid Leukemia

[0138] After the immunomagnetic beads and Ab-Oligo conjugates are prepared, the immunomagnetic beads are added to the peripheral blood. After the immunomagnetic beads are used to separate the extracellular vesicles in the peripheral blood, the Ab-Oligo conjugate is added to form the immunomagnetic beads-extracellular vesicle-Ab-Oligo immune complex. Then, primers are added to perform qPCR reaction. The results are as follows. Figure 13 As shown in Table 3.1 and Table 3.2, the amount of Ab-Oligo in sample A should be equal to the amount of Ab-Oligo in sample B plus the amount of Ab-Oligo in sample C. Ab-Oligo in B: (2 (24.2-18.55) x10) / 26x100%≈16%, Ab-Oligo in C: (2 (16.5-24.2)x16) / 26 x 100% ≈ 82%. B accounts for approximately 16% of A, C accounts for approximately 82% of A, and the sum of the two is approximately 98%. This indicates that only Ab-Oligo is a variable in the immunomagnetic bead-extracellular vesicle-Ab-Oligo immune complex, changing with the CD molecule content. However, negative control samples D, E, and F showed no amplification curves, further verifying that only Ab-Oligo is a variable. This method was then used to test clinical samples, and the results showed amplification curves for all three CD molecules in both AML patients and healthy subjects, demonstrating the feasibility of the method.

[0139] Table 3.1

[0140]

[0141] Table 3.2

[0142]

[0143] 3.7 Optimization of the combined detection method for multiple immune markers on the surface of extracellular vesicles in the peripheral blood of patients with acute myeloid leukemia

[0144] In order to optimize the combined detection method of multiple immune markers on the surface of extracellular vesicles in the peripheral blood of patients with acute myeloid leukemia, we set different cycle numbers for amplification reaction. Figure 14 As shown, the results showed that the Ct value was the largest at 45 cycles, and 45 cycles were finally selected as the reaction cycle number.

[0145] 3.8 Performance Verification of a Combined Detection Method for Multiple Immune Markers on the Surface of Extracellular Vesicles in Peripheral Blood of Patients with Acute Myeloid Leukemia

[0146] 3.8.1 Sensitivity of the combined detection method for multiple immune markers on the surface of extracellular vesicles in the peripheral blood of patients with acute myeloid leukemia

[0147] To evaluate the sensitivity of the combined detection method for multiple immune markers on the surface of extracellular vesicles in the peripheral blood of patients with acute myeloid leukemia, the concentration of extracellular vesicles in peripheral blood was analyzed by NTA technology. Peripheral blood with known concentrations was diluted into multiple concentration gradients: 0.274, 0.611, 1.098, 2.195, 4.39, 8.78×10 7 The Ct values ​​at different concentrations were analyzed using a qPCR instrument. The test results were plotted with the Ct value as the ordinate and the concentration as the abscissa to establish a standard curve, perform a linear fit, and derive a regression equation. Figure 15 As shown in the figure, the detection linear range of the established method for the combined detection of multiple immune markers on the surface of extracellular vesicles in the peripheral blood of patients with acute myeloid leukemia is 0.274-8.78×10 7 particles / mL, and the lowest concentration of extracellular vesicles that can be detected in peripheral blood is 0.274×107 particles / mL.

[0148] 3.8.2 Stability of the Combined Detection Method for Multiple Immune Markers on the Surface of Extracellular Vesicles in Peripheral Blood of Patients with Acute Myeloid Leukemia

[0149] To evaluate the combined detection method of multiple immune markers on the surface of extracellular vesicles in the peripheral blood of patients with acute myeloid leukemia, the concentration of extracellular vesicles in peripheral blood was analyzed by NS3000 nanoparticle size analyzer. 7 The three concentrations (particles / mL) were repeated 10 times. Coefficient of variation C·V = (standard deviation SD / mean) × 100%. Figure 16 As shown in the figure, the CV values ​​of low, medium and high concentrations of CD33, CD34 and CD9 extracellular vesicles were all less than 5%, indicating that the method has good stability.

[0150] 3.9 Clinical Validation of a Combined Detection Method for Multiple Immune Markers on the Surface of Extracellular Vesicles in Peripheral Blood of Patients with Acute Myeloid Leukemia

[0151] We used this method to test clinical samples from 20 patients with acute myeloid leukemia and 20 normal subjects. Figures 17 to 20 As shown, the results show that CD9, CD33, and CD34 can distinguish between patients with acute myeloid leukemia and healthy subjects. A receiver operating characteristic (ROC) curve was also plotted, with cut-off values ​​of 20.72, 20.47, and 20.31 for CD33, CD34, and CD9, respectively. When the Ct values ​​of a sample detected by this method are all below the calculated cut-off values, the patient can be preliminarily diagnosed as having acute myeloid leukemia.

[0152] In summary, acute myeloid leukemia is a highly malignant heterogeneous tumor. Early detection and precise typing and treatment of acute myeloid leukemia can reduce mortality. Detection technologies for extracellular vesicles in the peripheral blood of acute myeloid leukemia patients are mostly biased towards single-target detection. In order to simultaneously detect multiple CD molecules on extracellular vesicles in the peripheral blood of acute myeloid leukemia patients to meet the screening needs of acute myeloid leukemia. This study completed a combined detection method for multiple immune markers on the surface of extracellular vesicles in the peripheral blood of acute myeloid leukemia patients and verified it with clinical samples. It can distinguish between acute myeloid leukemia patients and normal people. Compared with traditional methods of detecting extracellular vesicles, it has certain clinical significance for distinguishing between acute myeloid leukemia and normal people, and the proposed method has certain prospects in the sensitive detection of multiple targets, which can help the accurate molecular typing of many complex diseases. A combined detection method for multiple immune markers on extracellular vesicles (EVs) from the peripheral blood of patients with acute myeloid leukemia (AML) was constructed and validated against clinical samples. The Ct values ​​for CD9, CD33, and CD34 on EVs from AML patients were lower than those from healthy controls. This was consistent with the flow cytometry results from clinically collected samples, demonstrating the practical value of this method for differentiating AML patients from healthy controls.

[0153] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0154] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.

Claims

1. A kit for the combined detection of multiple immune markers on the surface of extracellular vesicles in peripheral blood, characterized in that: The kit comprises: Immunomagnetic beads with CD63 antibody modified on the surface; Three different oligonucleotide chains are coupled to three antibodies respectively, forming three antibody oligonucleotide conjugates, wherein the three antibodies are CD9 antibody, CD33 antibody and CD34 antibody; and three pairs of primers and three fluorescent probes for amplifying three different oligonucleotide chains or their complementary paired sequences, wherein the fluorescent label in each fluorescent probe is different. The nucleotide sequence of the oligonucleotide chain coupled to the CD9 antibody is shown in SEQ ID NO: 1, the nucleotide sequence of the oligonucleotide chain coupled to the CD33 antibody is shown in SEQ ID NO: 5, and the nucleotide sequence of the oligonucleotide chain coupled to the CD34 antibody is shown in SEQ ID NO:

9.

2. The kit according to claim 1, wherein The primer pair sequences for amplifying the oligonucleotide chain coupled to the CD9 antibody are shown in SEQ ID NO: 2 and SEQ ID NO: 3; and / or, The primer pair sequences for amplifying the oligonucleotide chain conjugated to CD33 antibody are shown in SEQ ID NO: 6 and SEQ ID NO: 7; and / or, The sequences of the primer pair used to amplify the oligonucleotide chain conjugated to CD34 antibody are shown in SEQ ID NO: 10 and SEQ ID NO:

11.

3. The kit according to claim 1, wherein The fluorescent probe sequence for amplifying the oligonucleotide chain coupled to CD9 antibody is shown in SEQ ID NO: 4; and / or, The fluorescent probe sequence for amplifying the oligonucleotide chain coupled to the CD33 antibody is shown in SEQ ID NO: 8; and / or, The fluorescent probe sequence used to amplify the oligonucleotide chain coupled to the CD34 antibody is shown in SEQ ID NO:

12.

4. The kit according to any one of claims 1 to 3, wherein When preparing the oligonucleotide chain conjugate, the ratio of the antibody to the oligonucleotide chain is 1:3-15.

5. The kit according to claim 4, wherein The ratio of the antibody to the oligonucleotide chain is 1:3-5.

6. The kit according to any one of claims 1 to 3, wherein The CD63 antibody labeling amount in the immunomagnetic beads is 800ug / ml~1600ug / ml.

7. Use of a kit for the combined detection of multiple immune markers on the surface of peripheral blood extracellular vesicles as claimed in any one of claims 1 to 6 in the preparation of a product for detecting acute myeloid leukemia.

8. A method for jointly detecting multiple immune markers on the surface of extracellular vesicles of peripheral blood, characterized in that: A kit for the combined detection of multiple immune markers on the surface of peripheral blood extracellular vesicles according to any one of claims 1 to 6 comprises the following steps: Immunomagnetic beads modified with CD63 antibodies were added to the collected peripheral blood samples and mixed, and then the immunomagnetic beads-extracellular vesicle immune complexes were obtained by magnetic adsorption; mixing the three antibody-oligonucleotide conjugates with the immunomagnetic bead-extracellular vesicle immune complex to obtain an immunomagnetic bead-extracellular vesicle-antibody oligonucleotide conjugate immune complex; The immunomagnetic beads-extracellular vesicles-antibody oligonucleotide conjugate immune complex is amplified by qPCR to obtain an amplified product.

9. The method according to claim 8, wherein The number of cycles of the qPCR amplification was 43 to 47 times.

10. The method according to claim 8, wherein The concentration of extracellular vesicles in the peripheral blood sample was 0.274×10 7 Particles / mL~8.78×10 7 particles / mL.

11. The method according to claim 8, wherein The volume ratio of the added peripheral blood sample to the immunomagnetic beads is 5 μL:15 μL~17 μL, and the concentration of the immunomagnetic beads is 8 mg / ml~12 mg / ml.

Images