Monoclonal antibodies against DENV1 NS1 and their applications

Abstract

This application belongs to the field of biomedical technology, specifically relating to a monoclonal antibody against DENV1 NS1 and its application. The monoclonal antibody includes a capture antibody and a detection antibody; the amino acid sequences of the heavy chain complementarity-determining regions (CDR1, CDR2, and CDR3) of the capture antibody are shown in SEQ ID NO. 5-SEQ ID NO. 9 and SEQ ID NO. 29, respectively; the amino acid sequences of the heavy chain complementarity-determining regions (CDR1, CDR2, and CDR3) of the detection antibody are shown in SEQ ID NO. 19-SEQ ID NO. 23 and SEQ ID NO. 31, respectively. Both the capture antibody and the detection antibody provided by this invention exhibit high affinity and specific detection activity against the DENV1 NS1 antigen.

Description

Technical Field

[0001] This application belongs to the field of biomedical technology, specifically relating to a monoclonal antibody against DENV1 NS1 and its application. Background Technology

[0002] Dengue virus (DENV) is a single-stranded positive-sense RNA virus belonging to the Flaviviridae genus. It is transmitted from arthropods to mammals and can cause asymptomatic infection, dengue fever, and dengue hemorrhagic fever in humans. Globally, approximately 50 to 100 million people are infected with dengue fever annually, with about 20,000 deaths. The DENV genome encodes several non-structural proteins, including the NS1 protein, a highly conserved non-structural glycoprotein that plays a crucial role in viral replication and is present in the serum of patients during the acute phase of dengue infection, making it a good target for early diagnosis. However, screening for broad-spectrum NS1 antibodies is hampered by several factors, including the lack of antigenic stimulation from natural hexamers, low screening efficiency due to immunocompromised epitopes, and the limited availability of structural analysis techniques. This has resulted in a shortage of reported NS1 antibodies to date.

[0003] Monoclonal antibodies (mAbs) play a crucial role in disease diagnosis and treatment due to their high specificity and homogeneity. In the field of dengue virus diagnosis, monoclonal antibodies can be used to develop rapid diagnostic reagents, such as enzyme-linked immunosorbent assays (ELISA) and double-antibody sandwich ELISA, enabling rapid differential diagnosis of DENV infection. Furthermore, monoclonal antibodies can also be used to competitively bind to the DENV NS1 protein, inhibit vascular leakage symptoms, and / or provide in vivo protection against lethal damage from DENV infection, offering new directions for dengue vaccine development or the development of therapeutic drugs for DENV viral infections.

[0004] Obtaining high-affinity, high-specificity monoclonal antibodies against DENV1 NS1 is crucial for the highly sensitive and accurate detection of DENV1 NS1, and is also the key factor limiting the performance of immunoassay methods for DENV1 NS1. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide a monoclonal antibody against DENV1 NS1 and its application, specifically adopting the following technical solution:

[0006] In a first aspect, the present invention provides a monoclonal antibody against DENV1 NS1, the monoclonal antibody comprising a capture antibody (hereinafter referred to as D11) and a detection antibody (hereinafter referred to as D10).

[0007] The amino acid sequences of the heavy chain complementarity-determining regions CDR1, CDR2, and CDR3 of the capture antibody are shown in SEQ ID NO. 5, SEQ ID NO. 6, and SEQ ID NO. 7, respectively.

[0008] The amino acid sequences of the light chain complementarity-determining regions CDR1, CDR2, and CDR3 of the capture antibody are shown in SEQ ID NO. 8, SEQ ID NO. 29, and SEQ ID NO. 9, respectively.

[0009] The amino acid sequences of the heavy chain complementarity-determining regions CDR1, CDR2, and CDR3 of the detection antibody are shown in SEQ ID NO. 19, SEQ ID NO. 20, and SEQ ID NO. 21, respectively.

[0010] The amino acid sequences of the light chain complementarity-determining regions CDR1, CDR2, and CDR3 of the detection antibody are shown in SEQ ID NO. 22, SEQ ID NO. 31, and SEQ ID NO. 23, respectively.

[0011] As a further preferred embodiment, the amino acid sequences of the heavy chain complementarity-determining region and the light chain complementarity-determining region of the capture antibody are shown in SEQ ID NO. 1 and SEQ ID NO. 2, respectively;

[0012] The amino acid sequences of the heavy chain complementarity-determining region and the light chain complementarity-determining region of the detection antibody are shown in SEQ ID NO. 15 and SEQ ID NO. 16, respectively.

[0013] The specific amino acid sequence described above is as follows:

[0014] SEQ ID NO. 1:

[0015] QVQLQQSGAELMKPGASVKISCKASGYTFNTYWIEWVKQRPGHGLEWIGEILPGSRSFNYDEKFKGKATFTADTSSNTAYMQLSSLTSEDSAVYYCARGDYGDYAMDYWGQGTSVTVSS.

[0016] SEQ ID NO.2:

[0017] DIVMTQTPSSLSASLGDRVTISCSASQGIGNYLNWYQQKPDGTVKLLIYYTSSLHSGVPSRFSGSGSGTDFSLTISNLEPEDIATYYCQQYSKLYTFGGGTKLEIK.

[0018] SEQ ID NO. 15:

[0019] QVTLKESGPGILKPSQTLSLTCSFSGFSLSTSGLGVGWIRQPSGKGLEWLAHIWWDDEKFYNPSLKSRLTISKDTSRNQVFLKITSVDTADTATYFCARSPYYGMDYWGQGTSVIVSS。

[0020] SEQ ID NO. 16:

[0021] DIEMTQSPSSMYASLGERVTITCKASQDINSYLSWLQQKPGKSPKTLIYRANRLVDGVPSRFSGSGSGQDYSLTISSLEYEDMGIYYCLQYDELYTFGGGTKLEIK。

[0022] SEQ ID NO.5(CDR1):GYTFNTYW。

[0023] SEQ ID NO.6(CDR2):ILPGSRSF。

[0024] SEQ ID NO.7(CDR3):ARGDYGDYAMDY。

[0025] SEQ ID NO.8(CDR1):QGIGNY。

[0026] SEQ ID NO.29(CDR2):YTS。

[0027] SEQ ID NO.9(CDR3):QQYSKLYT。

[0028] SEQ ID NO.19(CDR1):GFSLSTSGLG。

[0029] SEQ ID NO.20(CDR2):IWWDDEK。

[0030] SEQ ID NO.21(CDR3):ARSPYYGMDY。

[0031] SEQ ID NO.22(CDR1):QDINSY。

[0032] SEQ ID NO.31(CDR2):RAN。

[0033] SEQ ID NO.23(CDR3):LQYDELYT。

[0034] In a second aspect, the present invention provides a polynucleotide for encoding the aforementioned monoclonal antibody against DENV1 NS1;

[0035] The nucleotide sequences encoding SEQ ID NO. 5, SEQ ID NO. 6, and SEQ ID NO. 7 are shown in SEQ ID NO. 10, SEQ ID NO. 11, and SEQ ID NO. 12, respectively.

[0036] The nucleotide sequences encoding SEQ ID NO. 8, SEQ ID NO. 29, and SEQ ID NO. 9 are shown in SEQ ID NO. 13, SEQ ID NO. 30, and SEQ ID NO. 14, respectively.

[0037] The nucleotide sequences encoding SEQ ID NO. 19, SEQ ID NO. 20, and SEQ ID NO. 21 are shown in SEQ ID NO. 24, SEQ ID NO. 25, and SEQ ID NO. 26, respectively.

[0038] The nucleotide sequences encoding SEQ ID NO. 22, SEQ ID NO. 31, and SEQ ID NO. 23 are shown in SEQ ID NO. 27, SEQ ID NO. 32, and SEQ ID NO. 28, respectively.

[0039] The nucleotide sequences encoding SEQ ID NO. 1 and SEQ ID NO. 2 are shown in SEQ ID NO. 3 and SEQ ID NO. 4, respectively;

[0040] The nucleotide sequences encoding SEQ ID NO. 15 and SEQ ID NO. 16 are shown in SEQ ID NO. 17 and SEQ ID NO. 18, respectively.

[0041] The specific nucleotide sequence described above is as follows:

[0042] SEQ ID NO. 3:

[0043] caggttcagctgcagcagtctggagctgagctgatgaagcctggggcctcagtgaagatatcctgcaaggcttctggctacacattcaatacctactggatagagtgggtaaagcagaggcctggacatggccttgagtggattggagagattttacctggaagtcgtagttttaactacgatgagaagttcaagggcaaggccacattcactgcagatacatcctccaacacagcctacatgcaactcagcagcctgacgtctgaggactctgccgtctattactgtgcaagaggggactatggagactatgctatggactactggggtcaaggaacctcagtcaccgtctcctct。

[0044] SEQ ID NO .4:

[0045] gacattgtgatgacccagactccatcctccctgtctgcctctctgggagacagagtcaccatcagttgcagtgcaagtcagggcattggcaattatttaaactggtatcagcagaaaccagatggaactgttaaactcctgatctattacacatcaagtttacactcaggagtcccatcaaggttcagtggcagtgggtctgggacagatttttctctcaccatcagcaacctggaacctgaagatattgccacttactattgtcagcagtatagtaagttgtacacgttcggaggggggaccaaactggaaataaaa。

[0046] SEQ ID NO .17:

[0047] caagttactctaaaagagtctggccctgggatattgaagccctcacagaccctcagtctgacttgttctttctctgggttttcactgagcacttctggtctgggtgtaggctggattcgtcagccttcagggaagggtctggagtggctggcgcacatttggtgggatgatgagaagttctataacccatccctgaagagccggctcacaatctccaaggatacctccagaaaccaggtattcctcaagatcaccagtgtggacactgcagatactgccacttacttctgtgctcgaagtccatactatggtatggactactggggtcaaggaacctcagtcatcgtctcctca。

[0048] SEQ ID NO .18:

[0049] gacattgagatgacacagtctccatcttccatgtatgcatctctaggagagagagtcactatcacttgcaaggcgagtcaggacattaatagctatttaagctggctccagcagaaaccagggaaatctcctaagaccctgatctatcgtgcaaacagattggtagatggggtcccatcaaggttcagtggcagtggatctgggcaagattattctctcaccatcagcagcctggagtatgaagatatgggaatttattattgtctacagtatgatgagttgtacacgttcggaggggggaccaagctggaaataaaa。

[0050] SEQ ID NO .10:ggctacacattcaatacctactgg。

[0051] SEQ ID NO .11:attttacctggaagtcgtagtttt。

[0052] SEQ ID NO .12:gcaagaggggactatggagactatgctatggactac。

[0053] SEQ ID NO.13: cagggcattggcaattat.

[0054] SEQ ID NO. 30: tacacatca.

[0055] SEQ ID NO.14:cagcagtatagtaagttgtacacg.

[0056] SEQ ID NO.24: gggttttcactgagcacttctggtctgggt.

[0057] SEQ ID NO.25: atttggtgggatgatgagaag.

[0058] SEQ ID NO.26: gctcgaagtccatactatggtatggactac.

[0059] SEQ ID NO.27: caggacattaatagctat.

[0060] SEQ ID NO. 32: cgtgcaaac.

[0061] SEQ ID NO.28:ctacagtatgatgatgagttgtacacg.

[0062] Thirdly, the present invention provides the application of the above-mentioned monoclonal antibody in the preparation of a kit for immunological detection.

[0063] Fourthly, the present invention provides a kit for immunological detection, the kit comprising the aforementioned monoclonal antibody against DENV1 NS1.

[0064] As a further preferred embodiment, the kit is an enzyme-linked immunosorbent assay (ELISA) kit.

[0065] As a further preferred embodiment, the kit is used to detect dengue virus infection.

[0066] Fifthly, the present invention provides a method for detecting DENV1 NS1 for non-diagnostic purposes, the method comprising using the above-described anti-DENV1 NS1 monoclonal antibody.

[0067] As a further preferred embodiment, the anti-DENV1 NS1 monoclonal antibodies are paired and the DENV1 NS1 content in the sample is detected using a double-antibody sandwich method.

[0068] As a further preferred embodiment, the above-mentioned double-antibody sandwich method specifically includes the following steps:

[0069] S1: Coat the capture antibody in a well plate and incubate at 37 °C for 2 h or at 4 °C overnight, then wash 3 times with 0.05% PBST.

[0070] S2: After blocking the well plate with skim milk for 2 h, wash three times with 0.05% PBST;

[0071] S3: Add the sample to be tested and the HRP-labeled detection antibody in sequence and incubate.

[0072] S4: After incubation, add TMB colorimetric solution. After 7 min of reaction, add 50 μl of 2 M H2SO4 to terminate the reaction. Take readings at a wavelength of 450 nm.

[0073] The beneficial effects of this invention are as follows:

[0074] This invention provides a monoclonal antibody against DENV1 NS1, comprising a capture antibody and a detection antibody. Experimental results show that both the capture and detection antibodies exhibit high affinity and specificity for the DENV1 NS1 antigen. This anti-DENV1 NS1 monoclonal antibody can be used in a double-antibody sandwich assay for the immunological analysis of DENV1 NS1, including enzyme-linked immunosorbent assay (ELISA) based on antigen-antibody specific reactions. The detection limit of the double-antibody sandwich ELISA reaches 6.17 ng / mL, with a linear detection range of 6.17-500 ng / mL, which is significant for the highly sensitive and accurate detection of DENV1 NS1. Furthermore, it has been reported that the concentration of NS1 in the blood of DENV1-infected individuals is approximately 0.01-50 μg / ml; the detection limit of this invention can achieve the detection of NS1 in serum samples. Attached Figure Description

[0075] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0076] Figure 1 These are SDS-PAGE identification images of DENV1 NS1 eukaryotic expression provided in some embodiments of this application;

[0077] Figure 2These are reduced SDS-PAGE identification images of capture antibody D11 and detection antibody D10 provided in some embodiments of this application;

[0078] Figure 3 These are verification diagrams of the effect of sandwich ELISA detection of DENV1 NS1 based on antibodies D11 and D10 provided in some embodiments of this application;

[0079] Figure 4 These are verification diagrams showing the effectiveness of sandwich ELISA detection of serum samples containing DENV1NS1 based on antibodies D11 and D10, provided by some embodiments of this application. Detailed Implementation

[0080] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0081] Example 1

[0082] Eukaryotic expression of DENV1 NS1 antigen

[0083] 1) Resuscitation of suspended cells:

[0084] 1. Preparation: Place the OPM-293 Hi-exp medium in a 37 ℃ water bath for 30 min and turn on the ultraviolet light in the clean bench for 30 min.

[0085] 2. Take a 125 mL cell culture flask, sterilize it with 75% ethanol, and place it in a clean bench. Use a sterile glass pipette to take 29 mL of preheated culture medium into the cell culture flask.

[0086] 3. Determine the exact location of the Expi293F™ cells to be thawed in the cryovial. After removal, rapidly thaw them in a water bath (approximately 1 min). After sterilization with 75% ethanol, place them in a clean bench and transfer the thawed cells to a cell culture flask using a pipette, gently mixing. Observe the cell state using 300 μl of the flask in a 24-well plate.

[0087] 4. Label the cell information on the cell culture flask, and then culture the cells in a cell culture shaker at 37 ℃, 8% CO2, and relative humidity ≥80% at a speed of 120 ± 5 rpm.

[0088] 2) Construction of NS1 eukaryotic expression plasmid: The base sequence corresponding to the NS1 protein was constructed into the PCMV3 expression vector using a seamless cloning method to facilitate subsequent eukaryotic expression. The vector plasmid DNA containing the target gene was prepared in advance using an endotoxin-free plasmid mini-prep kit, and sterilized by filtration through a sterile 0.22 μm aqueous filter membrane before being transferred to 2 mL centrifuge tubes. The base sequence used for NS1 is:

[0089]

[0090] 3) Plasmid transfection:

[0091] OPM-293 Hi-exp medium and Opti-MEM I serum-depleted medium were preheated in a 37 ℃ water bath for 30 min, and then exposed to UV light for 30 min in a clean bench. The viable cell density was increased to 4.5–5.5 × 10⁻⁶ cells / mL. 6 When the cell count is ≥95% and the cell viability is ≥95%, transfer all the cell culture medium from the cell culture flask to a 50 mL centrifuge tube, balance the medium, centrifuge at 300 g for 5 min, and discard the supernatant. Use a sterile pipette to aspirate 2-5 mL of OPM-293 Hi-exp medium, resuspend the cells by pipetting with a bent-tip pipette, and transfer to a container containing 94.622 mL of fresh, preheated OPM-293 Hi-exp medium. Sterilize and seal the flask, then return it to the cell shaker for further culture. Take a 50 mL sterile centrifuge tube and add OptiMEM type I serum-depleted medium, PEI, and DNA in the following order, mixing thoroughly by pipetting. Incubate at room temperature for 15 min. Remove the cell culture flask from the cell shaker and slowly add the prepared PEI-DNA complex to the flask using a circular motion. Sterilize and seal the flask, then return it to the cell shaker for further culture.

[0092] 4) Cells were cultured for 7 days at 37 ℃, 8% CO2, and relative humidity ≥80% in a shaker at 120 ±5 rpm. The obtained NS1 was purified using a nickel column and identified by SDS-PAGE. Figure 1 ).

[0093] Example 2

[0094] Preparation of DENV1 NS1 monoclonal antibody

[0095] (1) Animal immunization: DENV1 NS1 antigen was used as the immunogen and mixed with an equal volume of Freund's adjuvant. The mixture was vortexed until the immunogen was completely emulsified and immunized by multiple subcutaneous injections into the abdomen and back of BaLB / C mice. For the first immunization, Freund's complete adjuvant was used, and the immunogen dose was 120 μg / mouse. For subsequent immunizations, Freund's incomplete adjuvant was used, and the immunogen dose was gradually reduced. Immunization was performed once every 4 weeks for a total of 3 immunizations. Two weeks after the third immunization, tail blood was collected from the mice, and the serum antibody titer in the tail blood was determined by indirect ELISA. Three days before fusion, the mice with the highest titer were directly immunized with the immunogen at a booster dose of 50 μg / mouse.

[0096] (2) Preparation and screening of hybridoma cells: Selected immunized mice were euthanized by cervical dislocation, and the spleens were removed and ground in a clean bench. Mouse myeloma cells SP2 / 0 and spleen cells were mixed at a ratio of 1:10, and preheated 50% PEG was added for cell fusion. The mixture was then added to 96-well cell culture plates containing feeder cells. After fusion, the cell status was observed under a microscope. On the 5th day after fusion, a semi-quantitative medium change was performed, and the culture was continued until the 7th day, after which the medium was completely changed.

[0097] Approximately 7-10 days after cell fusion, the cell supernatant was aspirated from the cell culture plate. The level of secreted antibodies in the supernatant was measured using an indirect ELISA method. Positive cells were screened in the wells, and subcloning was performed using a limiting dilution method. The cells were coated with DENV1 NS1 protein. Blank control wells used PBS, negative controls used culture medium, and positive controls used serum from immunized mouse ocular blood. When the selected monoclonal cells showed a 100% positivity rate in a 96-well cell culture plate, they were considered positive monoclonal cells and were cryopreserved and expanded for further culture as appropriate.

[0098] (3) Preparation and purification of monoclonal antibody ascites: Balb / c mice approximately 8 weeks old were pre-stimulated with liquid paraffin one week in advance to promote the secretion and accumulation of nutrients in the peritoneal cavity. The obtained hybridoma cell line was expanded to the required number, centrifuged, carefully washed, resuspended in sterile 75% physiological saline, and injected into the mouse abdomen via intraperitoneal injection. After about one week, when the mouse abdomen was significantly distended, ascites was collected, centrifuged at 8000 r / min for 15 min, and the supernatant was collected as monoclonal antibody ascites.

[0099] The collected ascites fluid was purified using a Protein G affinity chromatography column. Before loading, the ascites fluid was filtered through a microporous membrane and used as the loading solution. The Protein G column was equilibrated with binding buffer (0.15 M NaCl, 20 mM Na2HPO4, pH 7.4). The filtered ascites fluid was then loaded onto the chromatography column. After equilibration, elution was performed using elution buffer (0.1 M citric acid, pH 2.5–3.0), and the antibody-rich eluent was collected. The eluent was acidic and the pH was immediately adjusted to neutral using neutralization buffer (1 M Tris-HCl, pH 9.0) to prevent antibody inactivation. Subsequent SDS-PAGE electrophoresis was used for identification, yielding capture antibody D11 and detection antibody D10 (see attached image). Figure 2 ).

[0100] (4) Determination of the heavy and light chain variable regions of monoclonal antibodies secreted by hybridoma cells

[0101] Hybridoma cell lines capable of stably secreting monoclonal antibodies were cultured to 1×10⁻⁶ cells / year. 6Cells / mL, discard the supernatant, add 1 mL Trizol reagent, gently vortex, then add 200 μL chloroform, invert and vortex for 40 s, and incubate on ice for 15 min; centrifuge at 4 °C, 12000 g for 15 min, transfer the supernatant to a new enzyme-free centrifuge tube, add an equal volume of isopropanol, and incubate on ice for 10 min; centrifuge at 4 °C, 12000 g for 10 min, discard the supernatant, add 1 mL 75% ethanol, centrifuge at 4 °C, 7500 g for 5 min, dry in a clean bench at low speed with ventilation for 5 min, add 30–50 μL enzyme-free sterile water, mix well to dissolve RNA. Using 1 μg total RNA as a template, reverse transcription is performed according to the Hifair III 1st Strand cDNA Synthesis Kit instructions. Primers for the upstream and downstream variable regions of the heavy and light chains of the monoclonal antibody were designed (primers are shown in Table 1 below). The cDNA obtained by reverse transcription was amplified using the following reaction parameters: 94 ℃ for 5 min, 94 ℃ for 10 s, 60 ℃ for 20 s, 72 ℃ for 30 s, for a total of 25 cycles, followed by 72 ℃ for 5 min. The amplified products were identified by 1% agarose gel electrophoresis, and the amino acid sequences of the products were determined to obtain the amino acid sequences of the monoclonal antibody against DENV1 NS1, including the capture antibody (hereinafter referred to as D11) and the detection antibody (hereinafter referred to as D10).

[0102] Table 1. Sequencing primers for the D10 and D11 light and heavy chain variable regions

[0103]

[0104] The capture antibody comprises the amino acid sequence: QVQLQQSGAELMKPGASVKISCKASGYTFNTYWIEWVKQRPGHGLEWIGEILPGSRSFNYDEKFKGKATFTADTSSNTAYMQLSSLTSEDSAVYYCARGDYGDYAMDYWGQGTSVTVSS (SEQ ID NO. 1) and the amino acid sequence: DIVMTQTPSSLSASLGDRVTISCSASQGIGNYLNWYQQKPDGTVKLLIYYTSSLHSGVPSRFSGSGSGTDFSLTISNLEPEDIATYYCQQYSKLYTFGGGTKLEIK (SEQ ID NO. 2);

[0105] The detection antibody comprises the amino acid sequence: QVTLKESGPGILKPSQTLSLTCSFSGFSLSTSGLGVGWIRQPSGKGLEWLAHIWWDDEKFYNPSLKSRLTISKDTSRNQVFLKITSVDTADTATYFCARSPYYGMDYWGQGTSVIVSS (SEQ ID NO. 15) and the amino acid sequence: DIEMTQSPSSMYASLGERVTITCKASQDINSYLSWLQQKPGKSPKTLIYRANRLVDGVPSRFSGSGSGQDYSLTISSLEYEDMGIYYCLQYDELYTFGGGTKLEIK (SEQ ID NO. 16).

[0106] A monoclonal antibody against DENV1 NS1, wherein the amino acid sequences CDR1, CDR2, and CDR3 of the complementarity-determining region of the heavy chain of the capture antibody are as follows:

[0107] CDR1: GYTFNTYW (SEQ ID NO.5);

[0108] CDR2: ILPGSRSF (SEQ ID NO.6);

[0109] CDR3: ARGDYGDYAMDY (SEQ ID NO. 7).

[0110] A monoclonal antibody against DENV1 NS1, wherein the amino acid sequences CDR1, CDR2, and CDR3 of the complementarity-determining region of the light chain of the capture antibody are as follows:

[0111] CDR1: QGIGNY (SEQ ID NO.8);

[0112] CDR2: YTS;

[0113] CDR3: QQYSKLYT (SEQ ID NO.9).

[0114] A monoclonal antibody against DENV1 NS1, wherein the amino acid sequences CDR1, CDR2, and CDR3 of the complementarity-determining region of the heavy chain of the antibody are as follows:

[0115] CDR1:GFSLSTSGLG (SEQ ID NO.19);

[0116] CDR2:IWWDDEK (SEQ ID NO.20);

[0117] CDR3: ARSPYYGMDY (SEQ ID NO. 21).

[0118] A monoclonal antibody against DENV1 NS1, wherein the amino acid sequences CDR1, CDR2, and CDR3 of the complementarity-determining region of the antibody's light chain are as follows:

[0119] CDR1: QDINSY (SEQ ID NO.22);

[0120] CDR2: RAN;

[0121] CDR3: LQYDELYT (SEQ ID NO. 23).

[0122] The amino acid sequence of the above immunogen is as follows:

[0123] DSGCVINWKGRELKCGSGIFVTNEVHTWTEQYKFQADSPKRLSAAIGKAWEEGVCGIRSATRLENIMWKQISNELNHILLENDMKFTVVVGDVAGILAQGKKMIRPQPMEHKYSWKSWGKAKIIGADVQNTTFIIDGPNTPECPDDQRAWNIWEVEDYGFGIFTTNIWLKLRDSYTQV CDHRLMSAAIKDSKAVHADMGYWIESEKNETWKLARASFIEVKTCIWPKSHTLWSNGVLESEMIIPKIYGGPISQHNYRPGYFTQTAGPWHLGKLELDFDLCEGTTVVVDEHCGNRGPSLRTTTVTGKIIHEWCCRSCTLPPLRFRGEDGCWYGMEIRPVKEKEENLVKSMVSA (SEQ ID NO.34)

[0124] Example 3

[0125] DENV1 NS1 monoclonal antibody is used for the determination of DENV1 NS1 antigen in a double-antibody sandwich ELISA (specific assay of DENV1 NS1 monoclonal antibody).

[0126] Monoclonal antibodies D11 and D10 were used in a double-antibody sandwich ELISA to detect DENV1 NS1 antigen. The specific method was as follows: D11 capture antibody was coated onto an ELISA plate and incubated overnight at 4 °C. The next day, the plate was washed three times with PBST (10 mM PBS, 0.05% Tween-20 (v / v)), blocked with PBS containing 5% skim milk powder, and incubated at 37 °C for 2 hours. 100 μL of DENV1NS1 antigen was added, and the plate was incubated at 37 °C for 0.5 hours. After washing the plate, HRP-labeled antibody D10 was added, and the plate was incubated at 37 °C for 0.5 hours. After washing the plate three times with PBST, TMB substrate was used for color development. After stopping the reaction, the OD value was read, specifically OD450.

[0127] Please see Figure 3 The experimental results showed that as the concentration of DENV1 NS1 antigen increased (500 ng / mL, 166.7 ng / mL, 55.6 ng / mL, 18.61 ng / mL, 6.17 ng / mL, and 0 ng / mL, respectively), the OD value of the plate wells increased in a gradient manner. However, no binding was observed in the wells where non-DENV1 NS1 antigen protein was added, indicating that the obtained monoclonal antibody has excellent binding performance and specificity for DENV1 NS1 antigen.

[0128] Example 4

[0129] The specific procedure for sensitivity detection of DENV1 NS1 monoclonal antibody is as follows:

[0130] (1) Coat 100 μL of 1 μg / mL D11 monoclonal antibody in a well plate. The dilution buffer used is 10 mM PBS. All subsequent buffers used are 10 mM PBS. Bind at 37 ℃ for 2 h or incubate overnight at 4 ℃. Wash 3 times with 0.05% PBST.

[0131] (2) Add 300 μL of 5% skim milk to block the well plate, incubate at 37 ℃ for 2 h, and wash 3 times with 0.05% PBST;

[0132] (3) Add 100 μL of diluted serum sample, incubate at 37 °C for 0.5 h, and wash 3 times with 0.05% PBST;

[0133] (4) Add 100 μL of 1 μg / mL HRP-labeled detection antibody D10, incubate at 37 °C for 0.5 h, and wash 3 times with 0.05% PBST;

[0134] (5) Add 100 μL of TMB colorimetric solution, react at 37 °C for 7 min, then add 50 μL of 2 M H2SO4 to terminate the reaction, and take the reading at a wavelength of 450 nm.

[0135] The results are as follows Figure 4 As shown, in the detection of actual serum samples, serum diluted 10 times, 100 times, and 1000 times showed a clear gradient. The dilution buffer was also 10 mM PBS, proving that this method can effectively detect NS1 in serum.

[0136] The embodiments of this application have been described above with reference to the accompanying drawings. Specific examples have been used to illustrate the principles and implementation methods of this application. The description of the above embodiments is only for the purpose of helping to understand the core ideas of this application. However, this application is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of this application without departing from the spirit and scope of the claims, and all of these forms are within the protection scope of this application.

Claims

1. A monoclonal antibody against DENV1 NS1, characterized in that, The monoclonal antibody includes capture antibody and detection antibody; The amino acid sequences of the heavy chain complementarity-determining regions CDR1, CDR2 and CDR3 of the capture antibody are shown in SEQ ID NO.5, SEQ ID NO.6 and SEQ ID NO.7, respectively; The amino acid sequences of the light chain complementarity-determining regions CDR1, CDR2, and CDR3 of the capture antibody are shown in SEQ ID NO. 8, SEQ ID NO. 29, and SEQ ID NO. 9, respectively. The amino acid sequences of the heavy chain complementarity-determining regions CDR1, CDR2, and CDR3 of the detection antibody are shown in SEQ ID NO.19, SEQ ID NO.20, and SEQ ID NO.21, respectively. The amino acid sequences of the light chain complementarity-determining regions CDR1, CDR2, and CDR3 of the detection antibody are shown in SEQ ID NO.22, SEQ ID NO.31, and SEQ ID NO.23, respectively.

2. The monoclonal antibody against DENV1 NS1 according to claim 1, characterized in that, The amino acid sequences of the heavy chain variable region and the light chain variable region of the capture antibody are shown in SEQ ID NO. 1 and SEQ ID NO. 2, respectively; The amino acid sequences of the heavy chain variable region and the light chain variable region of the detection antibody are shown in SEQ ID NO. 15 and SEQ ID NO. 16, respectively.

3. A polynucleotide, characterized in that, The polynucleotide is used to encode the monoclonal antibody against DENV1 NS1 as described in any one of claims 1-2; The nucleotide sequences encoding SEQ ID NO. 5, SEQ ID NO. 6, and SEQ ID NO. 7 are shown in SEQ ID NO. 10, SEQ ID NO. 11, and SEQ ID NO. 12, respectively. The nucleotide sequences encoding SEQ ID NO. 8, SEQ ID NO. 29, and SEQ ID NO. 9 are shown in SEQ ID NO. 13, SEQ ID NO. 30, and SEQ ID NO. 14, respectively. The nucleotide sequences encoding SEQ ID NO. 19, SEQ ID NO. 20, and SEQ ID NO. 21 are shown in SEQ ID NO. 24, SEQ ID NO. 25, and SEQ ID NO. 26, respectively. The nucleotide sequences encoding SEQ ID NO. 22, SEQ ID NO. 31, and SEQ ID NO. 23 are shown in SEQ ID NO. 27, SEQ ID NO. 32, and SEQ ID NO. 28, respectively. The nucleotide sequences encoding SEQ ID NO. 1 and SEQ ID NO. 2 are shown in SEQ ID NO. 3 and SEQ ID NO. 4, respectively; The nucleotide sequences encoding SEQ ID NO. 15 and SEQ ID NO. 16 are shown in SEQ ID NO. 17 and SEQ ID NO. 18, respectively.

4. The use of the monoclonal antibody according to claim 1 in the preparation of a kit for the immunological detection of DENV1 NS1.

5. A reagent kit for immunological detection, characterized in that, The kit includes the anti-DENV1 NS1 monoclonal antibody as described in claim 1.

6. The reagent kit according to claim 5, characterized in that, The kit is an enzyme-linked immunosorbent assay kit.

7. The reagent kit according to claim 5, characterized in that, The kit is used to detect dengue virus infection.

8. A method for detecting DENV1 NS1 for non-diagnostic purposes, characterized in that, The method includes using the anti-DENV1 NS1 monoclonal antibody as described in claim 1.

9. The method according to claim 8, characterized in that, The anti-DENV1 NS1 monoclonal antibodies were paired and the DENV1 NS1 content in the sample was detected using a double-antibody sandwich method.

10. The method according to claim 9, characterized in that, The double-anti-corrosion sandwich method specifically includes the following steps: S1: Coat the capture antibody in a well plate and incubate at 37 °C for 2 h or at 4 °C overnight, then wash 3 times with 0.05% PBST. S2: After blocking the well plate with skim milk for 2 h, wash three times with 0.05% PBST; S3: Add the test sample and HRP-labeled detection antibody sequentially and incubate. S4: After incubation, add TMB colorimetric solution. After 7 min of reaction, add 50 μl of 2 M H2SO4 to terminate the reaction. Take readings at a wavelength of 450 nm.

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