A kit for detecting the content of exosome CD82 protein

Abstract

The application belongs to the field of biological detection, and particularly relates to a kit for detecting the content of CD82 protein in exosomes. The kit comprises a standard sample, an enzyme-labeled plate coated with CA1 antibody, a working solution of HRP-labeled CA2 antibody, a buffer, a color developing solution and a reaction termination solution. The kit provided by the application can specifically detect the content of CD82 protein in blood exosomes, and can be used as an auxiliary diagnostic means for high-expression CD82 protein content gastric cancer. The kit has the advantages of simple detection method, strong accuracy, good stability and the like, and has a good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biological detection, specifically relating to a kit for detecting the content of exosome CD82 protein. Background Technology

[0002] Gastric cancer is a malignant tumor originating from renal tubular epithelial cells. It ranks second in incidence and third in mortality among malignant tumors in my country. The pathogenesis of gastric cancer is complex, and its incidence is increasingly affecting younger people. Due to the insensitivity of early diagnosis, many gastric cancer patients are diagnosed at an advanced stage, missing the optimal time for surgery. Current chemotherapy regimens for gastric cancer also lack targeted therapy and have many side effects. Early diagnosis and treatment are crucial to reducing the mortality rate of gastric cancer patients; therefore, finding new biomarkers and targets for gastric cancer is particularly important. Previous studies have shown that various biomarkers, such as CA19-9 and CEA, can serve as potential diagnostic tools for gastric cancer, but these biomarkers have low specificity. Among them, CA19-9 antigen is currently the most widely used tumor biomarker in clinical practice, but its specificity for diagnosing gastric cancer is only 70%-80%, which is insufficient to completely solve the problem. Therefore, there is an urgent need for a detection technology capable of detecting early-stage gastric cancer.

[0003] Blood exosomes are microspheres (50-150 nm) with cell membrane-like structures, secreted by cells into the peripheral blood and possessing biological activity. As intercellular communication mediators, exosomes promote physiological or pathological changes in cells by delivering cellular "cargo." CD82 is a transmembrane protein, an evolutionarily conserved molecule expressed in various tissue types. Studies have demonstrated the potential of exosomal CD82 protein as a marker for pancreatic cancer in proteomics. These findings are significant for understanding emerging tumor markers and tumorigenesis mechanisms and warrant further investigation. The potential of exosomal CD82 protein as a marker for gastric cancer has not yet been reported, and there are currently no commercially available products targeting exosomal CD82 protein for gastric cancer detection. Summary of the Invention

[0004] To overcome the shortcomings of the prior art, one of the objectives of this invention is to provide a kit for detecting the content of CD82 protein in exosomes.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] A kit for detecting CD82 protein content in exosomes, the kit comprising standards, an ELISA plate coated with CA1 antibody, HRP-labeled CA2 antibody working solution, buffer, chromogenic solution, and reaction termination solution;

[0007] The CA1 antibody is a monoclonal antibody against CD82 protein. The heavy chain amino acid sequence of the monoclonal antibody CA1 is shown in SEQ ID NO.1, and the light chain amino acid sequence is shown in SEQ ID NO.2.

[0008] The CA2 antibody is a monoclonal antibody against CD82 protein. The heavy chain amino acid sequence of the monoclonal antibody CA2 is shown in SEQ ID NO.3, and the light chain amino acid sequence is shown in SEQ ID NO.4.

[0009] Furthermore, the standard is CD82 recombinant protein.

[0010] Furthermore, the amino acid sequence of the CD82 recombinant protein is shown in SEQ ID NO.5.

[0011] Furthermore, the buffer solution is PBST; the colorimetric solution is TMB; and the reaction termination solution is sulfuric acid.

[0012] Furthermore, the concentration of the colorimetric solution is 2-3 mg / mL; the concentration of the reaction termination solution is 2-3 M.

[0013] The detection method of the kit for detecting CD82 protein content in exosomes described above includes the following steps:

[0014] (1) Take a blood sample, centrifuge it, and collect the plasma;

[0015] (2) The plasma from step (1) was separated and purified by sucrose density gradient centrifugation, and exosomes were extracted;

[0016] (3) The exosomes extracted in step (2) were lysed using an ultrasonic homogenizer;

[0017] (4) Use a kit to detect the CD82 protein content in exosomes to detect the content of CD82 in exosome lysates (3).

[0018] Further, step (3) specifically involves placing the extracted exosomes in ice water, using an ultrasonic disruptor to break down the exosomes, collecting the supernatant, and obtaining exosome lysates.

[0019] Further, the specific operation of step (4) is as follows: take out the enzyme-labeled plate coated with CA1 antibody, restore it to room temperature, wash with buffer, add the sample to be tested for incubation, and wash with buffer; add HRP-labeled CA2 antibody working solution for incubation, and wash with buffer; add chromogenic solution to the reaction well, develop color at room temperature in the dark for a period of time, and add stop solution to terminate the reaction; use an enzyme-labeled plate reader to perform dual-wavelength detection, measure the OD value at a reference wavelength of 450nm and the OD value at a reference wavelength of 630nm, and calculate the content of blood exosome CD82 protein according to the standard curve of the kit.

[0020] Compared with existing technologies, the main advantages of this invention are: This study experimentally demonstrates that blood exosomal CD82 protein is abnormally highly expressed in gastric cancer patients, indicating that exosomal CD82 protein has the potential to be a gastric cancer marker. Simultaneously, a kit for detecting exosomal CD82 protein content is provided, which can specifically detect the exosomal CD82 protein content in the blood of gastric cancer patients. As an auxiliary diagnostic method for gastric cancer with high exosomal CD82 protein expression, it has the advantages of simple detection method, high accuracy, and good stability, and has good application prospects. Attached Figure Description

[0021] Figure 1 Western blotting was used to detect CD82 protein levels in blood exosomes from gastric cancer patients and healthy individuals.

[0022] Figure 2 This is a schematic diagram of the process for a kit to detect CD82 protein content in exosomes;

[0023] Figure 3 A standard curve for a kit to specifically detect CD82 protein content in exosomals. Detailed Implementation

[0024] The technical solution of the present invention will be further described below with reference to specific embodiments. However, those skilled in the art should understand that the following embodiments are only for illustrating the present invention and should not be regarded as limiting the present invention. Specific conditions not specified in the embodiments are performed according to conventional conditions or conditions recommended by the manufacturer. Unless otherwise specified, the reagents or instruments used are all conventional products obtained through commercial channels.

[0025] Example 1

[0026] Isolation, purification, lysis, and identification of exosomes:

[0027] (1) Isolation and purification of exosomes: Blood samples from 5 patients with gastric cancer and 5 healthy individuals were collected. 5 mL of each sample was taken and thawed at 37℃. The samples were centrifuged at 3000g for 10 min to remove the precipitate, and the supernatant was transferred to a new centrifuge tube for collection. 1 mL of sucrose solution with concentration gradients of 0.05 g / mL, 0.1 g / mL, 0.2 g / mL, and 0.4 g / mL were sequentially injected into the centrifuge tubes. 2 mL of plasma was slowly added to the top layer of the sucrose solution. The tubes were then ultracentrifuged at 4℃ and 150000g for 6 h. The supernatant was aspirated and filtered through a 0.22 mm filter membrane to extract exosomes.

[0028] (2) Lysis and identification of exosomes: Take 100 μL of the centrifuge tube containing the purified exosomes from step (1) and place it in ice water. Use an ultrasonic homogenizer to break down the exosomes and collect the supernatant to obtain the exosome lysate. Transfer the lysed exosomes into centrifuge tubes and centrifuge at 4 °C and 16000 g for 60 minutes. Recover the supernatant to obtain the exosome lysate and store it at -80 °C. Take a small amount of the exosome lysate for Western blotting analysis and identification.

[0029] The results are as follows Figure 1 The image shows the Western blotting results of CD82 protein levels in exosomal exosomes from gastric cancer patients and healthy individuals. We observed higher expression levels of CD82 protein in exosomal exosomes in gastric cancer patients (lanes 1, 2, 3, 4, and 5) and lower expression levels in healthy individuals (lanes 6, 7, 8, 9, and 10), suggesting that CD82 protein levels in exosomal exosomes have the potential to be a biomarker for gastric cancer.

[0030] Example 2

[0031] Preparation of a kit for detecting CD82 protein content in blood exosomes:

[0032] S1. Obtaining monoclonal antibodies against recombinant CD82 protein:

[0033] (1) Immunization of experimental animals: Six healthy female mice in good growth condition, about 8 weeks old, were selected for immunization. CD82 recombinant protein was used as the immunogen, and one unimmunized healthy female mouse served as a negative control. The injection method was subcutaneous injection at multiple sites, with immunization once every 2 weeks for 3 consecutive immunizations. After each immunization, the antibody titer in the mouse serum was measured by enzyme-linked immunosorbent assay (ELISA). The mice with the highest antibody titer were selected for subsequent experiments.

[0034] (2) Hybridoma cell fusion: Female mice with the highest antibody titer from step (1) were euthanized by cervical dislocation, and their spleens were removed to prepare a spleen cell suspension. Myeloma cells and spleen cells were fused at a ratio of 5:1 using polyethylene glycol. Five days after cell fusion, the cells were cultured in HAT selective medium. Seven days later, the cell culture supernatant was collected, and positive hybridoma cells were screened using indirect ELISA.

[0035] (3) Subcloning and screening: The selected positive hybridoma cells were subcloned three times using the limiting dilution method. The single-clone stable cell line with the highest positive value was selected for expansion culture.

[0036] (4) Preparation of monoclonal antibodies: 1×10 6 The hybridoma cells obtained in step (3) were inoculated into the peritoneal cavity of female mice sensitized with paraffin oil. The state of the mouse abdomen was observed, and ascites was collected when the mouse abdomen was significantly distended. The antibody activity was accurately measured using a Biacore T200, and the two monoclonal antibodies with the highest activity were named CA1 and CA2. The protein concentration was determined by the BCA method, and the antibody concentration was adjusted to 5 mg / mL for later use. The antibodies were stored at -20℃.

[0037] Analysis of monoclonal antibody sequence structure: The obtained monoclonal antibodies CA1 and CA2 were further analyzed to determine their heavy chain and light chain variable region sequences. The heavy chain amino acid sequence of monoclonal antibody CA1 is shown in SEQ ID NO.1, and the light chain amino acid sequence is shown in SEQ ID NO.2; the heavy chain amino acid sequence of monoclonal antibody CA2 is shown in SEQ ID NO.3, and the light chain amino acid sequence is shown in SEQ ID NO.4.

[0038] SEQ ID NO.1:

[0039] QVQLQVSSGPTNCKPGASVKVSCKASSYNIKSYWSDWIKQKPGTLENYAYINDPENGD TNYTQSFQGKATLTADKSSSTSDMQLSSLTSEDTAVYYEARTYFRFAYLAWGTTLAVSS;

[0040] SEQ ID NO.2:

[0041] AYQMTQAAFFMSVSLGTVTVVTCKASQSVDTHVAWYQQKTGQHPKKLLYTGSTRLS GVPHARFPDRFSSAFTTDFIGTRISRVEIYYATYQGFWQSLHVPGTFGPGTNKLELK;

[0042] SEQ ID NO.3:

[0043] EVQLQQSSTELSQPGASRKISCATSGYYFSNYGIHWVRQRPGTLEEWIGEIRDKRFL STRLYSFKGKGRRFLSGASQTAYMYLQSLADTASAVYYCYCGTGGGRDVMDYGTLLAV SA;

[0044] SEQ ID NO.4:

[0045] DIVLTQSPASLPTSVSLHASISCRSSQVVGSNYGNTYLHWYLQKPTGLPKLLIYPAHVP DPARGSGTITTTITLTITITKISVEASNYCVQGWLQSLFGTYTTKLEIK;

[0046] S2. Preparation of ELISA plates coated with CA1 antibody:

[0047] CA1 was used as the capture antibody, and a 96-well ELISA plate was used as the solid-phase carrier. The capture antibody was diluted to 2 μg / mL with coating buffer (50 mM carbonate buffer) to obtain the coating solution. 200 μL of the coating solution was added to the ELISA plate, and the plate was sealed and coated overnight at 4°C. The coating solution was discarded, and the plate was washed three times with washing buffer. 200 μL of blocking buffer was added per well, and the plate was blocked at room temperature for 4 h. The liquid in the plate was discarded, and the plate was dried and stored at 4°C.

[0048] Preparation method of S3 and HRP labeled CA2 antibody working solution:

[0049] Using CA2 as the HRP-labeled antibody, the monoclonal antibody CA2 was dialyzed in 50 mM carbonate buffer for 24 h, with the buffer changed twice during this period, adjusting the concentration to 2 mg / mL. 5 mg of HRP powder was weighed and dissolved in 1 mL of ddH2O, and 200 μL of 0.1 M NaIO4 solution was added. The mixture was stirred at room temperature in the dark for 30 min to oxidize the glycosyl groups of HRP to aldehyde groups. The aldehyde-modified HRP solution was placed in a dialysis bag and dialyzed overnight in 10 mM sodium acetate buffer at 4 °C. Carbonate buffer was added to the overnight dialyzed solution to adjust the pH to 9.0, and then an equal volume of monoclonal antibody CA2 was immediately added. The mixture was gently stirred at room temperature in the dark for 2 h, followed by the addition of 0.1 mL of 4 mg / L NaBH4. After mixing, the mixture was incubated at 4 °C for 2 h to obtain a stable enzyme-labeled antibody. The above sample was then dialyzed overnight in 0.15 M pH 7.4 PBS at 4 °C to remove unbound antibody and other impurities. Centrifuge the dialysis solution to remove the precipitate, and the supernatant is the working solution of HRP-labeled CA2 antibody.

[0050] S4. Determination of standard curve for reagent kit:

[0051] Remove the ELISA plate coated with CA1 antibody and allow it to return to room temperature. Wash the plate three times with PBST buffer. Add 100 μL of CD82 recombinant protein standards at different dilutions (50 pg / mL, 100 pg / mL, 150 pg / mL, 200 pg / mL, 250 pg / mL, 300 pg / mL), incubate at 37°C for 2 h, and wash three times with PBST buffer. Add 100 μL of HRP-labeled CA2 antibody working solution to the reaction wells, incubate at 37°C for 2 h, wash five times with PBST buffer, add 100 μL of 3 mg / mL TMB chromogenic solution to the reaction wells, incubate at room temperature in the dark for 30 min, and stop the reaction by adding 100 μL of 3M sulfuric acid. Figure 2 The diagram shows the specific detection process of this kit. A microplate reader was used for dual-wavelength detection, measuring the OD values ​​at the maximum absorption wavelength of 450 nm and the reference wavelength of 630 nm. A standard curve was plotted with the standard concentration on the x-axis (pg / mL) and OD 450-OD 630 on the y-axis. The results are shown below. Figure 3 As shown. The standard curve for the kit used in this application for the detection of CD82 protein content in exosomes is y = 0.0069x + 0.0208, R0. 2 =0.9951.

[0052] S5. Preparation of a kit for detecting CD82 protein content in exosomes:

[0053] The kit of this invention is constructed by packaging the prepared enzyme-labeled plate coated with CA1 antibody, the working solution of HRP-labeled CA2 antibody, the standard CD82 recombinant protein, the buffer PBST, the chromogenic solution TMB, and the reaction termination solution sulfuric acid separately.

[0054] The amino acid sequence of the CD82 recombinant protein is shown in SEQ ID NO.5.

[0055] SEQ ID NO.5:

[0056] MGSACIKVTKYFLFLFNLIFFILGAVILGFGVWILADKSSFISVLQTSSSSLRMGAYVFIGVGAVTMLMGFLGCIGAVNEVRCLLGLYFAFLLLILIAQVTAGALFYFNMGKLKQEMGGIVTELI RDYNSSREDSLQDAWDYVQAQVKCCGWVSFYNWTDNAELMNRPEVTYPCSCEVKGEEDNSLSVRKGFCEAPGNRTQSGNHPEDWPVYQEGCMEKVQAWLQENLGIILGVGVGVAIVELLGMVLSI CLCRHVHSEDYSKVPKY.

[0057] Experimental Example 1

[0058] Blood sample testing:

[0059] Fifteen blood samples from healthy individuals (numbered 1-15) and sixteen blood samples from gastric cancer patients (numbered 16-30) were collected. Blood samples were thawed at 37°C, and exosome extraction and separation were performed according to the method described in Example 1. The purified blood exosome samples (numbered 1-30) were added to ELISA plates coated with CA1 antibody and incubated for 2 hours, followed by washing three times with PBST buffer. 100 μL of HRP-labeled CA2 antibody working solution was added to each well, and the plates were incubated at 37°C for 2 hours, followed by washing five times with PBST buffer. 100 μL of 3 mg / mL TMB chromogenic solution was added to each well, and the plates were incubated at room temperature in the dark for 30 minutes. The reaction was terminated by adding 100 μL of 3M sulfuric acid. Dual-wavelength detection was performed using an ELISA reader, measuring the OD values ​​at the maximum absorption wavelength of 450 nm and the reference wavelength of 630 nm. The results were repeated three times, and the average values ​​were calculated. The CD82 content of the blood exosomes was calculated based on the standard curve. The results are shown in Table 1.

[0060] Table 1 Clinical Sample Test Results

[0061]

[0062]

[0063] The results are shown in Table 1. The kit of the present invention can accurately detect the CD82 protein content in blood exosomes. As can be seen from the table, healthy individuals' blood exosomes contain trace amounts of CD82 protein, resulting in a negative result; compared with healthy individuals, the CD82 protein level in the blood exosomes of gastric cancer patients is significantly elevated, resulting in a positive result. This indicates that the kit of the present invention can use blood exosomes as samples to complete the detection of gastric cancer markers, meeting the needs of basic research and clinical diagnosis.

[0064] Experimental Example 2

[0065] Test kit stability:

[0066] Blood exosome CD82 protein standards (100 pg / mL, 200 pg / mL, and 300 pg / mL) were used as test samples. The stability of the ELISA plate coated with CA1 antibody and the HRP-labeled CA2 antibody working solution was tested at 37°C for 30 days, with the test sample content measured every 15 days. The ELISA plate coated with CA1 antibody and the HRP-labeled CA2 antibody working solution were stored at 4°C for 2 years, with the test sample content measured every 12 months. The test results are shown in Table 2.

[0067] Table 2 Clinical Sample Test Results

[0068]

[0069] The results are shown in Table 2. The kit showed good OD value gradient of the standard curve after 30 days of exposure at 37℃ and 2 years of low-temperature storage, and the detection concentration of blood exosome CD82 protein standard was also relatively stable, indicating that the kit has good stability.

[0070] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. The basic principles and main features of the present invention have been described above with specific implementation schemes. Based on the present invention, some modifications or substitutions can be made, but these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of protection claimed by the present invention.

Claims

1. A kit for detecting the content of exosome CD82 protein, characterized in that, The kit includes standards, an ELISA plate coated with CA1 antibody, HRP-labeled CA2 antibody working solution, buffer solution, chromogenic solution, and reaction termination solution. The CA1 antibody is a monoclonal antibody against CD82 protein. The heavy chain amino acid sequence of the monoclonal antibody CA1 is shown in SEQ ID NO.1, and the light chain amino acid sequence is shown in SEQ ID NO.

2. The CA2 antibody is a monoclonal antibody against CD82 protein. The heavy chain amino acid sequence of the monoclonal antibody CA2 is shown in SEQ ID NO.3, and the light chain amino acid sequence is shown in SEQ ID NO.

4.

2. The kit for detecting CD82 protein content in exosomes according to claim 1, characterized in that, The standard is CD82 recombinant protein.

3. The kit for detecting CD82 protein content in exosomes according to claim 2, characterized in that, The amino acid sequence of the CD82 recombinant protein is shown in SEQ ID NO.

5.

4. The kit for detecting CD82 protein content in exosomes according to claim 1, characterized in that, The buffer solution is PBST; the colorimetric solution is TMB; and the reaction termination solution is sulfuric acid.

5. The kit for detecting CD82 protein content in exosomes according to claim 4, characterized in that, The concentration of the colorimetric reagent is 2-3 mg / mL; the concentration of the reaction termination solution is 2-3 M.

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