Monoclonal antibody specifically binding to Chikungunya virus E2 protein and its application

Abstract

The invention discloses a monoclonal antibody capable of specifically binding to a chikungunya virus E2 protein. The monoclonal antibody is 1B7-H7, the amino acid sequence of a light chain variable region thereof is shown in SEQ ID NO:1, and the amino acid sequence of a heavy chain variable region is shown in SEQ ID NO:2; or the monoclonal antibody is 2A2-E10, the amino acid sequence of a light chain variable region is shown in SEQ ID NO:3, and the amino acid sequence of a heavy chain variable region is shown in SEQ ID NO:4; or the monoclonal antibody is 4D3-E8, the amino acid sequence of a light chain variable region is shown in SEQ ID NO:5, and the amino acid sequence of a heavy chain variable region is shown in SEQ ID NO:6; the monoclonal antibody is applied to the preparation of a reagent or a kit for specifically detecting a chikungunya virus E2 antigen, and has high specificity and sensitivity. The invention is conducive to the early diagnosis of the chikungunya virus.

Description

Technical Field

[0001] The invention relates to a monoclonal antibody capable of specifically binding to Chikungunya virus E2 protein and a DAS-ELISA kit for serological diagnosis of Chikungunya virus, belonging to the technical field of medical testing. Background Art

[0002] Chikungunya fever is a degenerative arthritis disease caused by Chikungunya virus (CHIKV). CHIKV is a single-stranded positive-strand RNA virus belonging to the Togaviridae family and the genus Alphavirus. It is mainly transmitted by Aedes aegypti and Aedes albopictus. The general population is susceptible to it. The acute phase of infection is characterized by sudden high fever (>39°C), accompanied by erythematous maculopapular rash, myalgia and arthralgia. The chronic phase is persistent or recurrent arthritis, which can last from several months to several years. According to research reports, 80%-93% of patients infected with CHIKV develop chronic diseases, and the economic burden is estimated to be £25,000 per patient per year. Currently, there is no vaccine or effective treatment for Chikungunya virus.

[0003] Yunnan is extremely suitable for the survival of Aedes albopictus due to its special geographical location and ecological environment. In addition, Yunnan borders high-risk areas for CHIKV, such as Laos and Myanmar, and faces the risk of cross-border introduction of CHIKV. Chikungunya virus, dengue virus, and Zika virus are all arboviruses, and there is overlap in epidemic areas and co-infection. In addition, the early stages of viral infection have similar clinical symptoms, and it is impossible to accurately determine which virus infection is caused based on clinical symptoms alone. Therefore, the situation of Chikungunya epidemic prevention and control in Yunnan Province is not optimistic. In order to prevent the introduction and spread of the epidemic, sufficient technical reserves must be made, and rapid detection is the basis.

[0004] At present, conventional diagnostic methods for detecting Chikungunya virus infection usually include virus isolation and culture, serological testing, nucleic acid molecular testing and other methods. Virus isolation and culture, RT-PCR, RT-qPCR, and multiplex PCR have the characteristics of high specificity, but are limited by factors such as the need for biosafety level 3 laboratories, professional operators, and long testing time. They are not conducive to rapid diagnosis and are not suitable for grassroots testing institutions to conduct CHIKV testing. Serological-based antigen-antibody testing not only has a short testing time (2-3 h), low testing cost, and does not require professional laboratory equipment and operators, but is also suitable for grassroots testing institutions such as customs to conduct large-scale screening for CHIKV infection, and also meets the needs of Yunnan Province and even China for CHIKV testing. Summary of the invention

[0005] The present invention provides a monoclonal antibody that can specifically bind to the E2 protein of the chikungunya virus, wherein the monoclonal antibody is 1B7-H7, the amino acid sequence of the light chain variable region is shown in SEQ ID NO:1, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:2; or the monoclonal antibody is 2A2-E10, the amino acid sequence of the light chain variable region is shown in SEQ ID NO:3, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:4; or the monoclonal antibody is 4D3-E8, the amino acid sequence of the light chain variable region is shown in SEQ ID NO:5, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:6.

[0006] Another object of the present invention is to use the above-mentioned monoclonal antibody in the preparation of a reagent or kit for specifically detecting the E2 antigen of the Chikungunya virus, wherein the kit is a DAS-ELISA kit for specifically detecting the E2 antigen of the Chikungunya virus, and the kit comprises one of the above-mentioned monoclonal antibodies and other conventional reagents for DAS-ELISA detection.

[0007] The purpose of the present invention is achieved through the following technical solutions:

[0008] The E2 protein of Chikungunya virus is a core protein that mediates the virus invasion of the host, has high immunogenicity, and is a protein that induces the body to produce a wide range of antibodies. The present invention uses the E2 protein (1-346aa) of the East / Central / South African type (Ross strain), which is the main epidemic strain in Yunnan Province and even in China, as a target antigen, constructs a prokaryotic expression vector, and induces the expression of the recombinant protein through the Rosetta (DE3) strain.

[0009] The present invention immunizes BALB / c mice and New Zealand white rabbits with prokaryotically expressed CHIKV-E2 recombinant protein to obtain three hybridoma cell lines (1B7-H7, 2A2-E10, 4D3-E8) specific to CHIKV-E2 protein and rabbit polyclonal antibody serum;

[0010] The present invention uses three hybridoma cell lines to inoculate mice to prepare monoclonal antibody ascites. After the monoclonal antibody is purified, the purity and antibody titer of the monoclonal antibody are evaluated, and the specificity of the monoclonal antibody is analyzed by indirect ELISA, IFA, Western Blot and other experiments.

[0011] The invention applies monoclonal antibodies to prepare a chikungunya virus E2 antigen detection kit with high sensitivity and specificity, which can directly detect CHIKVE2 antigen in the serum of CHIKV infected patients and realize early diagnosis of chikungunya virus infection. The kit is a detection kit based on a double antibody sandwich ELISA method, comprising a microporous reaction plate coated with a capture antibody, a sample treatment solution, a detection antibody, a positive control, a negative control, a concentrated washing solution, a color developing solution and a stop solution, wherein the capture antibody is a rabbit polyclonal antibody; the detection antibody is obtained by coupling one of monoclonal antibody 1B7-H7, monoclonal antibody 2A2-E10 and monoclonal antibody 4D3-E8 with horseradish peroxidase; the sample treatment solution, the concentrated washing solution, the color developing solution and the stop solution are all common reagents in the double antibody sandwich ELISA method, the positive control is a recombinant chikungunya virus E2 protein, and the negative control is a blank control that does not contain the recombinant chikungunya virus E2 protein.

[0012] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0013] The monoclonal antibody provided by the present invention can specifically bind to the Chikungunya E2 protein. The monoclonal antibody is used in a detection kit based on a double antibody sandwich ELISA method, wherein the capture antibody rabbit polyclonal antibody and the detection antibody are a group of antibodies that can specifically recognize different epitopes of the Chikungunya virus E2 protein and can only specifically bind to the Chikungunya virus. Therefore, the kit can accurately and quickly detect the Chikungunya virus, and has no cross reaction with other insect-borne viruses such as dengue virus and Zika virus. The kit has a high sensitivity to the Chikungunya virus, and the detection limit is 1×10 6 PFU / mL, the present invention is beneficial to the early diagnosis of Chikungunya virus and insect vector monitoring. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The results of PCR amplification of Chikungunya E2 protein, A: gel electrophoresis results, B: gene sequencing peak diagram, C: BLAST comparison results;

[0015] Figure 2 The results of protein detection after the induction expression and purification of CHIKV-E2 recombinant protein, where 1: bacteria before induction, 2: bacteria after induction, 3: crushed supernatant, 4: crushed precipitate, 5-9: purified protein (eluted with 100-500mM imidazole);

[0016] Figure 3 The results of SDS-PAGE test for monoclonal antibody and polyclonal antibody purification;

[0017] Figure 4 These are the results of ELISA tests for monoclonal antibodies and rabbit polyclonal antibodies;

[0018] Figure 5 Monoclonal antibody, rabbit polyclonal antibody Western Blot, IFA verification results, where A is the Western Blot test result, and B is the IFA test result;

[0019] Figure 6 The results of indirect immunofluorescence assay for detecting the immune response of monoclonal antibodies, rabbit polyclonal antibodies and cells infected with common arboviruses;

[0020] Figure 7 The results of antibody activity verification after three monoclonal antibodies were labeled with HRP;

[0021] Figure 8 To optimize the best capture antibody and detection antibody working concentration results;

[0022] Fig. 9 To determine the optimal blocking buffer experimental results;

[0023] Fig.10 To optimize the best temperature and time results for antigen incubation;

[0024] Fig.11 To verify the specificity of DAS-ELISA, A is the visualization result, and B is the OD450nm value;

[0025] Fig.12 Figure 3 is the test results of clinical samples, where Figure A shows the test results of 10 CHIKV simulated clinical samples, 10 DENV positive serum samples, and 10 negative serum samples; Figure B is the ROC curve of DAS-ELISA positive or negative results. DETAILED DESCRIPTION

[0026] The specific implementation of the present invention is further described below in conjunction with the examples, but the implementation and protection of the present invention are not limited thereto. It should be noted that if there are processes that are not particularly described in detail below, they can be implemented or understood by those skilled in the art with reference to the prior art; the reagents or instruments used without indicating the manufacturer are all conventional products that can be purchased commercially.

[0027] Example 1: Preparation of monoclonal antibodies and rabbit polyclonal antibodies

[0028] 1. Preparation of immune antigens

[0029] 1.1. Construction of pET-28a-CHIKV-E2 prokaryotic expression vector

[0030] Chikungunya virus (CHIKV, Ross strain) was taken out from the -80℃ refrigerator and inoculated into BHK cells. The cells were cultured in a cell culture incubator with BHK medium containing 2% serum for 48 hours. The supernatant was collected and centrifuged at 12000g for 10 minutes. The precipitate was discarded. RNA was extracted from the supernatant after centrifugation using a viral DNA / RNA Extraction Kit and reverse transcribed into cDNA using the HiScript® II 1st Strand cDNA Synthesis Kit. The extraction and reverse transcription were performed according to the instructions in the kit manual.

[0031] PCR amplification was performed using the cDNA of the Chikungunya virus as a template, and the primers were as follows:

[0032] CHIKV-E2-F: 5'-CGCGGATCCAGTATTAAGGACCACTTCAATGTC-3';

[0033] CHIKV-E2-R: 5'-CCCAAGCTTTCATAACTGCGGCCAATACTTATAC-3';

[0034] The reaction system (20 μL) was as follows: Premix Taq 10 μL, CHIKV-E2-F 1 μL, CHIKV-E2-R 1 μL, cDNA 2 μL, ddH2O 6 μL; the amplification program was as follows: pre-denaturation: 94°C, 5 min; denaturation: 95°C, 30 s; annealing: 58°C, 30 s; extension: 72°C, 2 min, 40 cycles; extension: 72°C, 10 min. The PCR product was verified by 2% agarose gel, and the agarose gel electrophoresis results showed that a 1038 bp CHIKV-E2 protein gene band was amplified; the target band that met the expected results was recovered by agarose gel electrophoresis DNA gel.

[0035] The gel-recovered product and pET28a were digested with BamHⅠ and Hind Ⅲ at 37℃ for 12h, then connected at 16℃ for 8h, and transformed into DH5α competent cells; the bacterial solution was spread on a plate containing kanamycin and cultured at 37℃ overnight, and a single colony was picked for PCR amplification. After agarose gel electrophoresis detection, the PCR product with the expected band size was sent to Kunming Shuoqing Biotechnology Co., Ltd. for sequencing; the sequencing result was a positive colony, and the BLAST sequence alignment was consistent; the bacterial solution with correct sequencing was expanded and cultured, and the plasmid was extracted with a plasmid mini extraction kit; the recombinant pET28a-CHIKV-E2 plasmid was then transformed into the Rosetta (DE3) expression strain, and a single clone was picked to preserve the strain, and the results are shown in Figure 1, a 1038 bp band was obtained by bacterial liquid PCR amplification. The bacterial liquid PCR results proved that the CHIKV-E2 protein recombinant expression vector had been successfully constructed, and the sequencing peak graph and NCBIBlast comparison results were 100% similar to the target fragment ( Figure 1 B, 1C).

[0036] 1.2 Induction, expression and purification of CHIKV-E2 protein

[0037] To express the recombinant CHIKV-E2 protein (E2 protein), we streaked the expression strain obtained in step 1.1 on LB plates in a "Z" pattern. Escherichia coli Rosetta (DE3) was grown overnight at 37°C in the presence of 50 g / mL kanamycin. After overnight culture (about 12 h), the positive transformants were transferred to 200 mL LB liquid medium and incubated at 37°C with shaking until the bacterial culture OD value reached 0.4-0.6, and then 0.5 mmol / L isopropylthio-D-glycosidase (IPTG) was added. 1 mL of bacterial culture was taken every 4 h for a total of 4 times, and then centrifuged at 6000g (Sorvall, Thermo scientific) for 15 minutes, lysed in loading buffer, and analyzed by 12% SDS-PAGE electrophoresis to observe whether E2 protein was induced.

[0038] The bacterial solution induced to express a large amount of E2 protein was ultrasonicated in ice for 10 minutes (set to open for 3 seconds pulse, close for 3 seconds pulse), and the ultrasonic frequency was set to 250Hz; after the disruption was completed, it was centrifuged at 10000g for 30min, and the supernatant and microspheres (inclusion bodies) were collected at the same time. The microspheres were denatured with 8mol / L urea at 4℃ for 4h, and then centrifuged at 10000g for 30min. The supernatant suspension was collected and impurities were removed by 0.45μm filter membrane; then the E2 antigen was purified by ProteinIso Ni-NTA Resin affinity chromatography. After the E2 protein was purified, the purified protein was detected by SDS-PAGE. The results are shown in Figure 2 After induction by IPTG, a large amount of E2 protein was expressed.

[0039] 2. Preparation of anti-CHIKV-E2 rabbit polyclonal antibody

[0040] Rabbit immunization: Take out the recombinant CHIKV-E2 protein with a measured concentration and immunize each rabbit with 1 mg of protein.

[0041] Immunization; 1 mg of protein was dissolved in 500 μL of sterile PBS and then an equal volume of adjuvant was added, mixed on a vortex shaker for 1 hour, then mixed by blowing and suction with a syringe and injected subcutaneously at multiple points; the experiment was repeated 14 days later, and after multiple immunizations, blood was collected from the ear vein to test the antiserum titer; when the antiserum titer reached the experimental standard, a booster immunization was performed and a large amount of blood was collected from the neck artery; the obtained polyantiserum was purified using a protein A Sepharose column.

[0042] Three days after the fourth immunization of New Zealand white rabbits, a large amount of rabbit blood was collected, and the serum was separated and purified. The purified antibodies were verified by SDS-PAGE, ELISA, and IFA. The results of SDS-PAGE showed that ( Figure 3 ) polyclonal antibodies have been successfully purified; ELISA test results show that ( Figure 4 ) The purified polyclonal antibody can react with the recombinant CHIKV-E2 protein; Figure 5 A is the Western Blot result of rabbit polyclonal antibody serum to detect the expression of recombinant protein, and the IFA result shows that ( Figure 5 B) The prepared rabbit polyclonal antibody can react with native CHIKV-E2 protein.

[0043] 3. Preparation of mouse monoclonal antibodies

[0044] 3.1 Immunization of mice

[0045] Female BALB / c mice aged 4-6 weeks were selected. The immunogen was the prokaryotically expressed recombinant CHIKV E2 protein prepared in step 1. For the first time, Freund's complete adjuvant was mixed with an equal volume of CHIKV E2 antigen for emulsification, and 30 μg was injected subcutaneously at multiple points in each mouse. Thereafter, Freund's incomplete adjuvant and recombinant CHIKV E2 antigen were used for immunization 4 times at intervals of 14 days. Blood was collected from the tail vein 3 days after the third and fourth immunizations, and the titer of the polyclonal antibody serum was detected by indirect ELISA. 100 μg of CHIKVE2 antigen was injected intraperitoneally into each mouse 3 days before fusion for booster immunization.

[0046] 3.2 Preparation and identification of hybridoma cells

[0047] Myeloma cells SP2 / 0 and spleen cells of immune mice were uniformly mixed at a ratio of 1:10 and added to a 50 mL centrifuge tube. PEG preheated at 37°C was added for fusion. The fusion product was added to 50 mL of RPMI-1640 medium containing 15% fetal bovine serum, centrifuged at 800 rpm for 10 min at room temperature, the supernatant was discarded, and the cells were suspended in 50 mL of HAT medium (containing 20% ​​FBS, 2% HAT, RPMI-1640). The cell suspension was added to four 96-well culture plates and incubated at 37°C and 5% CO2 The cells were cultured in a carbon dioxide incubator; on the fifth day after fusion, half of the HT medium was replaced, and the culture supernatant was taken on the 12th-13th day after cell fusion. The clones that were still strongly positive after double screening by indirect ELISA and IFA were cloned by limiting dilution method. The clones were cloned 2-3 times until the positive rate reached 100%, and the cell lines with high antibody secretion titer were selected for amplification and culture, and then stored in liquid nitrogen. After 3 rounds of subcloning, 3 anti-CHIKV-E2 monoclonal antibody cell lines were successfully screened out and named 1B7-H7, 2A2-E10, and 4D3-E8.

[0048] 3.3 Monoclonal Antibody Ascites Preparation and Purification

[0049] The monoclonal antibodies 1B7-H7, 2A2-E10 and 4D3-E8 of the present invention were prepared by an in vivo induction method, i.e., hybridoma cells 1B7-H7, 2A2-E10 and 4D3-E8 were inoculated into mice to prepare ascites. The specific method is as follows: 0.5 mL of Freund's incomplete adjuvant (Sigma) was injected intraperitoneally into each mouse to allow the tumor cells to grow in the form of ascites tumors in the peritoneal cavity; about 1-2 weeks after the mice were intraperitoneally injected with the adjuvant, about 1×10 6 The hybridoma cells in the logarithmic growth phase were suspended in serum-free RPMI 1640 medium and injected into the mouse peritoneal cavity. After 1-2 weeks, the ascites was collected and the supernatant was collected by centrifugation and stored at 4°C for future use. If stored for a long time, it should be placed in a -80°C refrigerator. Mouse monoclonal antibodies were purified using Protein A affinity purification filler from Beijing Quanshijin Biotechnology Co., Ltd. The specific operation method was carried out according to the product manual. The purified antibodies were tested by SDS-PAGE. The results are shown in Figure 3 The results showed that the monoclonal antibody was successfully purified; the purified antibody was verified by ELISA, Western Blot and IFA; the ELISA results are shown in Figure 4 The ascites of the three monoclonal antibodies could react with recombinant CHIKV-E2; Western Blot results showed Figure 5 The mouse monoclonal antibody prepared by A can react with the recombinant CHIKV-E2 protein; IFA results show that the mouse monoclonal antibody prepared can react with the natural CHIKV-E2 protein ( Figure 5B); After purification, the antibody was quantified with BCA protein analysis reagent, and the antibody after concentration determination was added with glycerol at a final concentration of 50% and stored at -80°C.

[0050] 3.4. Indirect immunofluorescence identification of the specificity of monoclonal antibodies and rabbit polyclonal antibodies

[0051] BHK cells were infected with four serotypes of DENV virus (DENV1, DENV2, DENV3, DENV4), ZIKV virus and CHIKV virus, respectively. After 48 h of infection, the cells were washed twice with pre-cooled 1× PBS, and then fixed with fixative (formaldehyde:acetone = 1:1) for 20 minutes. The cells were naturally air-dried and incubated at 37°C with the purified monoclonal antibody in step 3.3 or the rabbit polyclonal antibody in step 2 as the primary antibody and FITC-labeled goat anti-mouse IgG or goat anti-rabbit IgG as the secondary antibody. BHK cells not infected with the virus were set up as negative controls. The cell nuclei were stained with DAPI and the reaction was observed under a fluorescence microscope. The results are shown in Figure 2. Figure 6 The results showed that the three monoclonal antibodies had no cross-reaction with other insect-borne viruses and the specificity was 100%.

[0052] Example 2: Establishment of DAS-ELISA detection method

[0053] 1. Monoclonal antibodies 1B7-H7, 2A2-E10, 4D3-E8 were coupled with HRP to prepare antibody probes

[0054] In the experiment, LinKineTM HRP Labeling Kit from Abbkine Biotech was used for antibody labeling. The specific experimental steps are as follows:

[0055] (1) Pipette 200 μg of antibody into a 1.5 mL EP tube, add 10 μL of HRP labeling buffer, and mix gently;

[0056] (2) Take out the activated HRP solution from the kit, centrifuge it instantly, pipette 7.5 μL of the activated HRP solution into the EP tube, mix it gently, then add 100 μL of ultrapure water, mix it gently, wrap the EP tube with tin foil and keep it away from light at room temperature (20-25°C) for 2 hours;

[0057] (3) Add 1 mL of ultrapure water to HRP quencher powder and oscillate to make a 100× quencher solution. Dilute it 100 times to make a quencher working solution. Note: The 100× quencher solution should be stored at room temperature and used within 48 hours.

[0058] (4) After 2 hours of reaction in the dark, add 15 μL of quencher working solution, mix gently and place at 4°C in the dark for 2 hours. After the reaction is completed, add a certain volume of glycerol to make the concentration of the labeled antibody 1 mg / mL and store at -20°C before use;

[0059] Three monoclonal antibodies were successfully coupled with HRP to make antibody probes. Direct ELISA was used to detect the antibody activity of the three monoclonal antibodies after HRP labeling. The results are shown in Figure 7 The results showed that the three monoclonal antibodies conjugated with HRP were all active, and 2A2-E10 had the best activity.

[0060] 2. Establishment of DAS-ELISA detection method for monoclonal antibody 2A2-E10

[0061] (1) Coated capture antibody: Rabbit polyclonal antibodies used to capture specific antigens in the sample are fixed in the wells of the ELISA plate. Note: The polyclonal antibody is diluted 2-fold with the coating solution to a concentration of 20 μg / mL; 100 μL is added to each well and incubated overnight at 4°C.

[0062] Considering the cost of the kit, the optimal working concentrations of the capture antibody (rabbit polyclonal antibody) and the labeled antibody were determined. The concentration range of the rabbit polyclonal antibody was set at 40μg / mL-0.3125μg / mL. The DAS-ELISA test results showed that the optimal working concentration of the capture antibody was 20μg / mL ( Figure 8 A), the optimal dilution ratio of the detection antibody 2A2-E10 (HRP) is 1:250 ( Figure 8 B);

[0063] (2) Blocking: After coating overnight at 4°C, discard the liquid in the wells. Wash five times with PBS containing 0.05% Tween 20 (PBST). Block the ELISA microplate with 200 μL of 5% skim milk at 37°C and 60 rpm for 2 hours;

[0064] As an important reagent in immunology experiments, blocking solution can be used to block nonspecific binding sites. Blocking agents can effectively reduce nonspecific binding, which mainly comes from the binding of primary or secondary antibodies to antigens. Therefore, blocking helps to reduce background color and enhance the signal-to-noise ratio. In DAS-ELISA experiments, a good blocking solution is very important for improving detection sensitivity and accuracy. In this experiment, three common blocking buffers, 5% skim milk, 1% BSA, and 10% FBS, were used for the experiment. The experimental results showed that the best blocking effect was achieved using 5% skim milk, and its P / N value was greater than that of the other two blocking buffers ( Fig. 9 );

[0065] (3) Adding CHIKV-E2 protein: After blocking, discard the liquid in the wells, wash five times with PBS containing 0.05% Tween (PBST), dilute the E2 protein with 1% BSA, add 2 μg / well, and incubate at 37°C, 60 rpm for 2 h;

[0066] The improvement of DAS-ELISA detection sensitivity depends on the combination of capture antibody and antigen. The ability of antibody to capture antigen is related to the affinity of the antibody itself and the incubation time. In this experiment, the limitation of antibody itself was excluded and only the incubation time of antigen was considered and optimized. The incubation time was 30 min, 60 min, 90 min, 120 min, 150 min and 180 min at 37°C and room temperature respectively. The experimental results showed that the optimal incubation temperature was 37°C and the optimal incubation time was 2h ( Fig.10 );

[0067] (4) Add enzyme-labeled secondary antibody: After incubation for 2 hours, discard the liquid in the wells, wash 5 times with PBST, dilute 2A2-E10 (HRP) antibody to a concentration of 1:250 with 1% BSA, add 100 μL to each well, and incubate the ELISA plate in a constant temperature shaker at 37°C and 60 rpm for 2 hours;

[0068] (5) Color development: After 2 hours, wash the ELISA plate 5 times with PBST, add soluble TMB color development solution at 100 μL / well, and react in a constant temperature shaker at 37°C and 60 rpm for 20 min;

[0069] (6) Termination: Use 2 mol / L H2SO4 to terminate the reaction, add 50 μL to each well, and measure the optical density (OD) at 450 nm using a microplate reader.

[0070] 3. Composition of DAS-ELISA kit

[0071] a. Concentrated cleaning solution (100 mL): 10×PBS containing 5% Tween-20, i.e. 1L of solution contains 35.8g Na2HPO4·12H2O, 2.7g KH2PO3, 80g NaCl. After autoclaving at 121℃ for 20min, add 5mL Tween-20 and stir well. Dilute 10 times before use.

[0072] b. Sample diluent (50 mL): 1% BSA solution;

[0073] c. Specific detection antibody (100 μL): dilute with sample diluent at 1:250 when used;

[0074] d. Positive control (1.5 mL): CHIKV E2 antigen (20 μg / mL);

[0075] e. Negative control (1.5 mL): 1% BSA solution;

[0076] f. Color development solution (15 mL): Soluble single-component TMB substrate color development solution, can be used directly;

[0077] g. Stop solution (15 mL): 2 mol / L H2SO4;

[0078] h. Microwell reaction plate (8×12).

[0079] 2. The preparation method of the microwell reaction plate is as follows: the rabbit polyclonal antibody prepared in Example 1 is diluted to 20 μg / mL with CBS buffer, and 100 μL / well is coated on a polystyrene 96-well microwell plate, and the plate is kept at 4°C overnight. After patting dry, 200 μL of 5% skim milk blocking solution is added to each well, and the plate is blocked at 37°C for 2 hours to block nonspecific binding sites. The plate strips are patted dry, vacuum dried for 12-24 hours, and vacuum packed in aluminum film bags and stored at 4°C for future use.

[0080] Example 3: Evaluation of specificity and sensitivity of DAS-ELISA detection method

[0081] 1. Specificity experiment

[0082] (1) Sample selection and preparation

[0083] The plaque assay was used to determine the virus titers of CHIKV, four serotypes of DENV (DENV1, DENV2, DENV3, DENV4), and ZIKV. The virus titers were CHIKV: 3×10 10 PFU / mL, DENV-1:3.16×10 9 PFU / mL, DENV-2: 3.16×10 9 PFU / mL, DENV-3: 1.58×10 9 PFU / mL, DENV-4: 1.58×10 9 PFU / mL, ZIKV: 2.16×10 9 PFU / mL.

[0084] (2) Detection method

[0085] Take 100 μL of the above-mentioned virus sample to be tested and add it to the microwell reaction plate coated with rabbit polyclonal antibody for capturing antigen. At the same time, set up a negative control (1% BSA solution, without CHIKV E2 protein) and a positive control (CHIKV E2 antigen, concentration: 20 μg / mL). Incubate at 37°C for 2 hours, wash the strips after diluting 10 times with concentrated washing solution, wash the strips 5 times with 200 μL / well; add 100 μL / well specific detection antibody (2A2-E10-HRP), dilute it at 1:250, and incubate at 37°C for 2 hours; after washing the strips 5 times as above, add 100 μL / well substrate color development solution, color at room temperature in the dark for 20 minutes, add stop solution, 50 μL / well, stop the reaction, and measure the OD450nm value with an enzyme reader.

[0086] (3) Result determination: The blank well was adjusted to zero and the absorbance (A value) was measured at a wavelength of 450 nm. If the average value of the positive control was ≥ 0.50 and the average value of the negative control was ≤ 0.15, the experiment was considered valid. If the sample A value was ≥ the average value of the negative control A value × 3, it was considered positive, otherwise it was negative.

[0087] The kit of the present invention was used to detect inactivated CHIKV virus liquid, four serotypes of DENV, and ZIKV virus liquid. The test results showed that the kit of the present invention can specifically identify CHIKV virus and has no cross reaction with the other five flavivirus viruses ( Fig.11 ).

[0088] 2. Sensitivity test

[0089] (1) Sample selection and preparation

[0090] After inactivation of CHIKV virus, 1×10 10 PFU / mL was used for DAS-ELISA detection by diluting the sample tenfold from the beginning. At the same time, 100 μL of the diluted sample was taken to extract RNA using RNAisoPlus (Ta Ka Ra, China) and RT-qPCR was performed simultaneously to determine the sensitivity of the detection method;

[0091] (2) The detection method is the same as step 1

[0092] The kit of the present invention was used to detect the gradient dilution of inactivated CHIKV virus solution, and the results showed that the DAS-ELISA detection sensitivity was 1×10 6 PFU / mL, the results are shown in the table below;

[0093] The sensitivity of the kit of the present invention in detecting the dilution ratio of CHIKV virus culture fluid

[0094] ;

[0095] Table Notes: The results of the kit of the present invention are determined as follows: the sample detection value ≥ 3 times the average value of the negative control (calculated as 0.0628×3=0.1884) is positive (+), otherwise it is negative (-); the results of the Chikungunya virus nucleic acid detection kit are determined as follows: the test sample CT value ≤ 36 is positive.

[0096] Example 4: Application of the detection kit in clinical sample detection

[0097] 1. Sample collection

[0098] Ten CHIKV-positive cultures, 20 DENV-positive sera, and 20 normal controls were used for clinical testing and analysis; CHIKV-positive cultures consisted of 1×10 9 PFU / mL of virus culture fluid was mixed with normal human serum in a 1:1 ratio;

[0099] 2. Sample capture

[0100] Take 100 μL of the above-mentioned sample to be tested and add it to the ELISA microwell reaction plate coated with rabbit polyclonal antibody for capturing antigens, incubate at 37°C for 2 hours to capture the specific antigens in the sample;

[0101] 3. Add 2A2-E10-HRP

[0102] After incubation for 2 hours, the liquid in the wells was discarded, and the wells were washed 5 times with PBST. The detection antibody 2A2-E10 (HRP) was diluted to a concentration of 1:250 with 1% BSA (sample diluent), 100 μL was added to each well, and the ELISA plate was placed in a constant temperature shaker at 37°C and 60 rpm for incubation for 2 hours;

[0103] 4. Color reaction

[0104] After 2 h, the ELISA plate was washed 5 times with PBST, and soluble TMB colorimetric solution was added at 100 μL / well, and the reaction was carried out in a constant temperature shaker at 37°C and 60 rpm for 20 min;

[0105] 5. Termination of the reaction

[0106] The reaction was terminated with 2 mol / L H2SO4, 50 μL was added to each well, and the optical density (OD) was measured at 450 nm using a microplate reader.

[0107] The experimental results are shown in Fig.12 The results showed that the DAS-ELISA kit had good performance in clinical sample testing with a specificity of 100%. Fig.12 A shows that except for 10 cases that tested positive for CHIKV culture, all samples were negative. Sequence Listing <110> Kunming University of Science and Technology <120> Monoclonal antibody specifically binding to Chikungunya virus E2 protein and its application <160> 6 <170> SIPOSequenceListing 1.0 <210> 1 <211> 111 <212> PRT <213> Mouse (Mus musculus) <400> 1 Asp Ile Glu Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Asp Met Tyr 20 25 30 Gly Pro Ser Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr Ile Asp 65 70 75 80 Pro Val Glu Ala Asp Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Asn Ser 85 90 95 Glu Asp Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 2 <211> 119 <212> PRT <213> Mouse (Mus musculus) <400> 2 Glu Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Ser Leu Ser Leu Thr Cys Thr Val Thr Gly Tyr Phe Ile Cys Ser Asp 20 25 30 Tyr Ala Trp Asn Trp Ile Arg Gln Leu Pro Gly Asn Lys Leu Glu Trp 35 40 45 Met Gly Tyr Ile Ser Trp Ser Gly Leu Thr Ser Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe 65 70 75 80 Leu Gln Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys 85 90 95 Ala Arg Lys Gly Arg Ile Tyr Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Pro Leu Thr Val Ser Ser 115 <210> 3 <211> 108 <212> PRT <213> Mouse (Mus musculus) <400> 3 Asp Ile Gln Leu Thr Gln Pro Pro Ser Tyr Leu Ala Ala Ser Pro Gly 1 5 10 15 Glu Thr Ile Thr Ile Asn Cys Arg Ala Ser Lys Ser Tyr Ser Lys Tyr 20 25 30 Cys Leu Ala Trp Tyr Gln Glu Lys Pro Gly Lys Thr Asn Lys Leu Leu 35 40 45 Ile Tyr Ser Gly Ser Thr Leu Gln Ser Gly Ile Pro Ser Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Met Tyr Tyr Cys Gln Gln Gly Asn Met Tyr Pro 85 90 95 Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 4 <211> 122 <212> PRT <213> Mouse (Mus musculus) <400> 4 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Lys Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr His Asn Thr Tyr 20 25 30 Asn Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Ser Gly Ser Asn Met Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Gln Ser Ile 65 70 75 80 Leu Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Val Met Tyr 85 90 95 Tyr Cys Glu Arg Pro Asp Tyr Ser Gly Ser Ser Tyr Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Thr Leu Thr Val Ser Ser 115 120 <210> 5 <211> 107 <212> PRT <213> Mouse (Mus musculus) <400> 5 Asp Ile Gln Leu Thr Gln Pro Pro Ser Tyr Leu Ala Ala Ser Pro Gly 1 5 10 15 Glu Thr Ile Thr Ile Asn Cys Arg Ala Ser Lys Ser Cys Ser Lys Tyr 20 25 30 Leu Ala Trp Tyr Gln Glu Lys Pro Gly Lys Thr Asn Lys Leu Leu Ile 35 40 45 Tyr Ser Gly Ser Thr Leu Gln Ser Gly Ile Pro Ser Arg Phe Ala Gly 50 55 60 Leu Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Met Tyr Tyr Cys Gln Glu His Asn Glu Tyr His Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 6 <211> 116 <212> PRT <213> Mouse (Mus musculus) <400> 6 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Lys Gly 1 5 10 15 Ser Leu Lys Leu Ala Ala Ser Gly Phe Thr Arg Asn Thr Tyr Asn Met 20 25 30 Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Arg 35 40 45 Ile Gly Ser Gly Ser Asn Lys Tyr Ala Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Asp Arg Phe Thr Ile Ser Gln Ser Ile Leu Tyr Leu Gln Met Asn 65 70 75 80 Asn Leu Lys Thr Glu Asp Thr Val Met Tyr Tyr Cys Glu Arg Pro Cys 85 90 95 Tyr Ser Gly Ser Phe Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu 100 105 110 Thr Val Ser Ser 115

Claims

1. A monoclonal antibody that specifically binds to the E2 protein of Chikungunya virus, characterized in that: The monoclonal antibody is 1B7-H7, the amino acid sequence of its light chain variable region is shown in SEQ ID NO:1, and the amino acid sequence of its heavy chain variable region is shown in SEQ ID NO:2; or the monoclonal antibody is 2A2-E10, the amino acid sequence of its light chain variable region is shown in SEQ ID NO:3, and the amino acid sequence of its heavy chain variable region is shown in SEQ ID NO:4; or the monoclonal antibody is 4D3-E8, the amino acid sequence of its light chain variable region is shown in SEQ ID NO:5, and the amino acid sequence of its heavy chain variable region is shown in SEQ ID NO:

6.

2. Use of the monoclonal antibody according to claim 1 in preparing a reagent for specific detection of Chikungunya virus E2 antigen.

3. Use of the monoclonal antibody according to claim 1 in preparing a kit for specifically detecting Chikungunya virus E2 antigen.

4. The use according to claim 3, characterized in that: The kit is a DAS-ELISA kit for specifically detecting the E2 antigen of Chikungunya virus.

5. The use according to claim 4, characterized in that: The kit includes a microporous reaction plate coated with rabbit polyclonal antibody for capturing antigens, a sample processing solution, a monoclonal antibody combined with a marker, a positive control substance, a negative control substance, a concentrated washing solution, a color developing solution, and a stop solution.

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