Monkeypox virus rapid test strips and specific monoclonal antibodies

Abstract

The present application provides a monkeypox virus rapid test strip and a monkeypox virus-specific antibody therein, wherein the test strip is an immunochromatographic test strip, which contains monkeypox virus-specific monoclonal antibody 4C4 and monkeypox virus-specific monoclonal antibody 9D5. The monkeypox virus rapid test strip of the present application has good sensitivity and specificity for monkeypox virus, and improves the detection means of monkeypox virus.

Description

Field of the Invention

[0001] The present application belongs to the field of antibodies and biological detection. Specifically, the present application provides a rapid detection test strip for monkeypox virus and a specific monoclonal antibody. Background Art

[0002] Monkeypox is a zoonotic viral disease caused by infection with the monkeypox virus (MPXV). Its main clinical manifestations are fever, rash, and swollen lymph nodes.

[0003] Monkeypox virus is an enveloped double-stranded DNA virus belonging to the genus Orthopoxvirus of the Poxviridae family. It is one of the four orthopoxviruses that can cause disease in humans, the other three being Variola virus (VARV), Vaccinia virus (VACV) and Cowpox virus (CPXV). Monkeypox virus particles are brick-shaped or oval under an electron microscope. There are two forms of infection of virus particles: extracellular envelope virus (EV) and intracellular mature virion (MV). EV is formed by the virus acquiring an outer membrane in the cell. MV is the most abundant form and the most stable in the environment. It is the main form of virus transmission between hosts. At present, more than 20 structural proteins have been identified on the surface of MV and EV virus particles, many of which are target antigens recognized by neutralizing antibodies. A29L mainly binds to cell surface glycosaminoglycans, mediates the fusion of virus and cells, and is an important membrane protein of monkeypox virus MV. Its homologous proteins in vaccinia virus and cowpox virus are A27L and 162 respectively.

[0004] Currently, PCR is the main technology for monkeypox-specific diagnosis, but it is equipment-dependent and prone to contamination, and other detection methods other than nucleic acid detection have not yet been established. Therefore, it is necessary to pay close attention to the prevalence of monkeypox virus, strengthen virus detection and monitoring, and develop diagnostic reagents as soon as possible to cope with the possible prevalence of monkeypox virus. Summary of the invention

[0005] On the one hand, the present application provides a rapid test strip for monkeypox virus, wherein the test strip is an immunochromatographic test strip, which contains monkeypox virus-specific monoclonal antibody 4C4 and monkeypox virus-specific monoclonal antibody 9D5;

[0006] The amino acid sequence of the heavy chain CDR of the monkeypox virus-specific monoclonal antibody 4C4 is H-CDR1: SYTFSCYW, H-CDR2: ILPASGST, H-CDR3: SSALCTIITTRGMDC; the amino acid sequence of the light chain CDR of the monkeypox virus-specific monoclonal antibody 4C4 is L-CDR1: SSVNF, L-CDR2: STS, L-CDR3: HQWSSYPWT;

[0007] The amino acid sequence of the heavy chain CDR of the monkeypox virus-specific monoclonal antibody 9D5 is H-CDR1: GFTVTSYW, H-CDR2: ILPGCDST, H-CDR3: STALSTMITTRGMDC; the amino acid sequence of the light chain CDR of the monkeypox virus-specific monoclonal antibody 9D5 is L-CDR1: LSNVDSNGNTY, L-CDR2: KV, L-CDR3: FEHSHVPLT.

[0008] Further, the heavy chain amino acid sequence of the monkeypox virus-specific monoclonal antibody 4C4 is SEQ ID NO.1, and / or the light chain amino acid sequence of the monkeypox virus-specific monoclonal antibody 4C4 is SEQ ID NO.6;

[0009] The heavy chain amino acid sequence of the monkeypox virus-specific monoclonal antibody 9D5 is SEQ ID NO.11, and / or the light chain amino acid sequence of the monkeypox virus-specific monoclonal antibody 9D5 is SEQ ID NO.16.

[0010] Furthermore, the monkeypox virus-specific monoclonal antibody 4C4 is a scratching antibody on the test paper; and the monkeypox virus-specific monoclonal antibody 9D5 is a labeled antibody on the test paper.

[0011] Furthermore, the monkeypox virus-specific monoclonal antibody 9D5 is labeled with latex microspheres.

[0012] The markers that can be used in the immunochromatographic test paper of the present application are not limited to latex microspheres of various colors. After appropriate adjustment and verification, markers such as colloidal gold can also be used.

[0013] Furthermore, the test paper comprises a back plate, a nitrocellulose membrane, a latex microsphere pad, a sample pad, and absorbent paper; the latex microsphere pad is sprayed with a monkeypox virus-specific monoclonal antibody 9D5 marked with latex microspheres; and the nitrocellulose membrane is sprayed with a monkeypox virus-specific monoclonal antibody 4C4.

[0014] The structure of the immunochromatographic test paper of the present application is not limited to the above basic structure. On the basis of the unchanged antibody, other parts and structures of the immunochromatographic test paper based on the double antibody sandwich method can also be conventionally replaced. The materials of the back plate, membrane and pad part can be selected from various available types.

[0015] On the other hand, the present application provides a non-diagnostic method for detecting monkeypox virus, which uses the above-mentioned monkeypox virus rapid test strip for detection.

[0016] The non-diagnostic methods include, but are not limited to, methods for detecting whether the environment, equipment, food, and medicine are contaminated by viruses.

[0017] On the other hand, the present application provides a monkeypox virus-specific monoclonal antibody, wherein the amino acid sequence of the heavy chain CDR of the monoclonal antibody is H-CDR1: SYTFSCYW, H-CDR2: ILPASGST, H-CDR3: SSALCTIITTRGMDC; the amino acid sequence of the light chain CDR of the monoclonal antibody is L-CDR1: ESVDNYGLSF, L-CDR2: AA, L-CDR3: QQSKEVPWT; or the amino acid sequence of the heavy chain CDR of the monoclonal antibody is H-CDR1: GFTVTSYW, H-CDR2: ILPGCDST, H-CDR3: STALSTMITTRGMDC; the amino acid sequence of the light chain CDR of the monoclonal antibody is L-CDR1: LSNVDSNGNTY, L-CDR2: KV, L-CDR3: FEHSHVPLT.

[0018] Furthermore, the monoclonal antibody heavy chain amino acid sequence is SEQ ID NO.1, and the monoclonal antibody light chain amino acid sequence is SEQ ID NO.6; or, the monoclonal antibody heavy chain amino acid sequence is SEQ ID NO.11, and the monoclonal antibody light chain amino acid sequence is SEQ ID NO.16.

[0019] On the other hand, the present application provides the use of the above monoclonal antibody in the preparation of monkeypox virus detection reagents.

[0020] The detection reagent is not limited to the above-mentioned double antibody sandwich immunochromatographic test paper, and includes but is not limited to blocking immunochromatographic test paper, general ELISA method detection reagents, etc., all of which are within the protection scope of this application.

[0021] On the other hand, the present application provides a gene encoding a monkeypox virus-specific monoclonal antibody, wherein the gene comprises SEQ ID NO.5 and SEQ ID NO.10, or the gene comprises SEQ ID NO.15 and SEQ ID NO.20.

[0022] Those skilled in the art can use the above-mentioned genes to produce the above-mentioned antibodies through routine steps in the art such as constructing vectors, transformation, culturing, harvesting, and purification, wherein the vectors, host cells, etc. can be routinely constructed by those skilled in the art. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is the result of monoclonal antibody specificity test.

[0024] Figure 2 The specificity test results of paired monoclonal antibodies; 1: vaccinia virus A27L protein (1ug / ml), 2: vaccinia virus 162 protein (1ug / ml), 3: vaccinia virus (1*10 -5 TCID50 / ml), 4: Parapox virus (1*10 -5 TCID50 / ml), 5: Herpes simplex virus type 1 (1*10 -5 TCID50 / ml), 6: Herpes simplex virus type 2 (1*10 -5 TCID50 / ml), 7: varicella zoster virus (1*10 -5 TCID50 / ml), 8: measles virus (1*10 -3 TCID50 / ml), 9: Staphylococcus aureus (1*10 -9 CFU / ml).

[0025] Figure 3 This is the sensitivity test result of paired monoclonal antibodies. DETAILED DESCRIPTION

[0026] Example 1 Screening and identification of antibodies

[0027] Mouse immunization:

[0028] 50ug of A29L recombinant protein expressed by mammalian cells was mixed with an equal volume of MF59 adjuvant (200ul) and intraperitoneally immunized into 6-week-old female BALB / c mice. The immunization was repeated at 2 weeks and 4 weeks according to the above method. At the 5th week, the mouse serum was taken to test the antibody titer. The mouse with the highest titer was selected and boosted with 20ug of recombinant protein through the tail vein. The spleen of the mouse was taken 3 days later for the preparation of hybridoma cells.

[0029] Preparation and screening of hybridoma cell lines:

[0030] Three days after the booster immunization of mice, spleen cells of mice were taken and fused with myeloma cells SP2 / 0 through PEG1450 to prepare hybridoma cells. Ten days later, recombinant proteins A29L, A27L, 162 protein and vector tag protein were used for detection to screen out cell lines that only reacted with A29L but not with other proteins. After limiting dilution to a monoclonal state, the cell lines were expanded and frozen.

[0031] Ascites fluid preparation and antibody purification:

[0032] After the selected hybridoma cell lines were expanded and cultured, 0.2 mL (about 2×10-6 cells) was injected into the peritoneal cavity of female BALB / c mice. About 10 days later, when the abdomen of the mouse was obviously swollen, ascites was collected using a sterile syringe needle. The collected ascites was centrifuged at 12000r / min for 10 minutes, the upper layer of fat and the lower layer of sediment were removed, and the supernatant was collected. The ascites supernatant was diluted with binding buffer (0.02M PB, 150mM NaCl, pH 7.4) at a volume ratio of 1:10, filtered with a filter membrane, and then bound to the Protein G column (previously equilibrated with binding buffer). Then rinse with binding buffer for about 5 column volumes, elute with elution buffer (0.1M glycine, pH 2.7), and collect the elution peak. Then quickly neutralize with 1M Tris-HCl (pH 9.0) solution to dry the neutral range. The purified antibody was dialyzed into 0.01 M PBS, the concentration was determined and aliquoted for storage.

[0033] Characterization of purified monoclonal antibodies:

[0034] After multiple subcloning of the selected monoclonal cells, a total of 12 A29L-specific monoclonal antibodies were obtained. Indirect ELISA identified that they did not react with vaccinia virus A27L, vaccinia virus 162 protein, and tag protein. The monoclonal antibodies have good specificity and can be used for subsequent paired screening (such as Figure 1 shown).

[0035] A29L, A27L, 162 proteins and carrier tag proteins were reacted with monoclonal antibodies respectively. The results showed that all 12 screened monoclonal antibodies reacted specifically with A29L, and 10 of them had OD450mm values ​​>1, while they did not react with A27L, 162 proteins and controls, indicating that the monoclonal antibodies have good specificity.

[0036] Example 2 Screening of paired monoclonal antibodies

[0037] Preparation of latex microsphere test strips:

[0038] The purified 12 strains of A29L monoclonal antibodies were streaked separately: On different nitrocellulose (20mm X 300mm) membranes, the purified monoclonal antibodies 1.5mg / ml were sprayed horizontally at 1.0μL / cm using a streaking instrument to form a test line. At intervals of 6mm, sheep anti-mouse IgG was sprayed horizontally at 1.0μL / cm with a concentration of 1mg / ml to form a quality control line.

[0039] Preparation of antibody-bead labeling complex:

[0040] The 12 purified A29L monoclonal antibodies were labeled with microspheres respectively.

[0041] Washing of latex microspheres: Take a certain volume of red latex microspheres with a particle size of 300 mm (Suzhou Weidu Biotechnology Co., Ltd., DRO5C), pour into a clean centrifuge tube, add labeling buffer (50 mM MES, pH 6.0) at a ratio of 1:9. Centrifuge at 17000 r / min for 10 min, remove the supernatant, add 1 mL of labeling buffer, and add 1 mL of labeling buffer to resuspend the microspheres after centrifugation.

[0042] Activation of latex microspheres: Weigh 20 mg of NHS and EDC respectively, dissolve in 1 mL of labeling buffer to obtain 20 mg / ml NHS solution and EDC solution respectively: Take 20 uL of the prepared NHS solution and add it to the washed microspheres, mix quickly; then take 5 uL of the prepared EDC solution and add it to the microspheres, mix quickly, and incubate at room temperature for 20-30 min.

[0043] Labeling of latex microspheres: Centrifuge the activated latex microspheres obtained in step 2 at 17000r / min for 10min, add 1mL of labeling buffer to resuspend the microspheres, centrifuge again and add 1mL of labeling buffer to resuspend the microspheres. Take a certain amount of monoclonal antibody, add 100ul of microsphere buffer to every 100ug of antibody, mix quickly, and incubate at room temperature for 3h. Add blocking solution (20mg / mL BSA, dissolved with a final concentration of 100mM ethanolamine solution), incubate at room temperature for 1h, centrifuge at 17000r / min for 10min, discard the supernatant, and repeat washing twice to remove unbound antibodies. Resuspend the microspheres with 1mL of labeling buffer for the last time, which is the antibody-microsphere labeling complex, and place it at 4℃ for use.

[0044] Screening of paired monoclonal antibodies:

[0045] The NC membranes marked with different monoclonal antibodies were paired with the microsphere-labeled monoclonal antibodies one by one to screen out the paired monoclonal antibodies that can detect A29L recombinant protein but do not react with A27L, 162 protein and carrier tag protein. The A29L protein was diluted and tested, and the paired monoclonal antibodies with high detection sensitivity were selected for subsequent experiments.

[0046] The 12 monoclonal antibodies were stripped and labeled, and then paired with A29L, A27L, 162 protein and tag protein for detection. The combination that only reacted with A29L but not with other proteins was screened out: 4C4 stripping and 9D5 labeling, and then the sensitivity and specificity were studied. The 4C4-9D5 pairing was specifically analyzed with similar antigens such as vaccinia virus A27L protein, vaccinia virus 162 protein, and pathogen samples that cause similar symptoms such as vaccinia virus, parapox virus, herpes simplex virus 1 / 2, varicella zoster virus, measles virus, and Staphylococcus aureus, and it was found that none of them reacted with them ( Figure 2 ), indicating the detection specificity of this pairing. Clinical samples collected from non-monkeypox patients, such as oropharyngeal swabs, blood, herpes fluid and pustular fluid of varicella, were all negative.

[0047] Example 3 Preparation and Assembly of Monkeypox Virus Detection Test Strips

[0048] Preparation of latex microsphere pad: 6mm x 300mm glass fiber membrane was treated with PBS containing 1% BSA and 1% Tween-20, and the prepared latex microsphere labeled protein was sprayed at 1.2ml / strip, and dried at 40℃ for 2h for later use;

[0049] Assembly of latex microsphere test strips: With a 60mm x 300mm PVC backing plate as support, nitrocellulose membrane, latex microsphere pad, sample pad, and absorbent paper are attached to it. Two lines are coated on the nitrocellulose membrane and dried at 40℃ for 12 hours for use. The assembled large plate is cut into 4.05mm bare strips with a strip cutter and wrapped with a plastic card shell so that the sample pad is exposed to the sample addition hole position of the plastic card shell, and the quality control line and detection line are exposed to the result observation hole position. The latex microsphere test strip is assembled.

[0050] Example 4 Sensitivity and specificity test of monkeypox virus detection test strips

[0051] Positive samples are: Monkeypox virus A29L protein

[0052] Negative samples include: vaccinia virus A27L protein, vaccinia virus 162 protein, vaccinia virus, parapox virus, herpes simplex virus type 1 / 2, varicella zoster virus, measles virus, Staphylococcus aureus, and clinical samples such as oropharyngeal swabs, blood, varicella herpes fluid, and pustular fluid collected from non-monkeypox populations

[0053] The sample diluent was 0.01 mol / L PBS, pH 7.4, containing 0.1% Tween 20, 0.5% deoxycholic acid, and 0.05% Proclin 300.

[0054] The detection method is: dilute the positive and negative samples with diluent in a gradient manner, mix well and add 80ul to the sample well, and observe the results within 20 minutes.

[0055] Result determination: If both the T test line and the C quality control line have clearly visible red stripes, it is judged as positive. The darker the color of the T line, the stronger the positivity. If only the C quality control line has a clearly visible red stripe, it is judged as negative. If no red stripe appears on the C quality control line, it is judged as invalid.

[0056] The monkeypox virus A29L protein was diluted 10 times from 10 ng / ml, and it was found that it had good detection sensitivity for monkeypox recombinant antigen, and the detection limit for recombinant antigen was 10-100 pg / ml (see Figure 3 ). The specific sequence of the antibody is as follows:

[0057] Monoclonal antibody 4C4 heavy chain

[0058] Amino acid sequence

[0059] EVQLVESGAELMKPGASVKISCKATSYTFSCYWIEWVKQRPGHGLEWIGEILPASGSTNYNEKFKGKATFTADISSNTAYMQLSSLTSEDSAIYYCSSALCTIITTRGMDCWGQGTSVTVSS(SEQ ID NO.1)CDR1:SYTFSCYW(SEQ ID NO.2)

[0060] CDR2:ILPASGST (SEQ ID NO. 3)

[0061] CDR3:SSALCTIITTRGMDC(SEQ ID NO.4)

[0062] Nucleotide sequence

[0063] Gaggtgcagctggtggagtctggagctgagctgatgaagcctggggcctcagtgaagatatcctgcaag

[0064] gctactagctatacattcagttgctattggatagagtgggtaaagcagaggcctggacatggccttgag

[0065] tggattggagagatattacctgcaagtggtagtactaactacaatgagaagttcaagggcaaggccaca

[0066] ttcactgcagatatatcctccaacacagcctatatgcaactcagcagcctgacatctgaggactctgcc

[0067] atctattactgttcaagtgccctatgtactatcattacgacgagaggtatggactgctggggtcaaggaacctcagtcaccgtctcctca(SEQ ID NO.5)

[0068] Monoclonal antibody 4C4 light chain

[0069] Amino acid sequence

[0070] DVQMIQSPAIMSASLGEEITLTCSASSSVNFIHWYQQKSGTSPKLLIYSTSYLASGVPSRFSGSGSGTFYSLTISNVEAEDAADYYCHQWSSYPWTFGGGTKLEIKRTV(SEQ ID NO.6)

[0071] CDR1:SSVNF (SEQ ID NO.7)

[0072] CDR2: STS (SEQ ID NO. 8, since the WIPO Sequence software limits the sequence to at least 4 letters, the sequence 9 in the sequence list is counted as STSY)

[0073] CDR3: HQWSSYPWT (SEQ ID NO. 9)

[0074] Nucleotide sequence

[0075] Gatgttttgatgacccaaactccagcttctttggctgtgtctcaagggcagagggccaccatctcctgc

[0076] agagccagcgaaagtgttgataattatggccttagttttatgaactggttccaacagaaaccaggacag

[0077] ccacccaaactcctcatctatgctgcatccaaccaaggatccggggtccctgccaggtttagtggcagt

[0078] gggtctgggacagacttcagcctcaacatccatcctatggaggaggatgatactgcaatgtatttctgtcagcaaagtaaggaggttccgtggacgttcggtggaggcaccaagtggaaatcaaac(SEQ ID NO.10)

[0079] Heavy chain of monoclonal antibody 9D5

[0080] Amino acid sequence

[0081] EVKLVESGAELMKPGASVKISCKATGFTVTSYWIEWVKQRPGHGLEWIGEILPGCDSTNYNEKFKGKATFTADISSNTAYMQLSSLTSEDSAIYYCSTALSTMITTRGMDCWGQGTSVTVSS(SEQ ID NO.11)CDR1:GFTVTSYW(SEQ ID NO.12)

[0082] CDR2:ILPGCDST(SEQ ID NO.13)

[0083] CDR3:STALSTMITTRGMDC(SEQ ID NO.14)

[0084] Nucleotide sequence

[0085] Gaggtgaagctggtggagtctggagctgagctgatgaagcctggggcctcagtgaagatatcctgcaag

[0086] gctactggcttcacagtcactagctattggatagagtgggtaaagcagaggcctggacatggccttgag

[0087] tggattggagagattttacctggatgtgatagtactaactacaatgagaagttcaagggcaaggccaca

[0088] ttcactgcagatatatcctccaacacagcctatatgcaactcagcagcctgacatctgaggactctgcc

[0089] atctattactgttcaacagccctatcaactatgattacgacgagaggtatggactgctggggtcaaggaacctcagtcaccgtctcctca(SEQ ID NO.15)

[0090] Monoclonal antibody 9D5 light chain

[0091] Amino acid sequence

[0092] DVVVTQTPLSLPVSLGDQASISCRSSLSNVDSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFEHSHVPLTFGAGTKLELK(SEQ ID NO.16)

[0093] CDR1:LSNVDSNGNTY(SEQ ID NO.17)

[0094] CDR2: KVS (SEQ ID NO. 18, because the WIPO Sequence software limits the sequence to at least 4 letters, sequence 18 in the sequence list is counted as KVSN)

[0095] CDR3: FEHSHVPLT (SEQ ID NO. 19)

[0096] Nucleotide sequence

[0097] gatgttgtggtgactcaaactccactctccctgcctgtcagtcttggagatcaagcctccatctcttgc

[0098] agatctagtctgagcaatgtagatagtaatggaaacacctatttagagtggtacctgcagaagcctggc

[0099] cagtctccaaagctcctgatctacaaagtttccaaccgattttctggggtcccagacaggttcagtggc

[0100] agtggatcagggacagatttcacactcaagatcagcagagtggaggctgaagatctgggagtttattactgctttgaacattctcatgttccgctcacgttcggtgctgggaccaagctggagctgaaa(SEQ ID NO.20)。

Claims

1. A rapid test strip for monkeypox virus, characterized in that: The test strip is an immunochromatographic test strip, which contains monkeypox virus-specific monoclonal antibody 4C4 and monkeypox virus-specific monoclonal antibody 9D5; The amino acid sequence of the heavy chain CDR of the monkeypox virus-specific monoclonal antibody 4C4 is H-CDR1: SYTFSCYW, H-CDR2: ILPASGST, H-CDR3: SSALCTIITTRGMDC; the amino acid sequence of the light chain CDR of the monkeypox virus-specific monoclonal antibody 4C4 is L-CDR1: SSVNF, L-CDR2: STS, L-CDR3: HQWSSYPWT; The amino acid sequence of the heavy chain CDR of the monkeypox virus-specific monoclonal antibody 9D5 is H-CDR1: GFTVTSYW, H-CDR2: ILPGCDST, H-CDR3: STALSTMITTRGMDC; the amino acid sequence of the light chain CDR of the monkeypox virus-specific monoclonal antibody 9D5 is L-CDR1: LSNVDSNGNTY, L-CDR2: KVS, L-CDR3: FEHSHVPLT.

2. The test paper according to claim 1, wherein the heavy chain amino acid sequence of the monkeypox virus-specific monoclonal antibody 4C4 is SEQ ID NO.1, and / or the light chain amino acid sequence of the monkeypox virus-specific monoclonal antibody 4C4 is SEQ ID NO.6; the heavy chain amino acid sequence of the monkeypox virus-specific monoclonal antibody 9D5 is SEQ ID NO.11, and / or the light chain amino acid sequence of the monkeypox virus-specific monoclonal antibody 9D5 is SEQ ID NO.

16.

3. According to the test paper according to claim 1 or 2, the monkeypox virus-specific monoclonal antibody 4C4 is the marking antibody on the test paper; the monkeypox virus-specific monoclonal antibody 9D5 is the labeled antibody on the test paper.

4. The test paper according to claim 3, wherein the monkeypox virus-specific monoclonal antibody 9D5 is labeled with latex microspheres.

5. The test paper according to claim 4, wherein the test paper comprises a back plate, a nitrocellulose membrane, a latex microsphere pad, a sample pad, and a blotting paper; the latex microsphere pad is sprayed with a monkeypox virus-specific monoclonal antibody 9D5 marked with latex microspheres; and the nitrocellulose membrane is sprayed with a monkeypox virus-specific monoclonal antibody 4C4.

6. A non-diagnostic method for detecting monkeypox virus, characterized in that The method uses the detection test paper according to any one of claims 1-5 for detection.

7. A combination of monkeypox virus-specific monoclonal antibodies, characterized in that: The monkeypox virus-specific monoclonal antibody combination comprises monkeypox virus-specific monoclonal antibody 4C4 and monkeypox virus-specific monoclonal antibody 9D5; the amino acid sequence of the heavy chain CDR of the monkeypox virus-specific monoclonal antibody is H-CDR1: SYTFSCYW, H-CDR2: ILPASGST, H-CDR3: SSALCTIITTRGMDC; the amino acid sequence of the light chain CDR of the monkeypox virus-specific monoclonal antibody 4C4 is L-CDR1: SSVNF, L-CDR2: S TS, L-CDR3: HQWSSYPWT; the amino acid sequence of the heavy chain CDR of the monkeypox virus-specific monoclonal antibody 9D5 is H-CDR1: GFTVTSYW, H-CDR2: ILPGCDST, H-CDR3: STALSTMITTRGMDC; the amino acid sequence of the light chain CDR of the monkeypox virus-specific monoclonal antibody 9D5 is L-CDR1: LSNVDSNGNTY, L-CDR2: KVS, L-CDR3: FEHSHVPLT.

8. The monkeypox virus-specific monoclonal antibody combination according to claim 7, wherein the heavy chain amino acid sequence of the monkeypox virus-specific monoclonal antibody 4C4 is SEQ ID NO.1, and the light chain amino acid sequence of the monkeypox virus-specific monoclonal antibody 4C4 is SEQ ID NO.6; or, the heavy chain amino acid sequence of the monkeypox virus-specific monoclonal antibody 9D5 is SEQ ID NO.11, and the light chain amino acid sequence of the monkeypox virus-specific monoclonal antibody 9D5 is SEQ ID NO.

16.

9. Use of the monkeypox virus-specific monoclonal antibody combination according to claim 7 or 8 in the preparation of a monkeypox virus detection reagent.

10. The gene combination encoding the monkeypox virus-specific monoclonal antibody according to claim 7, characterized in that: The gene combination comprises a gene encoding monkeypox virus-specific monoclonal antibody 4C4 and a gene encoding monkeypox virus-specific monoclonal antibody 9D5; the gene encoding monkeypox virus-specific monoclonal antibody 4C4 comprises the nucleotide sequences shown in SEQ ID NO.5 and SEQ ID NO.10; the gene encoding monkeypox virus-specific monoclonal antibody 9D5 comprises the nucleotide sequences shown in SEQ ID NO.15 and SEQ ID NO.20.

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