Quality control diluent for exosome extraction, quality control for exosome extraction, and preparation method and application thereof

Abstract

The present application relates to the technical field of quality control products, and particularly relates to a quality control product diluent for exosome extraction, a quality control product for exosome extraction, a preparation method and application. The quality control product diluent comprises 10%-90% (v / v) animal serum, 10%-30% (v / v) buffer, 0.5%-2% (w / v) trehalose, 0.1%-1% (v / v) preservative, and the balance is water. The quality control product diluent for exosome extraction of the present application has simple components, is used for diluting the quality control product, and realizes stable detection of proteins. The quality control product for exosome extraction of the present application is suitable for detection technologies such as chemiluminescence or ELISA, and can also evaluate the recovery rate and extraction purity of different exosome extraction methods. In addition, by repeatedly extracting the mixed sample of the positive quality control product and the negative quality control product for multiple times, the repeatability of different exosome extraction methods can also be effectively evaluated.

Description

Technical Field

[0001] This invention relates to the field of quality control technology, and in particular to a quality control diluent for exosome extraction, a quality control product for exosome extraction, a preparation method therefor, and its application. Background Technology

[0002] Exosomes, as important nanoparticles for intercellular communication, contain abundant biological information, including proteins, lipids, and nucleic acids. This information has significant clinical value in the early diagnosis and treatment of diseases. As a core medium for intercellular communication, the miRNAs, functional proteins, and metabolites carried by exosomes have been shown to be closely related to pathological processes such as tumor metastasis, immune microenvironment regulation, and the progression of neurodegenerative diseases.

[0003] However, the routine exosome testing process lacks standardized quality control procedures to ensure the reliability of test results. In particular, a quality control system for exosome extraction used by clinical clients is currently lacking, necessitating the development of a widely applicable set of exosome extraction quality control standards to support the clinical application of exosome diagnostics. Summary of the Invention

[0004] One of the objectives of this invention is to provide a quality control diluent for exosome extraction, which is used to dilute the quality control sample and achieve stable protein detection.

[0005] The second objective of this invention is to provide a method for preparing a quality control diluent for exosome extraction, which is simple and easy to control.

[0006] The third objective of this invention is to provide a quality control product for exosome extraction, including a pairing system of negative and positive quality controls, to achieve routine quality control of the extraction performance of exosome extraction reagents. It can also evaluate the recovery rate and extraction purity of different exosome extraction methods, and is suitable for detection technologies such as chemiluminescence or ELISA, with great clinical application value and market promotion potential.

[0007] The fourth objective of this invention is to provide a method for preparing quality control products extracted from exosomes, establishing a pairing system between negative and positive quality control products, and the preparation method is simple, reproducible, and suitable for large-scale production.

[0008] The fifth objective of this invention is to provide a method for evaluating the performance of exosome extraction methods or reagent kits, which is simple to operate, has stable quality, is more suitable for client operation, and has high reliability of detection results.

[0009] One of the technical solutions adopted to achieve the objective of this invention is: a quality control diluent for exosome extraction, comprising: 10%-90% (v / v) animal serum; 10%-30% (v / v) buffer; 0.5%-2% (w / v) trehalose; 0.1%-1% (v / v) preservative; and the balance being water.

[0010] Wherein, v / v refers to the volume percentage concentration of the diluent; w / v refers to the mass / volume percentage concentration of the diluent, in g / ml.

[0011] Preferably, the buffer solution is a phosphate buffer. The phosphate buffer solution concentration is 10%-30% (v / v), for example 10%-20%, 10%, 20%, 30% (v / v), where v / v refers to the volume percentage concentration of the phosphate buffer solution relative to the diluent.

[0012] The type of phosphate buffer described in this invention can be conventional in the art, such as a phosphate buffer that does not contain potassium chloride or a buffer that contains only disodium hydrogen phosphate and sodium dihydrogen phosphate.

[0013] In this invention, when the phosphate buffer solution contains sodium dihydrogen phosphate, the sodium dihydrogen phosphate may be sodium dihydrogen phosphate dodecahydrate, sodium dihydrogen phosphate dihydrate, or anhydrous sodium dihydrogen phosphate. When the phosphate buffer solution contains disodium hydrogen phosphate, the disodium hydrogen phosphate may be disodium hydrogen phosphate dodecahydrate, disodium hydrogen phosphate dihydrate, or anhydrous disodium hydrogen phosphate.

[0014] Preferably, the phosphate buffer is a PBS buffer, specifically a 1×PBS buffer.

[0015] In this invention, the preparation of the phosphate buffer solution can be conventional in the art.

[0016] Preferably, the type of animal serum can be conventional in the art, and more preferably bovine serum, newborn calf serum, sheep serum, donkey serum, horse serum, rabbit serum, pig serum or chicken serum.

[0017] More preferably, the animal serum is serum from which endogenous exosomes have been removed.

[0018] Preferably, the animal serum content is 50%-90% (v / v), for example 90% (v / v), 85% (v / v), 80% (v / v), 75% (v / v), 70% (v / v), 65% (v / v), 60% (v / v), 55% (v / v), 50% (v / v), where v / v refers to the volume percentage concentration of animal serum in the diluent.

[0019] Preferably, the trehalose content is 0.5%-2% (w / v), such as 0.5% (w / v), 1% (w / v), 1.5% (w / v), 2% (w / v), etc., where w / v refers to the mass / volume percentage concentration of the trehalose in the diluted solution, in g / ml.

[0020] Preferably, the trehalose is D-trehalose.

[0021] Preferably, the content of PC300 is 0.1%-0.5% (v / v), for example 0.1% (v / v), 0.2% (v / v), 0.3% (v / v), 0.4% (v / v), 0.5% (v / v), where v / v refers to the volume percentage concentration of PC300 in the diluent.

[0022] PC300: A composite liquid biological preservative, the main components of which are 2-methyl-4-isothiazolin-3-one (MCI) and 5-chloro-2-methyl-4-isothiazolin-3-one (CMCI).

[0023] The second objective of this invention is achieved by the following technical solution: a method for preparing the quality control diluent for exosome extraction, comprising the following steps: a) removing endogenous exosomes from animal serum to obtain treated animal serum; b) mixing the obtained treated animal serum with buffer, trehalose, and preservative according to the specified amounts, and then adding water to make up to the final volume.

[0024] In this invention, the specific method for removing endogenous exosomes from animal serum is to centrifuge the animal serum at 100,000×g for more than 3 hours to remove endogenous exosomes and obtain the treated animal serum.

[0025] The third objective of this invention is achieved by the following technical solution: a quality control product for exosome extraction, comprising a positive control working solution and a negative control working solution; the positive control working solution comprises a labeled exosome standard and a diluent, and the negative control working solution comprises a labeled matrix and a diluent, wherein the diluent is the aforementioned diluent.

[0026] The positive control of this invention simulates the capture efficiency of real exosomes, while the negative control provides a free protein control, which can effectively evaluate the ability of different exosome extraction methods to distinguish between vesicles and free molecules.

[0027] Preferably, at least one of acridinium ester derivative AE-NHS, horseradish peroxidase HRP, and alkaline phosphatase AP is used for labeling exosomes and matrix.

[0028] Preferably, the matrix is ​​a protein; the protein is at least one of a protein monomer, a protein antibody, or a protein particle, wherein the size of the protein monomer and the protein antibody is less than 10 nm, the protein monomer is albumin, the protein antibody is an immunoglobulin, and the protein particle comprises a particulate structure composed of protein / lipid / nucleic acid, such as albumin aggregates, lipoprotein particles, etc.

[0029] The main factors affecting the purity of exosome extraction from blood include protein particles (albumin aggregates, lipoprotein particles, etc.) and free soluble proteins (including albumin, immunoglobulins, etc.). Single or mixed proteins / protein particles can be used as negative standards.

[0030] Preferably, the concentration of the positive control working solution is 0.1~5×10⁻⁶. 7 particles / mL.

[0031] Preferably, the concentration of the positive control working solution is 0.1~2×10⁻⁶. 7 particles / mL, for example, the concentration of the positive control working solution is 0.5 × 10⁻⁶. 7 particles / mL.

[0032] Preferably, the concentration of the negative control working solution is 0.01–0.1 μg / mL.

[0033] Preferably, the concentration of the negative control working solution is 0.05–0.1 μg / mL, for example, the concentration of the negative control working solution is 0.07 μg / mL.

[0034] The fourth objective of this invention is achieved through the following technical solution: a method for preparing the quality control product of the exosome extraction. The preparation method of the positive control working solution includes the following steps: A1. Obtain exosomes, label them with the aforementioned marker, and purify them to obtain labeled exosome standards; A2. Dilute the labeled exosome standard with the diluent to obtain the positive control working solution; The preparation method of the negative control working solution includes the following steps: B1. Directly couple the matrix to the marker, remove the free marker, and obtain the labeled matrix; B2. Dilute the labeled matrix with the diluent to obtain the negative control working solution.

[0035] Preferably, in step A1, the method for obtaining exosomes can be by purchasing or by cell culture extraction. When extracting exosomes by cell culture, in order to increase the yield of exosomes, a metal ion chelating agent or 0.5%-1% of fetal bovine serum that has been subjected to ultracentrifugation and exosome removal can be added.

[0036] The metal ion chelating agent is, for example, 2-4 mM EDTA (ethylenediaminetetraacetic acid).

[0037] Preferably, in steps A1 and B1, the marker used is at least one of acridinium ester derivative AE-NHS, horseradish peroxidase HRP, and alkaline phosphatase AP.

[0038] Preferably, the labeling is performed using a direct chemical coupling method. The labeling agent used is at least one of acrid ester derivative AE-NHS, horseradish peroxidase HRP, and alkaline phosphatase AP, which is applied to the surface of positive / negative standards.

[0039] The fifth objective of this invention is achieved through the following technical solution: the application of the quality control material for exosome extraction in evaluating the exosome extraction method.

[0040] The quality control materials of this invention can be applied to the performance evaluation and routine quality control of various exosome extraction protocols. By applying a specific extraction method to the exosome extraction quality control materials (including negative and positive controls), and calculating and comparing the detection values ​​(luminescence value or absorbance) of samples before and after treatment, key performance indicators such as extraction efficiency and purity of the method can be quantitatively evaluated, thereby reflecting the product quality of the corresponding exosome extraction kit. Furthermore, by repeatedly extracting mixed samples of positive and negative controls, the repeatability of different exosome extraction kits can be effectively evaluated.

[0041] The sixth objective of this invention is achieved by the following technical solution: a method for evaluating the performance of an exosome extraction kit, wherein the quality control material for the exosome extraction is used to evaluate the performance of the exosome extraction kit.

[0042] Preferably, the performance includes the extraction efficiency, extraction purity, and extraction repeatability of the exosome extraction kit.

[0043] Preferably, the evaluation of the extraction efficiency and / or purity of the exosome extraction kit includes the following steps: C1. Use the exosome extraction kit to be evaluated to extract exosomes from the positive control working solution and the negative control working solution, respectively. C2. Test the luminescence or absorbance of the positive control working solution and the negative control working solution before extraction and after extraction using the extraction kit as test values. C3. Based on the test values ​​obtained in step C2, calculate the exosome extraction efficiency and exosome extraction purity; C4. Based on the extraction efficiency and extraction purity calculated in step C3, comprehensively evaluate the performance of the exosome extraction kit in terms of extraction efficiency and extraction purity.

[0044] The quality control products of this invention are applicable to detection technologies such as chemiluminescence or ELISA; a pairing system of negative and positive quality control products is established to achieve routine quality control of exosome extraction reagents, and at the same time, the recovery rate and extraction purity of different exosome extraction kits can be evaluated.

[0045] Preferably, in step C2, the luminescence values ​​of the positive control working solution stock solution, the positive control working solution extract, the negative control working solution stock solution, and the negative control working solution extract are determined using chemiluminescence method, or the absorbance of the positive control working solution stock solution, the positive control working solution extract, the negative control working solution stock solution, and the negative control working solution extract is determined using ELISA method.

[0046] Preferably, in step C3, the formula for calculating the extraction efficiency is: , In the formula: —Average test value of the working solution stock solution for positive control samples; —Average test value of the working solution extract of the positive control sample; —The volume of the working solution stock solution for positive control samples; —Volume of extract collected from positive control working solution; —Extraction efficiency.

[0047] Preferably, in step C3, the formula for calculating the purity is: , In the formula: —Average test value of the original working solution for negative control samples; —Average test value of the working solution extract of the negative control sample; —The volume of the negative control working solution stock solution; —Volume of negative control working solution extract collected; —Purity of extraction.

[0048] Preferably, the evaluation of extraction repeatability includes evaluating the repeatability of extraction for the same sample and the repeatability for different samples using the coefficient of variation (CV). The method for evaluating the repeatability of extracting from the same sample includes the following steps: D1. Mix the positive control working solution and the negative control working solution evenly at a certain volume ratio; D2. Use exosome extraction method to extract from no less than 20 of the above mixed samples of the same proportion to obtain mixed quality control extract; D3. Measure the luminescence value or absorbance of the mixed quality control extract and calculate the average value of the test results; D4. Calculate the standard deviation and coefficient of variation To evaluate the repeatability of extraction from the same sample; The method for testing the repeatability of extracting different samples includes the following steps: E1. Mix the positive control working solution and the negative control working solution evenly at a certain volume ratio; E2. Use exosome extraction method to extract from no less than 20 of the above mixed standard samples in different proportions to obtain mixed quality control extract; E3. Measure the luminescence value or absorbance of the mixed quality control extract and calculate the average value of the test results; E4. Calculate the standard deviation and coefficient of variation To evaluate the repeatability of extraction from different samples.

[0049] Preferably, in steps D1 and E1, the positive control working solution and the negative control working solution are mixed at a volume ratio of 1:1-5.

[0050] Preferably, in steps D3 and E3, the luminescence value of the mixed quality control extract is determined by chemiluminescence method, and / or the absorbance of the mixed quality control extract is determined by ELISA method.

[0051] Preferably, in steps D4 and E4, the formula for calculating repeatability is as follows: ,in , In the formula: n —Determine the number of repetitions; —The value of the i-th measurement; —The average value of the measurement results; —Standard deviation; —Coefficient of variation.

[0052] The present invention has the following technical effects: The quality control diluent for exosome extraction of the present invention has a simple composition and is used to dilute quality control samples, thereby achieving stable detection of proteins.

[0053] The quality control materials for exosome extraction of this invention are suitable for detection technologies such as chemiluminescence or ELISA; they are compatible with different laboratories and instrument platforms, improving the comparability of clinical test results, establishing a pairing system of negative and positive quality control materials, and realizing routine quality control of exosome extraction protocols in clinical testing. Simultaneously, they can evaluate the recovery rate and purity verification of different exosome extraction methods. Compared with traditional exosome characterization methods (such as electron microscopy, Western blot, and nanoflow cytometry), they are simple to operate, provide stable quality, and are more suitable for client-side operation, possessing significant clinical application value and market promotion potential. This provides technical support for the standardized detection of exosomes, promoting their clinical application in areas such as early cancer diagnosis and treatment monitoring.

[0054] The present invention discloses a method for preparing quality control samples of exosome extraction, comprising exosome / vesicle acquisition and label conjugation. Through strict control of the preparation process and parameters (such as centrifugation conditions and labeling ratio), batch-to-batch consistency of the standard is ensured, and experimental errors are reduced. The aim is to provide a standardized and reproducible method for preparing quality control samples of exosome extraction, which is simple to operate, has good reproducibility, and is easy to scale up for production.

[0055] The exosome extraction quality control materials of this invention can be applied to the performance evaluation and routine quality control of various exosome extraction protocols. By applying a specific extraction method to the exosome extraction quality control materials (including negative and positive controls), and calculating and comparing the detection values ​​of samples before and after treatment, key performance indicators such as extraction efficiency and purity of the method can be quantitatively evaluated, thereby reflecting the product quality of the corresponding exosome extraction kit. Furthermore, by repeatedly extracting mixed samples of positive and negative controls, this method can also effectively evaluate the repeatability of different exosome extraction methods. Attached Figure Description

[0056] Figure 1 The results of NTA analysis of exosomes extracted from cell supernatant in Example 4 of this invention; Figure 2 The graph shows the extraction efficiency (Ee) and extraction purity (Ep) of exosomes extracted by precipitation method in Example 6 of this invention. Figure 3 The graph shows the extraction efficiency (Ee) and extraction purity (Ep) of exosomes extracted by ultracentrifugation in Example 7 of this invention. Figure 4The graph shows the extraction efficiency (Ee) and extraction purity (Ep) of exosomes extracted by ultrafiltration, size exclusion chromatography, and membrane affinity methods in Examples 8-10 of this invention. Detailed Implementation

[0057] The present invention will now be described in detail with reference to embodiments. This section provides a general description of the materials and experimental methods used in the experiments of the present invention. Although many materials and methods of operation used to achieve the objectives of the present invention are known in the art, the present invention is described in as much detail as possible herein. It will be apparent to those skilled in the art that, unless otherwise specified herein, the materials, equipment, and methods of operation used in the present invention are known in the art.

[0058] The materials used in the experiment are described below: The preparation method of the positive control working solution in this embodiment of the invention is as follows: PC300: A composite liquid biological preservative, the main components of which are 2-methyl-4-isothiazolin-3-one (MCI) and 5-chloro-2-methyl-4-isothiazolin-3-one (CMCI).

[0059] Acridine acid-NHS ester (Catalog No.: HS-11015005) (Helison Biotechnology Co., Ltd.)

[0060] Horseradish peroxidase (HRP).

[0061] Alkaline phosphatase (AP).

[0062] (1) Exosome extraction a) Use serum-free HEK293F cell culture medium and culture in suspension at 37 ℃, 5% CO2, and 120 rpm to maintain a cell density of 0.5~5×10⁻⁶ cells / year. 6 cells / mL; b) Collect exosomes; c) When using, add PBS to prepare a concentration range of 0.5~5×10⁻⁶. 11 positive standard reagent particles / mL; In step a), to increase exosome production, a metal ion chelating agent or 0.5% low-concentration serum can be added (this step is optional). (2) Exosome probe labeling (using acridine ester AE as an example) AE was labeled on the surface of positive / negative standards using a direct chemical coupling method. To improve labeling efficiency, an acrid ester derivative (AE-NHS) was selected for exocrine labeling, including the following steps: a) Positive standards were dispersed in PBS at pH 8.0 at concentrations ranging from 0.1 to 2 × 10⁻⁶. 7 particles / mL; b) AE-NHS is dissolved in dimethyl sulfoxide (DMSO) solution at a concentration range of 1-20 mM; c) Mix the positive standard solution and AE-NHS at a ratio of not less than 1:3 (w / w) (standard protein content (μg): AE-NHS (μg)); d) Reactions should be carried out in the dark for 0.5 to 2 hours; e) Add 10% lysine in 0.2 M NaHCO3 (pH=9.0) solution and incubate for 15-30 min to terminate the reaction; f) Purification was performed using a dialysis bag (25 kD) to remove free AE-NHS, yielding labeled exosome standards; (3) Preparation of working solution for exosome positive control: The labeled exosome standards were diluted to 0.1–2 × 10⁻⁶ using the quality control diluent. 7 Particles / mL, for storage.

[0063] The method for preparing the negative control sample in this embodiment of the invention is as follows: The main factors affecting the purity of exosome extraction from blood include lipoprotein particles and free soluble proteins (including albumin, immunoglobulins, and protein particles). Mixed proteins or protein particles can be used as negative standards.

[0064] The preparation process is similar to that of the positive control sample, but with the following differences: a) The marker is coupled to the matrix at a molar ratio of 1:1; b) After coupling, the free label is removed by ultrafiltration or gel filtration chromatography; c) Dilute the labeled matrix to 0.01-0.1 ug / ml using the quality control diluent and store it.

[0065] In the above methods, the diluent includes: 10%-90% (v / v) animal serum; 10%-30% (v / v) phosphate buffer; 0.5%-2% (w / v) trehalose; 0.1%-1% (v / v) PC300; and the remainder is water.

[0066] The method for preparing the quality control diluent for exosome extraction includes the following steps: a) Remove endogenous exosomes from animal serum to obtain processed animal serum; b) Mix the obtained treated animal serum with phosphate buffer, trehalose, and PC300 according to the specified amounts, and then add water to make up the volume.

[0067] The method of using positive and negative quality control samples in this embodiment of the invention includes the following steps: Positive control working solution and negative standard working solution were selected. After extraction using a specific exosome extraction method, the luminescence values ​​of the samples before and after extraction were directly tested using a fully automated chemiluminescence immunoassay analyzer, or the absorbance of the samples before and after extraction was detected by ELISA. The extraction efficiency, extraction purity and extraction repeatability were calculated to evaluate the applicability of the exosome extraction method in clinical testing.

[0068] (1) Extraction efficiency (Ee) The extraction efficiency test method and calculation formula are as follows: a) Analyze the working solutions of positive control samples labeled with three or more detection probes using chemiluminescence or ELISA, and calculate the average value of the test results. ); b) The working solution of the positive control sample was extracted three or more times using the exosome extraction method; c) Analyze the positive control extract using chemiluminescence or ELISA, and calculate the average value of the test results. ); d) Calculate Ee according to formula (1): (1) In the formula: —The average value of the stock solution test results for the positive control sample; —The average value of the working solution extract of the positive control sample; —The volume of the working solution stock solution for positive control samples; —Volume of extract collected from positive control working solution; —Extraction efficiency.

[0069] (2) Extraction purity (Ep) The extraction purity test method and calculation formula are as follows: a) Measure the working solutions of negative control samples labeled with three or more detection probes using chemiluminescence or ELISA, and calculate the average value of the test results. ); b) The negative control working solution was extracted three or more times using the exosome extraction method; c) The negative control extract was analyzed using chemiluminescence or ELISA, and the average value of the test results was calculated. ); d) Calculate Ep according to formula (2): (2) In the formula: —The average value of the negative control working solution stock solution test; —The average value of the working solution extract of the negative control sample; —The volume of the negative control working solution stock solution; —Volume of negative control working solution extract collected; —Purity of extraction.

[0070] (3) Extracting repeatability Extraction repeatability includes repeatability extraction from the same sample and repeatability extraction from different samples. It is generally evaluated using the coefficient of variation (CV). The test method and calculation formula are as follows: Extracting repeatability from the same sample: a) Mix the positive control working solution and the negative control working solution at a fixed ratio (not less than 1:1 and not more than 5:1; v / v). b) Use exosome extraction methods to extract from no fewer than 20 of the above-mentioned mixed quality control working solution samples with the same proportion; c) Use chemiluminescence or ELISA to analyze the extract of the mixed quality control working solution and calculate the average value of the test results; Extract repeatability from different samples: d) Mix the positive control working solution and the negative control working solution in different ratios (not less than 1:1 and not more than 5:1; v / v); e) Use exosome extraction methods to extract from no fewer than 20 of the above-mentioned mixed quality control working solution samples with different proportions. f) The extract of the mixed quality control working solution was tested using chemiluminescence or ELISA, and the average value of the test results was calculated. g) Calculate the standard deviation according to formulas (3) and (4) respectively. ) and coefficient of variation ( ).

[0071] (3) (4) In the formula: n —Determine the number of repetitions; —The value of the i-th measurement; —The average value of the measurement results; —Standard deviation; —Coefficient of variation.

[0072] Example 1 A quality control diluent for exosome extraction comprises: 90% (v / v) bovine serum; 5% (v / v) 1xPBS solution; 0.5% (w / v) trehalose; 0.1% (v / v) PC300; and the balance being water.

[0073] The method for preparing the quality control diluent for exosome extraction includes the following steps: a) removing endogenous exosomes by ultracentrifuging bovine serum at 100,000×g for 6h to obtain treated bovine serum; b) mixing the obtained treated bovine serum with 1xPBS solution, trehalose, and PC300 according to the measured amount, and then adding water to make up to the final volume.

[0074] Example 2 A quality control diluent for exosome extraction comprises: 50% (v / v) sheep serum; 20% (v / v) 1xPBS solution; 1% (w / v) trehalose; 0.5% (v / v) PC300; and the balance being water.

[0075] The method for preparing the quality control diluent for exosome extraction includes the following steps: a) removing endogenous exosomes by ultracentrifuging sheep serum at 100,000×g for 6h to obtain treated sheep serum; b) mixing the obtained treated sheep serum with 1×PBS solution, trehalose, and PC300 according to the measured amount, and then adding water to make up to the final volume.

[0076] Example 3 A quality control diluent for exosome extraction comprises: 10% (v / v) rabbit serum; 30% (v / v) 1×PBS solution; 2% (w / v) trehalose; 1% (v / v) PC300; and the balance being water.

[0077] The method for preparing the quality control diluent for exosome extraction includes the following steps: a) removing endogenous exosomes by ultracentrifuging rabbit serum at 100,000×g for 6h to obtain treated rabbit serum; b) mixing the obtained treated rabbit serum with 1xPBS solution, trehalose, and PC300 according to the measured amount, and then adding water to make up to the final volume.

[0078] Example 4 The preparation method of the positive control working solution includes the following steps: Cell supernatant exosome extraction: 1. Using serum-free medium specifically for HEK293F cells, HEK293F cells were cultured in suspension at 37 ℃, 5% CO2, and 120 rpm. The cells were cultured until the cell density reached 5 × 10⁶ cells / year. 6 When the cell count was 4.00 × g, the cell supernatant was collected by centrifugation for 30 min. 2. Exosomes were collected by ultracentrifugation. The pretreated HEK293F cell culture supernatant was centrifuged at 100,000×g for 100 min at 4 °C using an ultracentrifuge. 3. Discard the supernatant, resuspend the precipitate with PBS, and centrifuge again at 100,000×g for 120 min at 4 ℃; 4. Discard the supernatant, resuspend the precipitate in 100 μL PBS, which is the positive standard; 5. The cell supernatant exosomes were diluted 100-fold with PBS, and the collected exosome concentration was 5.91 × 10⁻⁶ by NTA assay. 8 particles / mL, such as Figure 1 As shown.

[0079] N-hydroxysuccinimide-activated acridine ester (AE-NHS) labeled positive standard: 1. Dilute the positive standard to 2×10⁻⁶ using PBS. 7 particles / mL; 2. N-hydroxysuccinimide-activated acridine ester (AE-NHS) was dissolved in dimethyl sulfoxide (DMSO) solution at a concentration of 1.25 mM; 3. Mix the positive standard solution and AE-NHS at a ratio of 1:3 (w / w) (standard protein content (μg): AE-NHS (μg)) and react in the dark for 30 min; 4. Add 10% lysine in 0.2 M NaHCO3 (pH=9.0) solution and incubate for 30 min to terminate the reaction; 5. Purify using a dialysis bag (25 kD) to remove free AE-NHS; Preparation of working solution for exosome positive control: The labeled positive standards were diluted to 0.5 × 10⁻⁶ using the diluents from Examples 1, 2, and 3, respectively. 7 Particles / mL were collected and stored to obtain positive control working solution 1, positive control working solution 2 and positive control working solution 3.

[0080] Example 5 The preparation method of the negative control working solution includes the following steps: N-hydroxysuccinimide-activated acridine ester (AE-NHS) labeled negative standard: 1. Dissolve BSA (bovine serum albumin) in PBS to a concentration of 0.07 ug / ml; 2. N-hydroxysuccinimide-activated acridine ester (AE-NHS) was dissolved in dimethyl sulfoxide (DMSO) solution at a concentration of 1.25 mM; 3. Mix BSA (bovine serum albumin) solution and AE-NHS at a ratio of 40:1 (w / w) (standard protein content (μg): AE-NHS (μg)) and react in the dark for 30 min; 4. Add 10% lysine in 0.2 M NaHCO3 (pH=9.0) solution and incubate for 30 min to terminate the reaction; 5. Purify using a dialysis bag (8 kD) to remove free AE-NHS; 6. Dilute the labeled negative standards to 0.07 ug / ml using the diluents from Examples 1, 2, and 3, respectively, and store them to obtain negative control working solution 1, negative control working solution 2, and negative control working solution 3.

[0081] Example 6 The extraction efficiency (Ee) and extraction purity (Ep) of exosomes extracted by precipitation method were tested using negative control working solution 1-3 and positive control working solution 1-3.

[0082] Experimental Materials and Methods: 1. Using the cell supernatant exosome extraction kit, the negative control working solution 1-3 and the positive control working solution 1-3 were extracted three times according to the instructions.

[0083] 2. The extract was tested using the Shine i2910 fully automated chemiluminescence immunoassay analyzer from Shenzhen Yingkai Biotechnology Co., Ltd.

[0084] 3. The extraction efficiency (Ee) and extraction purity (Ep) of the precipitation method were calculated.

[0085] Experimental results showed that the extraction efficiencies (Ee) of the precipitation method were 85.8%, 82.7%, and 83.2%, respectively, and the extraction purities (Ep) were 92.3%, 91.6%, and 91.5% (corresponding to positive control working solutions 1-3 and negative control working solutions 1-3), respectively. Figure 2 As shown, the negative and positive control samples of the present invention are compatible with the precipitation method for exosome extraction and can be used as a standardized reference system for quantitative characterization and efficacy verification of the precipitation method extraction process.

[0086] Example 7 The extraction efficiency (Ee) and purity (Ep) of exosomes extracted by ultracentrifugation were tested using negative control working solution 1-3 and positive control working solution 1-3. Experimental Materials and Methods: 1. The extraction efficiency (Ee) and purity (Ep) of exosomes were tested using ultracentrifugation with negative and positive quality controls. The specific steps for exosome extraction are as follows: a) Centrifuge at 100,000 × g for 100 min at 4 ℃; b) Discard the supernatant, resuspend the precipitate with PBS, and centrifuge again at 100,000×g for 120 min at 4 °C; c) Discard the supernatant and resuspend the precipitate in 100 μL PBS; 2. The extract was tested using the Shine i2910 fully automated chemiluminescence immunoassay analyzer from Shenzhen Yingkai Biotechnology Co., Ltd.

[0087] 3. The extraction efficiency (Ee) and extraction purity (Ep) of ultracentrifugation were calculated.

[0088] Experimental results showed that the extraction efficiencies (Ee) were 30.7%, 29.6%, and 27.7%, respectively, and the extraction purities (Ep) were 96.2%, 97.9%, and 97.7% (corresponding to positive control working solutions 1-3 and negative control working solutions 1-3), respectively. Figure 3 As shown, the results demonstrate that the quality control samples of this invention can be used to evaluate ultracentrifugation extraction processes.

[0089] Examples 6 and 7 respectively used three positive control working solutions and three negative control working solutions to quantitatively evaluate the purity and extraction efficiency of exosome extraction by precipitation method and ultracentrifugation method, verifying that the quality control products and quality control diluents of the present invention can be adapted to mainstream exosome extraction methods with different principles and have good universal applicability.

[0090] Example 8 The extraction efficiency (Ee) and extraction purity (Ep) of exosomes were tested by using negative control working solution 1 and positive control working solution 1.

[0091] Experimental Materials and Methods: 1. The extraction efficiency (Ee) and purity (Ep) of exosomes extracted using ultrafiltration were tested using negative and positive quality controls. The specific steps for extracting exosomes are as follows: a) The prepared negative and positive quality control samples were extracted three times using an ultracentrifuge, as follows: b) Use 100 kDa ultrafiltration centrifuge tubes and pre-wet the ultrafiltration membrane with sterile PBS; c) Take the working solution of each quality control sample and add it into an ultrafiltration tube. Centrifuge at 4℃ and 3000×g for 10 to 20 minutes to concentrate the sample by ultrafiltration and remove small molecule free impurities and soluble proteins. d) After concentration, add sterile PBS to the ultrafiltration tube for replacement washing, repeat the washing 2-3 times to further remove free proteins and small molecule interfering substances; e) Collect the concentrate in the ultrafiltration tube, which is the exosome extract obtained by ultrafiltration.

[0092] 2. The extract was tested using the Shine i2910 fully automated chemiluminescence immunoassay analyzer from Shenzhen Yingkai Biotechnology Co., Ltd.

[0093] 3. The extraction efficiency (Ee) and extraction purity (Ep) of ultrafiltration were calculated.

[0094] like Figure 4 As shown, the experimental results indicate that ultrafiltration has an extraction efficiency (Ee) of 82.9% and an extraction purity (Ep) of 85.9% for both negative and positive quality controls.

[0095] Example 9 Extraction efficiency (Ee) and extraction purity (Ep) of exosomes were tested using size exclusion chromatography column method by using negative control working solution 1 and positive control working solution 1.

[0096] Experimental Materials and Methods: 1. Using a size exclusion purification column specifically designed for exosomes, extract the negative control working solution 1 and the positive control working solution 1 three times each, following the product instructions.

[0097] 2. The extract was tested using the Shine i2910 fully automated chemiluminescence immunoassay analyzer from Shenzhen Yingkai Biotechnology Co., Ltd.

[0098] 3. The extraction efficiency (Ee) and extraction purity (Ep) of the size exclusion chromatography column method were calculated.

[0099] like Figure 4 As shown, the experimental results indicate that the size exclusion purification column method has an extraction efficiency (Ee) of 35.1% and an extraction purity (Ep) of 92.7% for both negative and positive quality controls.

[0100] Example 10 The extraction efficiency (Ee) and extraction purity (Ep) of exosomes were tested by using negative control working solution 1 and positive control working solution 1.

[0101] Experimental Materials and Methods: 1. Using an exosome membrane affinity purification kit, the negative control working solution 1 and the positive control working solution 1 were extracted three times each, according to the kit instructions.

[0102] 2. The extract was tested using the Shine i2910 fully automated chemiluminescence immunoassay analyzer from Shenzhen Yingkai Biotechnology Co., Ltd.

[0103] 3. The extraction efficiency (Ee) and extraction purity (Ep) of the membrane affinity method were calculated.

[0104] like Figure 4 As shown, the experimental results indicate that the membrane affinity method has an extraction efficiency (Ee) of 67.2% and an extraction purity (Ep) of 94.2% for both negative and positive quality controls.

[0105] Example 11 The exosome extraction methods applicable in Examples 6-10 were tested using a mixed solution of negative control working solution 1 and positive control working solution 1 to assess the extract repeatability of the same control working solution sample and the extract repeatability of different control samples, and the CV value was calculated. The preparation method for the control working solution solution used to test the extract repeatability of the same control working solution sample was as follows: positive control working solution 1 and negative control working solution 1 were mixed in a 1:1 ratio, and then diluted to 2 × 10⁻⁶ with control diluent. 7 The particle / mL test method for preparing the quality control solution to test the reproducibility of extraction from different quality control samples is as follows: positive quality control working solution 1 and negative quality control working solution 1 are mixed at different ratios of 1:1 to 1:5, and then diluted with quality control diluent to 2×10⁻⁶. 7 particles / mL.

[0106] Following the experimental protocols of Examples 6-10, extraction was repeated 10 times each, and the chemiluminescence signals after extraction were recorded and the standard deviations were calculated. ) and coefficient of variation ( ).

[0107] The experimental results are shown in Table 1. The membrane affinity method and size exclusion chromatography column method have good extraction repeatability for quality control samples, indicating that the patented method can be used to evaluate the stability of exosome extraction methods / extraction kits.

[0108] Table 1. CV values ​​of different exosome extraction methods for the same / different quality control samples.

[0109] Examples 6-10 used precipitation, ultracentrifugation, ultrafiltration, size exclusion chromatography, and membrane affinity methods to simultaneously extract and test the negative and positive quality controls prepared in this invention. The results showed that this set of negative and positive quality controls is applicable to exosome extraction processes based on different principles and can be used to evaluate the extraction specificity, separation and differentiation effects between vesicles and free impurity molecules, and detection repeatability of various exosome extraction methods.

[0110] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A quality control diluent for exosome extraction, characterized in that, include: 10%-90% (v / v) animal serum; 10%-30% (v / v) buffer; 0.5%-2% (w / v) trehalose; 0.1%-1% (v / v) preservative; balance water.

2. The quality control diluent for exosome extraction according to claim 1, characterized in that, The buffer solution is a phosphate buffer solution; Preferably, the preservative is PC300; Preferably, the animal serum is bovine serum, sheep serum, donkey serum, horse serum, rabbit serum, pig serum, or chicken serum.

3. A method for preparing a quality control diluent for exosome extraction as described in claim 1 or 2, characterized in that, The process includes the following steps: a) removing endogenous exosomes from animal serum to obtain treated animal serum; b) mixing the obtained treated animal serum with buffer, trehalose, and preservative according to the prescribed amounts, and then adding water to make up to the final volume.

4. A quality control product for exosome extraction, characterized in that, It includes a positive control working solution and a negative control working solution; the positive control working solution includes a labeled exosome standard and a diluent, and the negative control working solution includes a labeled matrix and a diluent, wherein the diluent is the diluent described in claim 1 or 2 or the diluent prepared by the preparation method of claim 3.

5. The quality control product for exosome extraction according to claim 4, characterized in that, At least one of acridinium ester derivative AE-NHS, horseradish peroxidase HRP, and alkaline phosphatase AP was used to label exosomes and matrix. And / or, the matrix is ​​a protein; And / or, the concentration of the positive control working solution is 0.1~5×10⁻⁶. 7 particles / mL; the concentration of the negative control working solution is 0.01–0.1 μg / mL; Preferably, the protein is at least one of a protein monomer, a protein antibody, or a protein particle.

6. A method for preparing a quality control product of exosome extraction as described in claim 4 or 5, characterized in that, The preparation method of the positive control working solution includes the following steps: A1. Obtain exosomes, label them with the aforementioned marker, and purify them to obtain labeled exosome standards; A2. Dilute the labeled exosome standard with the diluent to obtain the positive control working solution; The preparation method of the negative control working solution includes the following steps: B1. Directly couple the matrix to the marker, remove the free marker, and obtain the labeled matrix; B2. Dilute the labeled matrix with the diluent to obtain the negative control working solution.

7. A method for evaluating the performance of an exosome extraction method or kit, characterized in that: The performance of the exosome extraction method or exosome extraction kit was evaluated using the quality control material of exosome extraction as described in claim 4 or 5 or the quality control material of exosome extraction prepared by the preparation method as described in claim 6. Preferably, the performance of the exosome extraction kit includes the extraction efficiency, extraction purity, and extraction repeatability.

8. The method for evaluating the performance of an exosome extraction method or kit according to claim 7, characterized in that, The evaluation of the extraction efficiency and purity of the exosome extraction kit includes the following steps: C1. Use the exosome extraction kit to be evaluated to extract exosomes from the positive control working solution and the negative control working solution, respectively. C2. Test the luminescence or absorbance of the positive control working solution and the negative control working solution before extraction and after extraction using the extraction kit as test values. C3. Based on the test values ​​obtained in step C2, calculate the exosome extraction efficiency and exosome extraction purity; C4. Based on the extraction efficiency and extraction purity calculated in step C3, comprehensively evaluate the performance of the exosome extraction kit in terms of extraction efficiency and extraction purity; Preferably, in step C2, the luminescence values ​​of the positive control working solution stock solution, the positive control working solution extract, the negative control working solution stock solution, and the negative control working solution extract are determined using chemiluminescence immunoassay. And / or, the absorbance of the positive control working solution stock solution, the positive control working solution extract, the negative control working solution stock solution, and the negative control working solution extract was determined by ELISA.

9. The method for evaluating the performance of the exosome extraction kit according to claim 8, characterized in that, In step C3, the formula for calculating the extraction efficiency is: , In the formula: —Average test value of the working solution stock solution for positive control samples; —Average test value of the working solution extract of the positive control sample; —The volume of the working solution stock solution for positive control samples; —Volume of extract collected from positive control working solution; Extraction efficiency; And / or, in step C3, the formula for calculating the purity is: , In the formula: —Average test value of the original working solution for negative control samples; —Average test value of the working solution extract of the negative control sample; —Volume of the negative control working solution stock solution; —Volume of negative control working solution extract collected; —Purity of extraction.

10. The method for evaluating the performance of an exosome extraction method or kit according to claim 7, characterized in that, The evaluation of extraction repeatability includes extraction repeatability for the same sample and extraction repeatability for different samples, which is evaluated using the coefficient of variation (CV). The method for evaluating the repeatability of extracting from the same sample includes the following steps: D1. Mix the positive control working solution and the negative control working solution evenly at a certain volume ratio; D2. Use exosome extraction method to extract from no less than 20 mixed samples of the same proportion as above to obtain mixed quality control extract; D3. Measure the luminescence value or absorbance of the mixed quality control extract and calculate the average value of the test results; D4. Calculate the standard deviation and coefficient of variation To evaluate the repeatability of extraction from the same sample; The method for testing the repeatability of extracting different samples includes the following steps: E1. Mix the positive control working solution and the negative control working solution evenly at a certain volume ratio; E2. Use exosome extraction method to extract from no less than 20 of the above mixed standard samples in different proportions to obtain mixed quality control extract; E3. Measure the luminescence value or absorbance of the mixed quality control extract and calculate the average value of the test results; E4. Calculate the standard deviation and coefficient of variation To evaluate the repeatability of extraction for different samples; Preferably, in steps D1 and E1, the positive control working solution and the negative control working solution are mixed at a volume ratio of 1:1-5. Preferably, in steps D3 and E3, the luminescence value of the mixed quality control extract is determined by chemiluminescence method, and / or the absorbance of the mixed quality control extract is determined by ELISA method; Preferably, in steps D4 and E4, the formula for calculating repeatability is as follows: ,in , In the formula: n —Determine the number of repetitions; —The value of the i-th measurement; —The average value of the measurement results; —Standard deviation; —Coefficient of variation.

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