Monoclonal antibody for resisting DENV1 NS1 and application thereof

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to an anti-DENV1 NS1 monoclonal antibody and application thereof. The monoclonal antibody comprises a capture antibody and a detection antibody; amino acid sequences of a heavy chain complementarity determining region CDR1, a light chain complementarity determining region CDR2 and a light chain complementarity determining region CDR3 of the capture antibody are respectively as shown in SEQ ID NO.5 to SEQ ID NO.9 and SEQ ID NO.29; the amino acid sequences of a heavy chain complementarity determining region CDR1, a light chain complementarity determining region CDR2 and a light chain complementarity determining region CDR3 of the detection antibody are respectively as shown in SEQ ID NO.19 to SEQ ID NO.23 and SEQ ID NO.31. The capture antibody and the detection antibody provided by the invention have high affinity and specific detection activity on DENV1 NS1 antigen.

Description

Technical Field

[0001] The present application belongs to the field of biomedicine technology, and specifically relates to a monoclonal antibody against DENV1 NS1 and its application. Background Art

[0002] Dengue virus (DENV) is a single-stranded positive-sense RNA virus belonging to the genus Flavivirus. It is transmitted to mammals through arthropods and can cause latent infection, dengue fever, and dengue hemorrhagic fever in humans. Globally, about 50 to 100 million people are infected with dengue fever each year, and about 20,000 people die. The DENV genome encodes a variety of non-structural proteins, including NS1 protein, which is a highly conserved non-structural glycoprotein that plays an important role in viral replication and is present in the serum of patients during the acute infection period of dengue fever. It is a good early diagnostic target. However, the screening of NS1 broad-spectrum antibodies is limited by the lack of natural hexamer antigen stimulation, the low screening efficiency due to the immunological weakness of conservative epitopes, and the lack of widespread popularization of structural analysis technology, resulting in a shortage of NS1 antibodies that have been reported so far.

[0003] Monoclonal antibodies (mAbs) play an important role in the diagnosis and treatment of diseases 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 assay (ELISA) and double antibody sandwich ELISA, which can achieve rapid differential diagnosis of DENV infection. In addition, monoclonal antibodies can also be used to compete for binding to DENV NS1 protein, inhibit vascular leakage symptoms and / or protect against fatal damage caused by DENV infection in vivo, providing a new direction for the development of dengue vaccine or therapeutic drugs for DENV virus infectious diseases.

[0004] How to obtain high-affinity and specific monoclonal antibodies against DENV1 NS1 is crucial for the highly sensitive and accurate detection of DENV1 NS1, and is also the key to limiting the performance of immunoassay methods for DENV1 NS1. Summary of the invention

[0005] The purpose of the present invention is to solve the deficiencies of the prior art and provide a monoclonal antibody against DENV1 NS1 and its application, specifically adopting the following technical solutions: In a first aspect, the present invention provides a monoclonal antibody against DENV1 NS1, wherein the monoclonal antibody comprises a capture antibody (abbreviated as D11 in the present invention) and a detection antibody (abbreviated as D10 in the present invention); The amino acid sequences of the heavy chain complementary 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 complementary 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 complementary 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 complementary 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.

[0006] As a further preferred embodiment, the amino acid sequences of the heavy chain complementary determining region and the light chain complementary determining region of the capture antibody are shown as SEQ ID NO. 1 and SEQ ID NO. 2, respectively; The amino acid sequences of the heavy chain complementary determining region and the light chain complementary determining region of the detection antibody are shown in SEQ ID NO. 15 and SEQ ID NO. 16, respectively.

[0007] The above amino acid sequence is as follows: SEQ ID NO. 1: QVQLQQSGAELMKPGASVKISCKASGYTFNTYWIEWVKQRPGHGLEWIGEILPGSRSFNYDEKFKGKATFTADTSSNTAYMQLSSLTSEDSAVYYCARGDYGDYAMDYWGQGTSVTVSS.

[0008] SEQ ID NO.2: DIVMTQTPSSLSASLGDRVTISCSASQGIGNYLNWYQQKPDGTVKLLIYYTSSLHSGVPSRFSGSGSGTDFSLTISNLEPEDIATYYCQQYSKLYTFGGGTKLEIK.

[0009] SEQ ID NO. 15: QVTLKESGPGILKPSQTLSLTCSFSGFSLSTSGLGVGWIRQPSGKGLEWLAHIWWDDEKFYNPSLKSRLTISKDTSRNQVFLKITSVDTADTATYFCARSPYYGMDYWGQGTSVIVSS.

[0010] SEQ ID NO. 16: DIEMTQSPSSMYASLGERVTITCKASQDINSYLSWLQQKPGKSPKTLIYRANRLVDGVPSRFSGSGSGQDYSLTISSLEYEDMGIYYCLQYDELYTFGGGTKLEIK.

[0011] SEQ ID NO.5 (CDR1): GYTFNTYW.

[0012] SEQ ID NO.6 (CDR2): ILPGSRSF.

[0013] SEQ ID NO.7 (CDR3): ARGDYGDYAMDY.

[0014] SEQ ID NO.8 (CDR1): QGIGNY.

[0015] SEQ ID NO.29 (CDR2): YTS.

[0016] SEQ ID NO.9 (CDR3): QQYSKLYT.

[0017] SEQ ID NO.19 (CDR1): GFSLSTSGLG.

[0018] SEQ ID NO.20 (CDR2): IWWDDEK.

[0019] SEQ ID NO.21 (CDR3): ARSPYYGMDY.

[0020] SEQ ID NO.22 (CDR1): QDINSY.

[0021] SEQ ID NO.31 (CDR2): RAN.

[0022] SEQ ID NO.23 (CDR3): LQYDELYT.

[0023] In a second aspect, the present invention provides a polynucleotide for encoding the above-mentioned monoclonal antibody against DENV1 NS1; 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 as 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 as SEQ ID NO.27, SEQ ID NO.32, and SEQ ID NO.28, respectively.

[0024] 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 SEQ ID NO. 17 and SEQ ID NO. 18, respectively.

[0025] The above nucleotide sequence is as follows: SEQ ID NO. 3: caggttcagctgcagcagtctggagctgagctgatgaagcctggggcctcagtgaagatatcctgcaaggcttctggctacacattcaatacctactggatagagtgggtaaagcagaggcctggacatggccttgagtggattggagagattttacctggaagtcgtagttttaactacgatgagaagttcaagggcaaggccacattcactgcagatacatcctccaacacagcctacatgcaactcagcagcctgacgtctgaggactctgccgtctattactgtgcaagaggggactatggagactatgctatggactactggggtcaaggaacctcagtcaccgtctcctct。

[0026] SEQ ID NO .4: gacattgtgatgacccagactccatcctccctgtctgcctctctgggagacagagtcaccatcagttgcagtgcaagtcagggcattggcaattatttaaactggtatcagcagaaaccagatggaactgttaaactcctgatctattacacatcaagtttacactcaggagtcccatcaaggttcagtggcagtgggtctgggacagatttttctctcaccatcagcaacctggaacctgaagatattgccacttactattgtcagcagtatagtaagttgtacacgttcggaggggggaccaaactggaaataaaa。

[0027] SEQ ID NO .17: caagttactctaaaagagtctggccctgggatattgaagccctcacagaccctcagtctgacttgttctttctctgggttttcactgagcacttctggtctgggtgtaggctggattcgtcagccttcagggaagggtctggagtggctggcgcacatttggtgggatgatgagaagttctataacccatccctgaagagccggctcacaatctccaaggatacctccagaaaccaggtattcctcaagatcaccagtgtggacactgcagatactgccacttacttctgtgctcgaagtccatactatggtatggactactggggtcaaggaacctcagtcatcgtctcctca。

[0028] SEQ ID NO .18: gacattgagatgacacagtctccatcttccatgtatgcatctctaggagagagagtcactatcacttgcaaggcgagtcaggacattaatagctatttaagctggctccagcagaaaccagggaaatctcctaagaccctgatctatcgtgcaaacagattggtagatggggtcccatcaaggttcagtggcagtggatctgggcaagattattctctcaccatcagcagcctggagtatgaagatatgggaatttattattgtctacagtatgatgagttgtacacgttcggaggggggaccaagctggaaataaaa。

[0029] SEQ ID NO .10:ggctacacattcaatacctactgg。

[0030] SEQ ID NO .11:attttacctggaagtcgtagtttt。

[0031] SEQ ID NO .12:gcaagaggggactatggagactatgctatggactac。

[0032] SEQ ID NO .13:cagggcattggcaattat。

[0033] SEQ ID NO. 30: tacacatca.

[0034] SEQ ID NO.14: cagcagtatagtaagttgtacacg.

[0035] SEQ ID NO.24: gggttttcactgagcacttctggtctgggt.

[0036] SEQ ID NO.25: atttggtggggatgatgagaag.

[0037] SEQ ID NO.26: gctcgaagtccatactatggtatggactac.

[0038] SEQ ID NO.27: caggacattaatagctat.

[0039] SEQ ID NO. 32: cgtgcaaac.

[0040] SEQ ID NO.28:ctacagtatgatgatgagttgtacacg.

[0041] In a third aspect, the present invention provides use of the above monoclonal antibody in preparing a kit for immunological detection.

[0042] In a fourth aspect, the present invention provides a kit for immunological detection, comprising the above-mentioned monoclonal antibody against DENV1 NS1.

[0043] As a further preferred embodiment, the kit is an enzyme-linked immunosorbent assay kit.

[0044] As a further preferred embodiment, the kit is used for detecting dengue virus infection.

[0045] In a fifth aspect, the present invention provides a method for detecting DENV1 NS1 for non-diagnostic purposes, the method comprising using the above-mentioned anti-DENV1 NS1 monoclonal antibody.

[0046] As a further preferred embodiment, the anti-DENV1 NS1 monoclonal antibodies are paired in a double antibody sandwich method to detect the DENV1 NS1 content in the sample.

[0047] As a further preferred embodiment, the above double antibody sandwich method specifically comprises the following steps: S1: The capture antibody was coated in the well plate, and the plate was allowed to bind for 2 h at 37 °C or overnight at 4 °C, and then washed three times with 0.05% PBST. S2: After adding skim milk to block the plate for 2 h, wash it three times with 0.05% PBST; S3: adding the sample to be tested and the HRP-labeled detection antibody in sequence for incubation; S4: After incubation, TMB colorimetric solution was added. After 7 min reaction, 50 μl 2 M H2SO4 was added to terminate the reaction and the reading was performed at a wavelength of 450 nm.

[0048] The beneficial effects of the present invention are: The present invention provides a monoclonal antibody against DENV1 NS1, including a capture antibody and a detection antibody. The experimental results of the present invention show that both the capture antibody and the detection antibody have high affinity and specific detection activity for the DENV1 NS1 antigen. The monoclonal antibody against DENV1 NS1 can be used for immunological analysis of DENV1 NS1 in a double antibody sandwich manner, including enzyme-linked immunosorbent assay based on antigen-antibody specific reaction immunological analysis detection type. The detection limit based on the double antibody sandwich ELISA can reach 6.17 ng / mL, and the linear detection range is 6.17-500 ng / mL, which is of great significance for the highly sensitive and accurate detection of DENV1 NS1. In addition, it is reported that the concentration of NS1 in the blood of DENV1 infected people is about 0.01-50 μg / ml. The detection limit of the present detection method invention can achieve the detection of NS1 in serum samples. . BRIEF DESCRIPTION OF THE DRAWINGS

[0049] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0050] Figure 1 It is a SDS-PAGE identification diagram of DENV1 NS1 eukaryotic expression provided by some embodiments of the present application; Figure 2 It is a reduced SDS-PAGE identification diagram of the capture antibody D11 and the detection antibody D10 provided in some embodiments of the present application; Figure 3 This is a diagram for verifying the effect of sandwich ELISA for detecting DENV1 NS1 based on antibodies D11 and D10 provided in some embodiments of the present application; Figure 4This is a verification diagram of the effect of sandwich ELISA based on antibodies D11 and D10 for detecting actual serum samples containing DENV1NS1 provided in some embodiments of the present application. DETAILED DESCRIPTION

[0051] The following will be combined with the drawings in the embodiments of the present application to clearly and completely describe the technical solutions in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of this application.

[0052] Example 1 Eukaryotic expression of DENV1 NS1 antigen 1) Recovery of suspended cells: 1. Preparation: Preheat the OPM-293 Hi-exp culture medium in a 37°C water bath for 30 minutes and turn on the ultraviolet light on the clean bench for 30 minutes.

[0053] 2. Take a 125 mL cell culture flask, disinfect it with 75% ethanol and place it on a clean bench. Use a sterile glass pipette to take 29 mL of preheated culture medium into the cell culture flask.

[0054] 3. Determine the specific location of the Expi293F TM cells to be revived in the cryopreservation tank, take them out and thaw them quickly in a water bath (about 1 min), disinfect them with 75% ethanol and place them in a clean bench, transfer the thawed cells to the cell culture bottle with a pipette, and shake them gently. Take 300 μl in a 24-well plate to observe the cell status.

[0055] 4. Label the cell information on the cell culture flask, and then culture it in a cell culture shaker at 37°C, 8% CO2, and relative humidity ≥80% at a speed of 120 ± 5 rpm.

[0056] 2) Construction of NS1 eukaryotic expression plasmid: The base sequence corresponding to the NS1 protein was constructed into the pCMV3 expression vector by 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 miniprep kit, filtered and sterilized with a sterile 0.22 μm water filter membrane, and placed in a 2 mL centrifuge tube. The base sequence used for NS1 is: 3) Plasmid transfection: Place OPM-293 Hi-exp medium and Opti-MEM I reduced serum medium in a 37°C water bath for 30 minutes and turn on the ultraviolet light for 30 minutes. 6 When the cell viability is ≥95%, transfer all the cell culture medium in the cell culture bottle to a 50 mL centrifuge tube, balance it and centrifuge it at 300 g for 5 minutes, and discard the supernatant. Use a sterile pipette to draw 2-5 mL of OPM-293 Hi-exp medium, use an elbow dropper to blow and resuspend the cells, and transfer 94.622 mL of fresh and preheated OPM-293 Hi-exp medium, sterilize and seal the bottle mouth, and return it to the cell shaker for continued culture. Take a 50 mL sterile centrifuge tube, add OptiMEM I type reduced serum medium, PEI, and DNA in order, and mix them by blowing and mixing, and incubate at room temperature for 15 minutes. Remove the cell culture bottle from the cell shaker, slowly add the prepared PEI-DNA complex to the cell culture bottle in a circular manner, sterilize and seal the bottle mouth, and return it to the cell shaker for continued culture.

[0057] 4) Incubate the cells at 120 ± 5 rpm in a cell culture shaker at 37 °C, 8% CO2, and relative humidity ≥ 80% for 7 days. Purify the NS1 using a nickel column and identify it by SDS-PAGE ( Figure 1 ).

[0058] Example 2 Preparation of DENV1 NS1 monoclonal antibody (1) Animal immunization: DENV1 NS1 antigen was used as an immunogen, mixed with an equal volume of Freund's adjuvant, vortexed until the immunogen was completely emulsified, and immunized by multiple subcutaneous injections on the abdomen and back of BaLB / C mice. Freund's complete adjuvant was used for the first immunization, and the immunogen dose was 120 μg / mouse; Freund's incomplete adjuvant was used for subsequent immunizations, and the immunogen dose was gradually reduced, with immunization once every 4 weeks, for a total of 3 immunizations. Two weeks after the third immunization, the tail blood of the mice was collected, and the serum antibody titer in the tail blood was determined by indirect ELISA. Three days before fusion, the immunogen was directly used to boost the mice with the highest titer, with an immunization dose of 50 μg / mouse.

[0059] (2) Preparation and screening of hybridoma cells The selected immune mice were killed by cervical dislocation, and the spleen was removed and ground on 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, and then added dropwise to a 96-well cell culture plate with feeder cells. After fusion, the cell state was observed under a microscope, and semi-quantitative medium was replaced on the 5th day after fusion. Culture was continued until the 7th day, after which the full medium was replaced.

[0060] About 7 to 10 days after cell fusion, the cell supernatant in the cell culture plate was aspirated, and the secretion of antibodies in the cell supernatant was determined by indirect ELISA. The positive cell plate wells were screened and subcloned by limiting dilution. DENV1 NS1 protein was coated, the blank control well was PBS, the negative control was culture medium, and the positive control was the eye blood serum of immunized mice. When the positive rate of the selected monoclonal cells in the 96-well cell culture plate reached 100%, it was judged as a positive monoclonal clone, and it was frozen and expanded in time.

[0061] (3) Preparation and purification of monoclonal antibody ascites Balb / c mice about 8 weeks old were injected with liquid paraffin one week in advance for pre-stimulation to promote the secretion and accumulation of nutrients in the mouse peritoneal cavity. The hybridoma cell line was expanded to the required number, centrifuged, carefully washed, resuspended in sterile 75% saline, and injected into the mouse abdomen by intraperitoneal injection. After the mouse abdomen was significantly swollen in about one week, the ascites was collected and centrifuged at 8000 r / min for 15 min. The supernatant was collected as the monoclonal antibody ascites.

[0062] The collected ascites was purified using a Protein G affinity chromatography purification column. Before loading, the ascites was filtered through a microporous membrane and used as the loading solution. The Protein G column was equilibrated with a binding buffer (0.15 M NaCl, 20 mM Na2HPO4, pH 7.4). The filtered ascites was loaded onto the chromatography column. After equilibration, the elution was performed using an elution buffer (0.1 M citric acid, pH 2.5-3.0) to collect the antibody-rich eluate. The eluate was acidic and the pH should be immediately adjusted to neutral using a neutralization buffer (1 M Tris-HCl, pH 9.0) to prevent the inactivation of the antibody. SDS-PAGE electrophoresis was then used for identification to obtain the capture antibody D11 and the detection antibody D10 (if attached). Figure 2 ).

[0063] (4) Determination of the heavy and light chain variable regions of monoclonal antibodies secreted by hybridoma cells The hybridoma cell line that can stably secrete monoclonal antibodies was cultured to 1×10 6cells / mL, discard the supernatant, add 1 mL Trizol reagent, shake gently, add 200 μL chloroform, shake upside down for 40 s, and place on ice for 15 min; centrifuge at 4 ℃ and 12000 g for 15 min, transfer the supernatant to a new enzyme-free centrifuge tube, add an equal volume of isopropanol, and place on ice for 10 min; centrifuge at 4 ℃ and 12000 g for 10 min, discard the supernatant, add 1 mL 75% ethanol, centrifuge at 4 ℃ and 7500 g for 5 min, dry at low speed in the clean bench for 5 min, add 30-50 μL enzyme-free sterile water, mix and dissolve RNA. Reverse transcription was performed using 1 μg total RNA as a template according to the instructions of the Hifair III 1st Strand cDNA Synthesis Kit. The upstream and downstream primers of the heavy and light chain variable regions of the monoclonal antibody were designed respectively (the primers are shown in Table 1 below), and the cDNA obtained by reverse transcription was amplified, and the reaction parameters were: 94 ° C 5 min, 94 ° C 10 s, 60 ° C 20 s, 72 ° C 30 s, a total of 25 cycles, and then 72 ° C 5 min. The obtained amplification products were identified by 1% agarose gel, and the obtained products were sequenced to obtain the amino acid sequence of the monoclonal antibody against DENV1 NS1, including the capture antibody (abbreviated as D11 in the present invention) and the detection antibody (abbreviated as D10 in the present invention); Table 1 Sequencing primers for the variable regions of light and heavy chains of D10 and D11 The capture antibody includes the amino acid sequence: QVQLQQSGAELMKPGASVKISCKASGYTFNTYWIEWVKQRPGHGLEWIGEILPGSRSFNYDEKFKGKATFTADTSSNTAYMQLSSLTSEDSAVYYCARGDYGDYAMDYWGQGTSVTVSS (SEQ ID NO. 1) and the amino acid sequence: DIVMTQTPSSLSASLGDRVTISCSASQGIGNYLNWYQQKPDGTVKLLIYYTSSLHSGVPSRFSGSGSGTDFSLTISNLEPEDIATYYCQQYSKLYTFGGGTKLEIK (SEQ ID NO. 2); The detection antibody comprises an amino acid sequence: QVTLKESGPGILKPSQTLSLTCSFSGFSLSTSGLGVGWIRQPSGKGLEWLAHIWWDDEKFYNPSLKSRLTISKDTSRNQVFLKITSVDTADTATYFCARSPYYGMDYWGQGTSVIVSS (SEQ ID NO. 15) and an amino acid sequence: DIEMTQSPSSMYASLGERVTITCKASQDINSYLSWLQQKPGKSPKTLIYRANRLVDGVPSRFSGSGSGQDYSLTISSLEYEDMGIYYCLQYDELYTFGGGTKLEIK (SEQ ID NO. 16).

[0064] A monoclonal antibody against DENV1 NS1, wherein the amino acid sequences CDR1, CDR2, and CDR3 of the complementary determining regions of the heavy chain of the capture antibody are respectively: CDR1: GYTFNTYW (SEQ ID NO.5); CDR2: ILPGSRSF (SEQ ID NO.6); CDR3: ARGDYGDYAMDY (SEQ ID NO. 7).

[0065] A monoclonal antibody against DENV1 NS1, wherein the amino acid sequences CDR1, CDR2, and CDR3 of the complementary determining regions of the light chain of the capture antibody are respectively: CDR1: QGIGNY (SEQ ID NO.8); CDR2: YTS; CDR3: QQYSKLYT (SEQ ID NO.9).

[0066] Monoclonal antibody against DENV1 NS1, wherein the amino acid sequences CDR1, CDR2, and CDR3 of the heavy chain of the detection antibody are: CDR1:GFSLSTSGLG (SEQ ID NO.19); CDR2:IWWDDEK (SEQ ID NO.20); CDR3: ARSPYYGMDY (SEQ ID NO. 21).

[0067] Monoclonal antibody against DENV1 NS1, wherein the amino acid sequences CDR1, CDR2, and CDR3 of the complementary determining regions of the light chain of the detection antibody are: CDR1: QDINSY (SEQ ID NO.22); CDR2: RAN; CDR3: LQYDELYT (SEQ ID NO. 23).

[0068] The amino acid sequence of the above immunogen is as follows: DSGCVINWKGRELKCGSGIFVTNEVHTWTEQYKFQADSPKRLSAAIGKAWEEGVCGIRSATRLENIMWKQISNELNHILLENDMKFTVVVGDVAGILAQGKKMIRPQPMEHKYSWKSWGKAKIIGADVQNTTFIIDGPNTPECPDDQRAWNIWEVEDYGFGIFTTNIWLKLRDSYTQV CDHRLMSAAIKDSKAVHADMGYWIESEKNETWKLARASFIEVKTCIWPKSHTLWSNGVLESEMIIPKIYGGPISQHNYRPGYFTQTAGPWHLGKLELDFDLCEGTTVVVDEHCGNRGPSLRTTTVTGKIIHEWCCRSCTLPPLRFRGEDGCWYGMEIRPVKEKEENLVKSMVSA (SEQ ID NO.34) Example 3 DENV1 NS1 monoclonal antibody is used for double antibody sandwich ELISA to determine DENV1 NS1 antigen (specificity determination of DENV1 NS1 monoclonal antibody) Monoclonal antibodies D11 and D10 were used for double antibody sandwich ELISA to detect DENV1 NS1 antigen. The specific method was as follows: D11 capture antibody was coated on the 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 DENV1NS1 antigen was added, incubated at 37°C for 0.5 hour, and after washing the plate, HRP-labeled antibody D10 was added and incubated at 37°C for 0.5 hour. After washing the plate three times with PBST, TMB substrate was used for color development, and the OD value was read after the reaction was terminated, specifically OD450.

[0069] See also Figure 3The above experimental results showed that with the increase of DENV1 NS1 antigen concentration (the antigen concentrations were 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 values ​​of the plate wells increased gradiently, while no binding was observed in the wells to which non-DENV1 NS1 antigen proteins were added, indicating that the obtained monoclonal antibody had excellent binding performance and specificity to the DENV1 NS1 antigen.

[0070] Example 4 The sensitivity test of DENV1 NS1 monoclonal antibody is as follows: (1) Coat 100 μL of 1 μg / mL D11 monoclonal antibody in the well plate. The dilution buffer used was 10 mM PBS. The subsequent buffer used was 10 mM PBS. Incubate at 37°C for 2 h or at 4°C overnight, and then wash three times with 0.05% PBST. (2) Add 300 μL of 5% skim milk to block the plate, incubate at 37 °C for 2 h, and wash three times with 0.05% PBST; (3) Add 100 μL of diluted serum sample, incubate at 37 °C for 0.5 h, and wash three times with 0.05% PBST; (4) Add 100 μL of 1 μg / mL HRP-labeled detection antibody D10, incubate at 37 °C for 0.5 h, and wash three times with 0.05% PBST; (5) Add 100 μL TMB colorimetric solution and incubate at 37 °C for 7 min. Then add 50 μL 2 M H2SO4 to terminate the reaction and read the result at 450 nm.

[0071] The results are as follows Figure 4 As shown, in the actual serum sample detection, the serum diluted 10 times, 100 times, and 1000 times showed an obvious gradient, and the dilution buffer was also 10 mM PBS, proving that this method can effectively detect NS1 in serum.

[0072] The embodiments of the present application are described above in conjunction with the accompanying drawings. Specific examples are used herein to illustrate the principles and implementation methods of the present application. The description of the above embodiments is only used to help understand the core idea of ​​the present application, but the present application is not limited to the above-mentioned specific implementation methods. The above-mentioned specific implementation methods are merely illustrative and not restrictive. Under the inspiration of the present application, ordinary technicians in this field can also make many forms without departing from the scope of protection of the purpose of the present application and the claims, all of which are within the protection of the present application.

Claims

1. A monoclonal antibody against DENV1 NS1, characterized in that The monoclonal antibody comprises a capture antibody and a detection antibody; The amino acid sequences of the heavy chain complementary 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 complementary 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 complementary 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 complementary 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 anti-DENV1 NS1 monoclonal antibody according to claim 1, characterized in that The amino acid sequences of the heavy chain complementary determining region and the light chain complementary determining 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 complementary determining region and the light chain complementary determining 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 anti-DENV1 NS1 monoclonal antibody according to 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 as 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 SEQ ID NO. 17 and SEQ ID NO. 18, respectively.

4. Use of the monoclonal antibody according to claim 1 in preparing a kit for immunological detection.

5. A kit for immunological detection, characterized in that: The kit comprises the anti-DENV1 NS1 monoclonal antibody of claim 1.

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

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

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

9. The method according to claim 6, characterized in that The anti-DENV1 NS1 monoclonal antibodies are paired in a double antibody sandwich method to detect the DENV1 NS1 content in the sample.

10. The method according to claim 7, characterized in that The double antibody sandwich method specifically comprises the following steps: S1: The capture antibody was coated in the well plate, and the plate was allowed to bind for 2 h at 37 °C or overnight at 4 °C, and then washed three times with 0.05% PBST. S2: After adding skim milk to block the plate for 2 h, wash it three times with 0.05% PBST; S3: adding the sample to be tested and the HRP-labeled detection antibody in sequence for incubation; S4: After incubation, TMB colorimetric solution was added. After 7 min reaction, 50 μl 2 M H2SO4 was added to terminate the reaction and the reading was performed at a wavelength of 450 nm.

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