CD81 monoclonal antibody, detection kit and application of CD81 monoclonal antibody

Abstract

The invention relates to the technical field of biological medicine detection, in particular to a CD81 monoclonal antibody, a detection kit and application of the CD81 monoclonal antibody. According to the detection kit disclosed by the invention, the expression level of the CD81 protein in peripheral blood can be detected with high sensitivity by utilizing the independently researched and developed CD81 monoclonal antibody and the biotin-labeled nucleic acid aptamer; and the CD81 monoclonal antibody and the nucleic acid aptamer have high specificity, so that the interference of non-specific binding is reduced, and the detection accuracy is improved. According to the kit, by detecting the expression level of the CD81 protein in peripheral blood, early prediction and diagnosis can be carried out before preeclampsia clinical symptoms appear, and precious time is provided for clinical intervention. In addition, the kit is simple and convenient in operation flow and easy to popularize and apply in clinical laboratories.

Description

Technical Field

[0001] This invention relates to the field of biomedical detection technology, specifically to a CD81 monoclonal antibody, a detection kit, and their applications. Background Technology

[0002] Preeclampsia (PE) is a serious complication unique to pregnancy, posing a significant threat to maternal and infant health. Currently, early diagnosis and prediction of PE remain challenging, with traditional methods relying heavily on the appearance of clinical symptoms and signs, often by which time the optimal intervention window has been missed. Therefore, developing a sensitive and specific early diagnostic biomarker is crucial for improving the prognosis of PE.

[0003] CD81 is a four-transmembrane protein widely involved in cell recognition, signal transduction, and cell adhesion. Previous research by the applicant found that CD81 expression levels in the placenta gradually decrease with increasing gestational age, but in the placenta of patients with PE (polycystic urealysis), its expression level continuously increases with gestational age. Further immunoblotting assays confirmed a significant increase in CD81 expression in the peripheral blood of PE patients, suggesting that peripheral blood CD81 levels may serve as a potential biomarker for early prediction and diagnosis of PE. However, the immunoblotting assays used in this study have low throughput and long processing times, making them unsuitable for large-scale clinical testing.

[0004] Currently, most commercially available peripheral blood protein detection kits employ a protein-antibody sandwich method. This strategy requires highly specific paired antibodies that do not occupy epitopes. CD81 protein itself has a very small molecular weight, and the predicted epitopes for antibody detection are all located in the extracellular loop, and the number of epitopes is limited. Therefore, even after obtaining multiple monoclonal antibodies using peptide immunization and cell immunization, it has still been impossible to find a pair of antibodies with high sensitivity that recognizes native CD81 in peripheral blood.

[0005] Nucleic acid aptamers are synthetically produced single-stranded oligonucleotides that specifically bind to target molecules. They are typically 25-60 nucleotides in length and can recognize a variety of target molecules, including proteins, bacteria, viruses, cells, and tissues. Nucleic acid aptamers have low molecular weights, no immunogenicity or toxicity, and can be synthesized, structurally modified, and labeled using chemical methods. They can reversibly denature and renature, and can be stored and transported at room temperature. Due to their advantages such as high specificity, high affinity, good stability, ease of modification, low cost, and broad target range, they are also known as "chemical antibodies." The binding of nucleic acid aptamers to target molecules mainly depends on the diversity of the single-stranded nucleic acid structure and spatial conformation. They can adaptively fold to form stable three-dimensional spatial structures through the pairing of certain complementary bases within the chain, as well as electrostatic interactions and hydrogen bonding. Compared with monoclonal antibodies, nucleic acid aptamers have higher specificity and sensitivity to natural proteins. Summary of the Invention

[0006] Objective of the Invention: The technical problem to be solved by this invention is to address the shortcomings of existing technologies by providing a CD81 monoclonal antibody, a detection kit, and their applications. This invention provides a peripheral blood CD81 detection kit. This kit utilizes a self-developed CD81 antibody as a capture antibody, combined with a biotin-labeled CD81 nucleic acid aptamer, to quantitatively analyze the expression level of CD81 protein in peripheral blood through colorimetric analysis, thereby achieving early prediction and diagnosis of peripheral pulmonary embolism (PE). This invention employs a self-developed CD81 antibody as a capture antibody and then uses a biotin-labeled nucleic acid aptamer that recognizes CD81 as a detection antibody (chemical antibody), ensuring high specificity and high sensitivity in detection.

[0007] To address the aforementioned technical problems, this invention discloses a CD81 monoclonal antibody, a detection kit, and their applications. The specific technical solution is as follows: In a first aspect, the present invention provides a CD81 monoclonal antibody, wherein the amino acid sequence of the heavy chain variable region of the CD81 monoclonal antibody is shown in SEQ ID NO.1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.2. In some embodiments of the present invention, the amino acid sequence of the heavy chain constant region of the CD81 monoclonal antibody is shown in SEQ ID NO.3, and the amino acid sequence of the light chain constant region is shown in SEQ ID NO.4.

[0008] In a second aspect, the present invention provides an ELISA kit for detecting the expression level of CD81 protein, the ELISA kit comprising a capture antibody and a detection antibody, wherein the capture antibody is the CD81 monoclonal antibody described in the first aspect.

[0009] The detection antibody is a nucleic acid aptamer. The nucleotide sequence of the nucleic acid aptamer is shown in SEQ ID NO. 5.

[0010] The nucleic acid aptamer is attached with a fluorescent label, which is either biotinylated or CY5-labeled. In some embodiments of the present invention, the fluorescent label is biotinylated.

[0011] The ELISA kit includes an enzyme-labeled secondary antibody, coating buffer, blocking solution, diluent, washing solution, positive control, and negative control.

[0012] The enzyme-labeled secondary antibody is HRP-streptavidin; the positive control is 293F cell lysis buffer; and the negative control is PBS buffer. In some embodiments of the present invention, the coating buffer formulation includes: 1.59 g Na2CO3, 2.93 g NaHCO3, 0.2 g NaN3, with H2O added to 1000 mL, pH 9.6. The blocking solution formulation includes: 3.0 g BSA added to 100 mL PBS. The diluent formulation includes: 0.39 g NaH2PO4·2H2O, 1.27 g Na2HPO4, 0.85 g NaCl, 200 mg NaN3, with H2O added to 1000 mL, used to dilute proteins, antibodies, or samples; and the washing buffer is PBST buffer.

[0013] Thirdly, the present invention provides the use of the CD81 monoclonal antibody described in the first aspect or the ELISA kit described in the second aspect in the preparation of diagnostic products for preeclampsia. The products include reagents or kits.

[0014] The product is used to detect the expression level of CD81 protein in the sample to be tested.

[0015] Fourthly, this invention provides an ELISA method for detecting CD81 protein expression levels for non-diagnostic purposes, using the ELISA kit described in the second aspect, comprising the following detection conditions: a CD81 monoclonal antibody coating concentration of 5–20 µg / mL; a test sample dilution ratio of 1:1–80 v / v; incubation at 4–37 °C for 1–12 h; and a detection antibody concentration of 500–4000 nM; incubation at 4–37 °C for 1–12 h. In some embodiments of this invention, the test sample includes pregnant woman serum. In other embodiments of this invention, the CD81 monoclonal antibody coating concentration is 10 µg / mL; the test sample dilution ratio is 1:5 v / v; and the detection antibody concentration is 2000 nM.

[0016] In some embodiments of the present invention, the ELISA method for detecting CD81 protein expression levels for non-diagnostic purposes includes the following steps: (1) Add 5~20 µg / mL of CD81 monoclonal antibody coating solution to the microplate and coat overnight at 4 ℃; (2) Discard the coating solution, wash, add blocking solution, and block at 37 ℃ for 2 h; (3) Discard the blocking solution, wash, add the sample to be tested, set up positive control and negative control, and incubate at 4~37 ℃ for 1~12 h; (5) Discard the sample solution to be tested, wash, add 500~4000 nM of fluorescently labeled nucleic acid aptamer, and incubate at 4~37℃ for 1~12 h; (6) Discard the nucleic acid adaptor solution, wash, add HRP-streptavidin, and incubate at room temperature for 1 hour; (7) Discard HRP-streptavidin, wash, add chromogenic solution and incubate in the dark for 10-15 minutes; add stop solution to terminate the reaction, and read the OD in the wells using a microplate reader. 450 value.

[0017] Beneficial effects: 1. High sensitivity: This kit uses a self-developed CD81 monoclonal antibody and biotin-labeled nucleic acid aptamers, which can detect the expression level of CD81 protein in peripheral blood with high sensitivity.

[0018] 2. High specificity: Both CD81 antibody and nucleic acid aptamer have high specificity, reducing interference from non-specific binding and improving detection accuracy.

[0019] 3. Early diagnosis: By detecting the expression level of CD81 protein in peripheral blood, this kit can make early predictions and diagnoses before the clinical symptoms of PE appear, providing valuable time for clinical intervention.

[0020] 4. Easy to operate: This kit is easy to operate and can be easily promoted and applied in clinical laboratories. Attached Figure Description

[0021] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0022] Figure 1 The results of the competitive binding assay of CD81 antibody-nucleic acid aptamer are shown. A represents the flow cytometry results of CD81 nucleic acid aptamer after binding 4-C1-E4-F2 cells to 293F cells; B represents the flow cytometry results of CD81 nucleic acid aptamer directly binding to 293F cells.

[0023] Figure 2 To analyze the CD81 expression level of 293F cells transfected with siCON or siCD81 using immunoblotting.

[0024] Figure 3 To analyze the expression level of CD81 in the peripheral blood of normal individuals and PE patients using immunoblotting.

[0025] Figure 4The expression level of CD81 in peripheral blood of normal individuals and PE patients was detected by antibody-aptamer sandwich ELISA. In the figure, A represents the standard curve of antibody-aptamer sandwich ELISA, and B represents the CD81 concentration obtained by antibody-aptamer sandwich ELISA. Detailed Implementation

[0026] Example 1 Antibody-Nucleic Acid Aptamer Competition Binding Experiment The self-designed CD81 monoclonal antibody 4-C1-E4-F2 (its heavy chain variable region amino acid sequence is shown in SEQ ID NO.1, its heavy chain constant region amino acid sequence is shown in SEQ ID NO.3, its light chain variable region amino acid sequence is shown in SEQ ID NO.2, and its light chain constant region amino acid sequence is shown in SEQ ID NO.4) was paired with a nucleic acid aptamer (its nucleotide sequence is shown in SEQ ID NO.5). First, it was necessary to determine whether there was a competitive relationship between the CD81 protein antigen epitopes bound by the antibody and the nucleic acid aptamer. If there was no competition, then a sandwich ELISA reaction could be performed. It is known that 293F cells can express CD81 protein. This study used 293F cells as the target molecule and conducted a competitive binding experiment between the prepared CD81 monoclonal antibody 4-C1-E4-F2 and the CD81 nucleic acid aptamer.

[0027] Preparation process of CD81 monoclonal antibody 4-C1-E4-F2: First, a CHO recombinant cell line stably expressing CD81 protein (its amino acid sequence is shown in SEQ ID NO. 6) was constructed using genetic engineering technology, and cell lines highly expressing CD81 were screened using flow cytometry. Subsequently, Balb / c mice were immunized multiple times using this recombinant cell line, with Freund's complete adjuvant and Freund's incomplete adjuvant to enhance the immune response. Spleen cells from immunized mice were collected and electrofused with myeloma cells to prepare hybridoma cells. Subclonal screening was performed using limiting dilution to obtain hybridoma cell lines that stably secrete monoclonal antibodies. The supernatant of hybridoma cells was screened by indirect ELISA to identify the high-affinity monoclonal antibody 4-C1-E4-F2 targeting the extracellular macrocyclic region of CD81 protein. Finally, the antibody was produced and purified using large-scale cell culture technology for subsequent experiments.

[0028] The CD81 aptamer with the nucleotide sequence shown in SEQ ID NO.5 was synthesized by Sangon Biotech Co., Ltd., and labeled with biotin or CY5 fluorescence as needed for the experiment. The CD81 monoclonal antibody-aptamer competitive binding experiment was performed according to the following steps: (1) HEK-293F cells were pre-cooled on ice for 20 minutes, washed three times with 1×PBS, and then resuspended in 1×PBS to obtain 293F cell suspension, 200 μL per tube, with a final concentration of 1×10⁻⁶. 6 per mL.

[0029] (2) Take one vial of aptamer powder, briefly separate it, and add 1 mL of CY5 fluorescently labeled aptamer binding solution (2.5 mM MgCl2 in 1×PBS solution, prepared by adding 0.2308 g MgCl2 to 5 mL of 10×PBS and then adding pure water to 50 mL) to prepare a 1000 nM stock solution. After standing for 2 minutes, denature at 95 ℃ for 5 minutes, and then renature on ice for 5 minutes. According to experimental requirements, further dilute to a 200 nM aptamer application solution for later use.

[0030] (3) Dilute the monoclonal antibody 4-C1-E4-F2 to 10 µg / mL using 1×PBS.

[0031] (4) Take two tubes of 293F cell suspension prepared in step (1), add 1 μL of CD81 monoclonal antibody 4-C1-E4-F2 to the first tube, incubate at room temperature for 20 minutes, centrifuge at 500 g for 3 minutes, and wash 3 times with 200 μL of 1×PBS. Do not add antibody to the second tube as a control group.

[0032] (5) Add 200 nM aptamer application solution to the two tubes of 293F cell suspension in step (4), gently vortex for 3-5 seconds, and incubate on ice in the dark for 20 minutes.

[0033] (6) Gently tap to mix, use flow cytometry to measure fluorescence intensity, and calculate the average fluorescence intensity of 293F cells bound to 4-C1-E4-F2 antibody and 293F cells without antibody binding.

[0034] The results showed that when CD81 aptamers were used to detect 293F cells bound by the 4-C1-E4-F2 antibody and 293F cells without antibody binding, there was no significant difference in the mean fluorescence signal between the two groups (1.74 vs 1.60, p > 0.05), indicating that there is no competitive relationship between the aptamer and the 4-C1-E4-F2 antibody. Figure 1 ).

[0035] Example 2: Composition of the Peripheral Blood CD81 Sandwich ELISA Detection Kit The peripheral blood CD81 sandwich ELISA detection kit of the present invention mainly comprises the following components: 1. CD81 capture antibody: The independently developed high-specificity CD81 monoclonal antibody 4-C1-E4-F2 is used to capture CD81 protein in peripheral blood samples.

[0036] 2. Biotin-labeled CD81 nucleic acid aptamer (detection antibody): A biotin-labeled CD81-specific nucleic acid aptamer (nucleotide sequence as shown in SEQ ID NO.5) is used to bind to the captured CD81 protein.

[0037] 3. Protein dilution buffer (0.01 mol / L, pH 7.2): 0.39 g NaH2PO4·2H2O, 1.27 g Na2HPO4, 0.85 g NaCl, 200 mg NaN3, add H2O to 1000 mL, used to dilute proteins, antibodies or samples.

[0038] 4. Washing buffer (PBST, pH 7.4): 2.0 g NaCl, 0.2 g KH2PO4, 2.9 g Na2HPO4·12H2O, 0.2 g KCl, 0.2 g NaN3, 0.5 mL Tween 20, add H2O to 1000 mL, used to wash unbound antibodies and nucleic acid aptamers to reduce background interference.

[0039] 5. 3% BSA blocking solution: 3.0 g bovine serum albumin (BSA) added to 100 mL 1×PBS, prepared fresh before use.

[0040] 6. CBS coating buffer (pH 9.6): 1.59 g Na2CO3, 2.93 g NaHCO3, 0.2 g NaN3, add H2O to 1000 mL.

[0041] 7. Enzyme-labeled secondary antibody HRP-labeled streptavidin (Solarbio, catalog number SE068) and TMB two-component chromogenic solution (Solarbio, catalog number PR1210).

[0042] 8. Standards: The supernatant of HEK293 cells overexpressing CD81 and the lyophilized CD81 powder obtained by immunoprecipitation (IP) were used as standards. The specific preparation method was as follows: After HEK293 cells overexpressed CD81, the cell culture supernatant was first collected, centrifuged at 1000-1500 rpm for 5-10 minutes to remove cell debris, and the supernatant was retained with a protease inhibitor added to prevent degradation. Subsequently, the supernatant was pretreated with pre-cooled lysis buffer, and Protein A / G agarose beads were added for pre-cleansing at 4°C for 1 hour to remove non-specific bindings. Next, CD81-specific antibody 4-C1-E4-F2 was added to the supernatant, and the cells were incubated at 4°C for 2-4 hours to form antigen-antibody complexes. Fresh Protein A / G beads were then added and incubated for another hour. The bead-antibody-antigen complexes were collected by centrifugation at 4°C and washed 3-5 times with washing buffer to remove impurities. Finally, CD81 protein was eluted with SDS-containing buffer, the eluent was transferred to a lyophilization bottle, pre-frozen at -80°C overnight, and then lyophilized in a lyophilizer to obtain CD81 dry powder.

[0043] The detection method of the peripheral blood CD81 sandwich ELISA detection kit of the present invention includes the following steps: 1. Sample collection and processing: Peripheral blood samples were collected from the subjects and treated with protein diluent to release CD81 protein.

[0044] 2. Capture CD81 protein: Add the treated sample to a microplate containing CD81 capture antibody and incubate to allow the CD81 protein to bind to the capture antibody.

[0045] 3. Washing: Wash the microplate with washing buffer to remove unbound material.

[0046] 4. Labeling: Add biotin-labeled CD81 aptamers and incubate to allow them to bind to the captured CD81 protein.

[0047] 5. Wash again: Wash the microplate with washing buffer to remove unbound nucleic acid aptamers.

[0048] 6. Detection: Add the detection substrate (HRP-labeled streptavidin) and use a microplate reader to detect the OD value in the microplate.

[0049] 7. Data Analysis: Plot a standard curve based on the standard and calculate the concentration of CD81 protein in the sample.

[0050] Example 3: Preparation and Optimization of CD81 Antibody-aptamer Sandwich ELISA The reagents used in the sandwich ELISA experiment described in this embodiment are shown in Example 2.

[0051] 1. Optimization of reaction conditions for antibody-aptamer sandwich ELISA detection of serum CD81 protein (1) Orthogonal experiments were used to preliminarily determine the optimal CD81-specific antibody coating concentration, CD81 nucleic acid aptamer working concentration, and sample serum dilution ratio for sandwich ELISA detection. The orthogonal experimental conditions were as follows: the coating antibody concentrations were 5 µg / mL, 10 µg / mL, and 20 µg / mL, and the aptamer working concentrations were 4000 nM, 2000 nM, and 500 nM, respectively. Pregnant women's serum was diluted with protein diluent at volume ratios of 1:5 and 1:20, respectively. 293F cell lysis buffer was used as a positive control, and 1×PBS was used as a negative control. Reproduced wells were used for detection. The 293F cell lysis buffer was prepared as follows: 293F cells in good growth condition were collected into centrifuge tubes, centrifuged, and the supernatant was discarded. An appropriate amount of cell lysis buffer containing protease inhibitors was added, the cells were resuspended, and the cells were fully lysed on ice using an ultrasonic disruptor or cell scraper. Then, the cells were centrifuged at low temperature and high speed, and the supernatant was taken as the 293F cell lysis buffer.

[0052] (2) After determining the optimal specific antibody coating concentration, aptamer working concentration, and serum dilution ratio, an orthogonal experiment was conducted to determine the incubation time and temperature for the antibody-aptamer sandwich reaction. The reaction temperatures were 4℃, 25℃, and 37℃, and the incubation times were 1 hour, 4 hours, and overnight, respectively.

[0053] 2. CD81 antibody-aptamer sandwich ELISA detection procedure (1) Dilute the capture antibody 4-C1-E4-F2 with CBS coating buffer to a final concentration of 5, 10 or 20 µg / mL, add 100 μL / well to the microplate, and coat overnight at 4 ℃.

[0054] (2) Remove the coating solution, wash 3 times with PBST, add 230 μL / well, add 200 μL / well of 3% BSA blocking solution, block at 37 ℃ for 2 hours, remove the blocking buffer, and wash 3 times with PBST.

[0055] (3) Add serum samples (undiluted, 1:5 or 1:20 v / v dilution) and standards (100 μL / well) to each well. Use 293F cell lysis buffer as a positive control and 1×PBS as a negative control. Perform replicate detection. After incubation at 37 ℃ for 1 h, remove the liquid from the wells, wash three times with PBST, and add 230 μL / well.

[0056] (4) Take one vial of biotin-labeled nucleic acid aptamer powder, briefly separate it, and add aptamer binding solution to prepare a 1000 nM stock solution. Denature at 95°C for 5 minutes, cool on ice for 5 minutes, fold at 37°C for 15 minutes, and set aside.

[0057] (5) According to the experimental requirements, use aptamer binding solution to dilute the aptamer stock solution to the working concentration (4000 nM, 2000 nM or 500 nM), 100 μL / well, incubate at 37 ℃ for 1 h, remove the liquid in the well, wash 3 times with PBST, 230 μL / well.

[0058] (6) Add 100 μL / well of HRP-streptavidin (diluted with PBST at 1:10000 v / v) and incubate at room temperature for 1 hour; remove the liquid from the well and wash twice with PBST, 230 μL / well.

[0059] (7) Add 100 μL / well of TMB chromogenic buffer and incubate at room temperature in the dark for 10-15 minutes. Add 50 μL / well of stop buffer and read the OD values ​​in the wells using a microplate reader. 450 value.

[0060] The optimal reaction conditions for 4-C1-E4-F2 antibody-aptamer sandwich ELISA detection were determined through the above orthogonal experiments: ELISA plates were coated with 5 µg / mL, 10 µg / mL, and 20 µg / mL of 4-C1-E4-F2 antibody, respectively, and ELISA sandwich reactions were performed using 4000 nM, 2000 nM, and 500 nM nucleic acid aptamers as detection antibodies. As shown in Table 1, in the three sets of data with different concentration combinations, the detection values ​​at a serum dilution ratio of 1:5 were all higher than those at a dilution ratio of 1:20, and most were higher than those at undiluted serum. However, when the 4-C1-E4-F2 concentration was 10 µg / mL, the detection value at a serum dilution ratio of 1:5 was close to that at a 4-C1-E4-F2 concentration of 20 µg / mL. Similarly, when the nucleic acid aptamer concentration was 2000 nM, the detection value at a serum dilution ratio of 1:5 was close to that at a nucleic acid aptamer concentration of 4000 nM. This result indicates that using 10 µg / mL 4-C1-E4-F2 as the capture antibody and 2000 nM nucleic acid aptamer as the detection antibody, with a serum dilution of 1:5 v / v, the detection effect was relatively good and the reagent consumption was low. Orthogonal experiments were conducted using the above optimal reaction conditions for incubation temperature and time. The results showed that incubation at 4℃, 25℃, and 37℃ for 1 hour, 4 hours, or overnight had little effect on the detection results. Therefore, 4℃ incubation for 1 hour was conventionally selected as the reaction condition (Table 2).

[0061] Table 1. Results of the orthogonal experiment on antibody coating concentration, working concentration of nucleic acid aptamer, and serum dilution ratio.

[0062] Table 2 Results of the orthogonal experiment of incubation temperature and time

[0063] Example 4: Specificity and sensitivity analysis of the reagent kit Specificity analysis: After transfecting 293F cells with the CD81 interference sequence, the supernatant was collected for detection. Specifically, the control sequence (siCON, Ribobio, Guangzhou) and the interference sequence (siCD81, sequence 5'-CCGCCTGTGTATATAACGTTT-3') were transfected into 293F cells. After 72 hours, the cell culture supernatant was collected, and impurities were removed by low-temperature high-speed centrifugation. The CD81 antibody-aptamer sandwich ELISA assay was then performed using the optimal reaction conditions obtained from the orthogonal experiment in Example 3. Simultaneously, cells were collected for immunoblotting to verify the interference effect of siCD81. The immunoblotting results showed that the CD81 interference sequence (siCD81) significantly inhibited the protein expression level of CD81 in 293F cells. Figure 2 The results of CD81 antibody-aptamer sandwich ELISA in detecting the cell supernatants of the two groups also showed similar trends (Table 3). This fully demonstrates that CD81 antibody-aptamer sandwich ELISA has high specificity for detecting CD81.

[0064] Table 3. Determination of CD81 content in 293F cell supernatant

[0065] Sensitivity analysis: The 293F cell lysis buffer dilution method was used for detection. Specifically, 10^7 healthy 293F cells were collected into centrifuge tubes. After centrifugation and discarding the supernatant, an appropriate amount of cell lysis buffer containing protease inhibitors was added to resuspend the cells. The cells were then lysed thoroughly on ice using an ultrasonic homogenizer, followed by low-temperature high-speed centrifugation, and the supernatant was collected. Subsequently, the cells were diluted with PBS at volume ratios of 1:5, 1:10, 1:20, 1:40, and 1:80. The CD81 antibody-aptamer sandwich ELISA assay was performed under the optimal reaction conditions described in Example 3, and a commercial CD81 detection kit (LS-F7468, LSBio, USA) was used as a control group to analyze the cell lysis buffer.

[0066] The results of the 293F cell lysate assay showed that the CD81 antibody-aptamer sandwich ELISA exhibited good sensitivity. Even when the 293F cell lysate was diluted to 1:80, the protein was still detectable. In contrast, the control kit, when diluted to 1:20, showed detection values ​​close to the blank value (Table 4).

[0067] Table 4. Determination of CD81 content in 293F cell lysates

[0068] Example 5: Clinical application study of the reagent kit 1. Source of clinical samples The sample was obtained from a prospective longitudinal cohort of preeclampsia at Gulou Hospital Affiliated to Nanjing University School of Medicine (ethics no. 2016-113-01). All pregnant women signed informed consent forms. Detailed records were kept of the pregnant women's basic information, including age, parity, delivery, method of conception, smoking history, past medical history (including chronic hypertension, diabetes, autoimmune diseases, nephritis, hepatitis, tuberculosis, heart disease, etc.), and family history. All pregnant women were followed up after delivery, including monitoring their weight, blood pressure, prenatal ultrasound examinations, prenatal screening and diagnostic results, and pregnancy complications (Table 5). Serum samples from 8 preeclampsia patients and 8 normal controls at the same gestational age were selected for antibody-aptamer sandwich ELISA analysis.

[0069] Table 5 Basic Clinical Information of Pregnant Women from Maternal Serum Sources

[0070] 2. Immunoblotting detection of CD81 expression in the serum of the above 16 cases. (1) Centrifuge the serum at 12000 g for 30 minutes and take 2 μL of supernatant. Add 4 μL of loading buffer (Regan, China) and 14 μL of 1× PBS to each serum sample and boil at 100 ℃ for 10 minutes to denature the protein.

[0071] (2) Take 15 μL of denatured serum for polyacrylamide gel electrophoresis at 100 V for 120 min.

[0072] (3) Rinse the nitrocellulose membrane with transfer buffer 30 minutes in advance, and use a transfer apparatus to transfer the protein onto the nitrocellulose membrane. Assemble the transfer system according to the following structure: negative electrode - sponge - three-layer filter paper - gel - membrane - three-layer filter paper - sponge - positive electrode.

[0073] (4) The transferred membrane was washed three times with 1×TBST and placed on a low-speed horizontal shaker at room temperature for 5 minutes each time. After adding 3% BSA blocking solution, it was blocked at 4°C for 2 hours.

[0074] (5) Discard the blocking solution, wash three times with 1×TBST, add CD81 antibody (1:1000, Santa Cruz, sc-7637) or internal control transferrin antibody (1:1000, Abcam, ab277635), place on a low-speed horizontal shaker, and incubate overnight at room temperature. Wash three times with 1×TBST, place on a low-speed horizontal shaker, and incubate at room temperature for 5 min each time.

[0075] (6) Add goat anti-mouse IgG secondary antibody (for detecting CD81) or goat anti-rabbit IgG secondary antibody (for detecting transferrin), place on a low-speed horizontal shaker, and incubate at room temperature for 1 hour.

[0076] (7) Wash the membrane three times with 1×TBST, place it on a low-speed horizontal shaker at room temperature, and shake for 5 minutes each time. After adding the substrate chromogenic solution, take pictures using an imaging analysis system.

[0077] The results showed that CD81 expression in maternal serum was confirmed by immunoblotting in 8 normal control maternal serum samples (1, 2, 7, 8, 9, 10, 11, 12) and 8 preeclampsia patients (3, 4, 5, 6, 13, 14, 15, 16). Furthermore, the expression level of CD81 in maternal serum of the preeclampsia group was significantly higher than that of the control group (P = 0.0003). Figure 3 Immunoblotting confirmed that CD81 expression was elevated in the peripheral blood of PE patients.

[0078] 3. CD81 antibody-aptamer sandwich ELISA detection procedure (1) Dilute the capture antibody to a final concentration of 10 µg / mL using CBS coating buffer, add 100 μL / well to the microplate, and coat overnight at 4 °C.

[0079] (2) Remove the coating solution, wash 3 times with PBST, add 230 μL / well, add 200 μL / well of 3% BSA blocking solution, block at 37 ℃ for 2 hours, remove the blocking buffer, and wash 3 times with PBST.

[0080] (3) The lyophilized protein supernatant of 293F cells with high CD81 expression was used as a standard. The standard was diluted with 1×PBS. The initial concentration of S1 was 100 ng / mL, the concentration of S2 was 50 ng / mL, the concentration of S3 was 25 ng / mL, the concentration of S4 was 12.5 ng / mL, the concentration of S5 was 6.25 ng / mL, the concentration of S6 was 3.125 ng / mL, the concentration of S7 was 1.5625 ng / mL, and the concentration of S8 was 0.78125 ng / mL.

[0081] (4) Add serum samples diluted 1:5 (using protein diluent) and standards (100 μL / well) to each well. Use 293F cell lysis buffer as a positive control and 1×PBS as a negative control. Perform replicate detection. After incubation at 4 °C for 1 h, remove the liquid from the wells, wash three times with PBST, and add 230 μL / well.

[0082] (5) Take one vial of biotin-labeled aptamer powder, briefly separate it, and add aptamer binding solution to prepare a 1000 nM stock solution. Denature at 95°C for 5 minutes, cool on ice for 5 minutes, fold at 37°C for 15 minutes, and set aside.

[0083] (6) According to the experimental requirements, the aptamer stock solution was diluted to the optimal concentration of 2000 nM using aptamer binding solution, 100 μL / well, and incubated under the optimal reaction conditions. The liquid in the well was removed, and the well was washed 3 times with PBST, 230 μL / well.

[0084] (7) Add 100 μL / well of HRP-streptavidin (diluted with PBST at 1:10000 v / v) and incubate at room temperature for 1 hour; remove the liquid from the well and wash twice with PBST, 230 μL / well.

[0085] (8) Add 100 μL / well of TMB chromogenic buffer and incubate at room temperature in the dark for 10-15 minutes. Add 50 μL / well of stop buffer and read the OD values ​​in the wells using a microplate reader. 450 value.

[0086] Antibody-aptamer sandwich ELISA results of peripheral blood CD81 in normal pregnant women and PE patients are as follows: Figure 4 The standard curve shows that the detection range of this kit is 0.78 ng / mL-100 ng / mL, and the R value of the curve is... 2 ≥0.99 ( Figure 4 (A) Results from serum samples from 8 normal pregnant women and 8 PE patients showed that the kit could identify and quantify peripheral blood CD81 levels. Furthermore, the CD81 levels in the peripheral blood of PE patients were all above 20 ng / mL, while the CD81 levels in the peripheral blood of normal pregnant women were all below 20 ng / mL. The CD81 levels in the peripheral blood of PE patients were significantly higher than those in the peripheral blood of normal pregnant women (…). Figure 4 (B) This result is consistent with the results of the immunoblotting assay described above. These results indicate that this antibody-aptamer sandwich ELISA can be used to detect CD81 levels in peripheral blood.

[0087] This invention provides a concept and method for CD81 monoclonal antibody, detection kit, and their applications. Many methods and approaches exist for implementing this technical solution; the above description is merely a preferred embodiment of the invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention. All components not explicitly stated in this embodiment can be implemented using existing technologies.

Claims

1. A CD81 monoclonal antibody, characterized in that, The amino acid sequence of the heavy chain variable region of the CD81 monoclonal antibody is shown in SEQ ID NO.1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.

2.

2. An ELISA kit for detecting CD81 protein expression levels, characterized in that, The ELISA kit includes a capture antibody and a detection antibody, wherein the capture antibody is the CD81 monoclonal antibody as described in claim 1.

3. The ELISA kit according to claim 2, characterized in that, The detection antibody is a nucleic acid aptamer.

4. The ELISA kit according to claim 3, characterized in that, The nucleotide sequence of the nucleic acid aptamer is shown in SEQ ID NO.

5.

5. The ELISA kit according to claim 3, characterized in that, The nucleic acid aptamer is attached with a fluorescent label, which may include biotin or CY5 label.

6. The ELISA kit according to claim 2, characterized in that, The ELISA kit includes enzyme-labeled secondary antibody, coating buffer, blocking buffer, diluent, washing buffer, positive control, and negative control.

7. The ELISA kit according to claim 6, characterized in that, The enzyme-labeled secondary antibody is HRP-streptavidin; the positive control is 293F cell lysis buffer; and the negative control is PBS buffer.

8. The use of the CD81 monoclonal antibody of claim 1 or the ELISA kit of any one of claims 2 to 7 in the preparation of preeclampsia diagnostic products.

9. The application according to claim 8, characterized in that, The product is used to detect the expression level of CD81 protein in the sample to be tested.

10. An ELISA method for detecting CD81 protein expression levels for non-diagnostic purposes, characterized in that, The detection was performed using the ELISA kit according to any one of claims 2 to 7, including the following detection conditions: the coating concentration of CD81 monoclonal antibody was 5 to 20 µg / mL; The dilution factor of the sample to be tested is 1:1~80 v / v, and the reaction conditions are 4~37 ℃ incubation for 1~12 h; The concentration of the detection antibody was 500~4000 nM, and the incubation conditions were 4~37 ℃ for 1~12 h.

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