Anti-hantavirus NP protein antibody pair and application thereof

Abstract

The invention relates to the technical field of biology, in particular to an anti-hantavirus NP protein antibody pair and application thereof. The protein detection limit of a double-antibody sandwich ELISA detection system established on the basis of the antibody pair is 1ng / mL, the antibody pair has good performance in the aspects of linear range, specificity, accuracy and the like, and an effective tool is provided for early clinical diagnosis, epidemiological monitoring, virus load evaluation and related scientific research of hantavirus.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more particularly to an antibody pair against hantavirus NP protein and its application. Background Technology

[0002] Hantaviruses are segmented negative-sense RNA viruses belonging to the genus *Hantavirus* in the family Bunyaviridae. They can cause serious human diseases such as hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS). These diseases progress rapidly and have a high mortality rate. Early symptoms of infection (such as fever, headache, and myalgia) are nonspecific and easily confused with common influenza-like illnesses. Therefore, developing rapid and accurate early diagnostic techniques is crucial for timely clinical intervention, reducing mortality, and controlling the spread of epidemics.

[0003] Currently, serological testing is a commonly used technique for diagnosing hantavirus infection, relying on specific antibodies produced by the body after infection for identification. However, antibodies need time to accumulate to a detectable threshold in the early stages of infection, and often cannot be effectively detected within several days after infection due to insufficient antibody levels, creating a "detection blind spot" during the diagnostic window period, leading to missed detection or delayed diagnosis of early-stage infections. While nucleic acid testing has high sensitivity, it has stringent requirements for laboratory environment, equipment, and operator expertise, and the testing process is time-consuming. Furthermore, the survival time of viral RNA in patient body fluids is limited. These factors collectively restrict its widespread application in primary healthcare institutions or large-scale field screening scenarios. Therefore, there is an urgent need to develop a detection system that can directly, sensitively, and specifically detect hantavirus to overcome the current limitations of hantavirus testing. Summary of the Invention

[0004] In view of this, the present invention proposes an antibody pair against hantavirus NP protein and its application.

[0005] The technical solution of this invention is implemented as follows:

[0006] In a first aspect, the present invention provides an antibody pair against hantavirus NP protein, the antibody pair comprising a first monoclonal antibody and a second monoclonal antibody; each monoclonal antibody comprises a light chain variable region (VL) and a heavy chain variable region (VH); the light chain variable region comprises light chain CDR1, light chain CDR2, and light chain CDR3 (LCDR1~LCDR3); the heavy chain variable region comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 (HCDR1~HCDR3).

[0007] The sequence information of the first monoclonal antibody is as follows:

[0008] The amino acid sequence of the light chain CDR1 is shown in SEQ ID NO: 3;

[0009] The amino acid sequence of the light chain CDR2 is shown in SEQ ID NO: 4;

[0010] The amino acid sequence of the light chain CDR3 is shown in SEQ ID NO: 5;

[0011] The amino acid sequence of heavy chain CDR1 is shown in SEQ ID NO: 8;

[0012] The amino acid sequence of the heavy chain CDR2 is shown in SEQ ID NO: 9;

[0013] The amino acid sequence of the heavy chain CDR3 is shown in SEQ ID NO: 10;

[0014] The sequence information of the second monoclonal antibody is as follows:

[0015] The amino acid sequence of the light chain CDR1 is shown in SEQ ID NO: 17;

[0016] The amino acid sequence of the light chain CDR2 is shown in SEQ ID NO: 18;

[0017] The amino acid sequence of the light chain CDR3 is shown in SEQ ID NO: 19;

[0018] The amino acid sequence of heavy chain CDR1 is shown in SEQ ID NO: 22;

[0019] The amino acid sequence of the heavy chain CDR2 is shown in SEQ ID NO: 23;

[0020] The amino acid sequence of the heavy chain CDR3 is shown in SEQ ID NO: 24.

[0021] Furthermore, the amino acid sequence of the light chain variable region of the first monoclonal antibody is shown in SEQ ID NO: 2, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 7;

[0022] The amino acid sequence of the light chain variable region of the second monoclonal antibody is shown in SEQ ID NO: 16, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 21.

[0023] Furthermore, the amino acid sequence of the light chain of the first monoclonal antibody is shown in SEQ ID NO: 1, and the amino acid sequence of the heavy chain is shown in SEQ ID NO: 6;

[0024] The amino acid sequence of the light chain of the second monoclonal antibody is shown in SEQ ID NO: 15, and the amino acid sequence of the heavy chain is shown in SEQ ID NO: 20.

[0025] Secondly, the present invention provides the use of the antibody pair in the preparation of a kit for detecting Hantavirus.

[0026] Thirdly, the present invention provides a nucleic acid molecule that encodes the amino acid sequence of the first monoclonal antibody or the second monoclonal antibody.

[0027] Furthermore, the nucleic acid molecule encoding the variable region sequence of the light chain of the first monoclonal antibody is shown in SEQ ID NO: 12;

[0028] The nucleic acid molecule encoding the heavy chain variable region sequence of the first monoclonal antibody is shown in SEQ ID NO: 14;

[0029] The nucleic acid molecule encoding the variable region sequence of the light chain of the second monoclonal antibody is shown in SEQ ID NO: 26;

[0030] The nucleic acid molecule encoding the heavy chain variable region sequence of the second monoclonal antibody is shown in SEQ ID NO: 28.

[0031] Furthermore, the nucleic acid molecule encoding the light chain sequence of the first monoclonal antibody is shown in SEQ ID NO: 11;

[0032] The nucleic acid molecule encoding the heavy chain sequence of the first monoclonal antibody is shown in SEQ ID NO: 13;

[0033] The nucleic acid molecule encoding the light chain sequence of the second monoclonal antibody is shown in SEQ ID NO: 25;

[0034] The nucleic acid molecule encoding the heavy chain sequence of the second monoclonal antibody is shown in SEQ ID NO: 27.

[0035] Fourthly, the present invention provides a kit for detecting Hantavirus, comprising the aforementioned antibody pair.

[0036] Furthermore, the detection kit is an enzyme-linked immunosorbent assay (ELISA) kit, an ELISA kit, an immunohistochemistry kit, an immunofluorescence kit, an immunoblotting kit, or a flow cytometry kit.

[0037] Furthermore, the kit is a double-antibody sandwich enzyme-linked immunosorbent assay kit, wherein the first monoclonal antibody is a capture antibody, the second monoclonal antibody is a detection antibody, and the second monoclonal antibody is conjugated with a detection marker.

[0038] The beneficial effects of the present invention include at least the following:

[0039] The monoclonal antibody pair against the NP protein of hantavirus provided by this invention can specifically bind to different antigenic epitopes of the NP protein. The double-antibody sandwich ELISA detection system established based on this antibody pair has a detection limit of 1 ng / mL and exhibits good performance in terms of linearity, specificity, and accuracy. This method provides an effective tool for the early clinical diagnosis, epidemiological surveillance, viral load assessment, and related scientific research of hantavirus. Attached Figure Description

[0040] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying 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.

[0041] Figure 1 The construction and identification of plasmids for prokaryotic expression and purification of Hantavirus NP protein are shown below. A and B are schematic diagrams of the construction of recombinant expression plasmids pET-28a-HTNV-NP and pET-28a-SEOV-NP, respectively. C shows the results of bacterial PCR identification of the recombinant plasmids; lanes 1-5: HTNV-NP recombinant bacteria; lanes 6-9: SERV-NP recombinant bacteria. D and E show the SDS-PAGE identification results of purified HTNV-NP and SERV-NP proteins, respectively. F shows the Western blot results of Hantavirus NP protein verified by His-tagged antibody; lane 1: HTNV-NP; lane 2: SERV-NP.

[0042] Figure 2 The results are from the rabbit serum antibody titer assay.

[0043] Figure 3 The results of specific detection of 12 monoclonal antibody culture supernatants based on prokaryotic and eukaryotic NP proteins are shown. In this paper, A represents the detection results using prokaryotically expressed HTNV-NP and SEIV-NP proteins as coating antigens, and B represents the detection results using eukaryotically expressed HTNV-NP and SEIV-NP proteins as coating antigens.

[0044] Figure 4 ELISA titer and EC of rabbit monoclonal antibodies 50 Measurement results;

[0045] Figure 5 To screen for the optimal antibody pair for double-antibody sandwich ELISA (DAS-ELISA) using the checkerboard method;

[0046] Figure 6 A schematic diagram illustrating the principle of DAS-ELISA detection of Hantavirus NP protein;

[0047] Figure 7 The performance validation of the DAS-ELISA detection method is as follows: A is the DAS-ELISA standard curve plotted using prokaryotically expressed HTNV-NP, SEOV-NP, and DBSV-NP proteins; B is the determination of the cut-off value of the DAS-ELISA detection method; C is the specificity assessment of the DAS-ELISA detection method; and D is the clinical sample validation results of the DAS-ELISA detection method. Detailed Implementation

[0048] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall be followed. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0049] Example 1: Expression, purification, and identification of NP recombinant protein

[0050] This embodiment uses both prokaryotic and eukaryotic expression systems to prepare recombinant NP proteins of Hantavirus (HTNV and SEOV types) to obtain NP antigens with different conformations. Although the prokaryotic system has high expression levels, the expression product is prone to forming inclusion bodies, which may lead to partial epitope masking; while eukaryotic expression can obtain soluble proteins with correct folding conformations, which is beneficial for the full exposure of antigenic epitopes.

[0051] 1. Prokaryotic protein expression

[0052] Based on the NP protein coding sequences of Hantavirus HTNV type (strain HTNV-JM119, NP protein GenBank accession number: PX833970) and SEOV type (strain SEOV-XY134, NP protein GenBank accession number: PX833971) (Table 1), we first cloned the NP protein gene into the prokaryotic expression vector pET-28a and introduced a 6×His tag at its C-terminus.

[0053] Table 1

[0054]

[0055] The recombinant plasmid was transformed into *E. coli* BL21(DE3), and expression was induced using 0.5 mM IPTG. After induction, the bacterial cells were collected, sonicated, and the His-tagged recombinant NP protein was purified using a Ni-NTA affinity chromatography column. The purified protein was renatured by dialyzing and dissolved in PBS. The following results were obtained: prokaryotic HTNV-NP protein with a final concentration of approximately 0.80 mg / mL and a total volume of 4 mL, with a purity ≥90% as determined by SDS-PAGE; and prokaryotic SEEV-NP protein with a final concentration of approximately 0.54 mg / mL and a total volume of 4 mL, with a purity ≥90% as determined by SDS-PAGE. Figure 1 ).

[0056] 2. Eukaryotic protein expression

[0057] The NP protein coding sequence of the above HTNV was inserted into the Not I and Xba I multiple cloning sites of the pFastBacI vector (the NP protein coding sequence of SEOV was inserted into the EcoRI and Not I multiple cloning sites of the pFastBacI vector), and a Strep II tag was added to the C-terminus of the NP protein to obtain the recombinant expression vector.

[0058] The recombinant expression vector was transformed into DH10Bac competent cells to obtain recombinant baculoviruses containing the target gene fragment. These recombinant baculoviruses were then transfected into SF9 cells for protein expression. The successfully packaged recombinant baculoviruses from SF9 adherent cells were inoculated into 200 mL of High 5 insect cells for amplified expression. After 96 hours of expression, cells were collected, lysed, and purified using Strep-Tactin affinity chromatography to obtain: eukaryotic HTNV-NP protein (final concentration approximately 0.218 mg / mL, total volume 5 mL, purity ≥ 90% as determined by SDS-PAGE); and eukaryotic SEEV-NP protein (final concentration approximately 0.281 mg / mL, total volume 3 mL, purity ≥ 90% as determined by SDS-PAGE).

[0059] Example 2: Preparation of rabbit monoclonal antibody against NP protein

[0060] 1. Animal immunization

[0061] Healthy New Zealand white rabbits were used. The purified NP recombinant protein was thoroughly emulsified and mixed with an equal volume of Freund's adjuvant. Two rabbits were then immunized with multiple subcutaneous injections. Each rabbit received 0.5 mg of the prepared HTNV+SEOV NP prokaryotic protein at a 1:1 mass ratio. Booster immunizations were administered every week for a total of four immunizations. The initial immunization used Freund's complete adjuvant, while subsequent booster immunizations used Freund's incomplete adjuvant. After the final immunization, serum was collected and the antibody titer in rabbit serum was detected by indirect ELISA. The detection steps were as follows: The purified prokaryotic and eukaryotic recombinant proteins were used as coating agents and diluted to a concentration of 1 μg / mL with 0.05M coating buffer. 100 μL of the solution was added to each well of a 96-well microplate and incubated overnight at 4°C. The coating buffer was discarded after washing the plate twice with PBS + Tween 20 (500 μL + 500 mL PBS). The plate was then dried with PBST for 4 min each time. 100 μL of blocking buffer (5% BSA) was added to each well and the plate was blocked at 37°C for 2 h. Afterwards, discard the liquid, wash the plate three times with PBST for 3 minutes each time, and blot dry. Dilute rabbit serum with PBS at ratios of 1 / 1000, 1 / 2000, 1 / 4000, 1 / 8000, 1 / 16000, 1 / 32000, 1 / 64000, and 1 / 128000. Set up negative and blank controls. Add 100 μL of diluted serum to each well and incubate at 37°C for 1 hour. Discard the primary antibody, wash the plate five times with PBST for 3 minutes each time, and blot dry on the last wash. Dilute HRP-labeled goat anti-rabbit IgG secondary antibody with PBST at a ratio of 1:4000, add 100 μL to each well, and incubate at 37°C for 1 hour. Discard the secondary antibody, wash the plate five times with PBST for 3 minutes each time, blot dry, add 100 μL of TMB single-component chromogenic solution (Solepro) to each well, and incubate at 37°C in the dark for 5 hours. After min, add 50 μL of 2M H2SO4 ELISA stop solution to each well to terminate the reaction, and measure the OD value using an ELISA reader at a wavelength of 450 nm.

[0062] The results of rabbit serum antibody titer determination are as follows: Figure 2 As shown.

[0063] 2. Preparation of monoclonal antibodies

[0064] (1) Isolation of single B cells:

[0065] Rabbits with the highest serum titers were selected, and their spleens and peripheral blood were aseptically collected 3 days after the last booster immunization. Lymphocytes were separated by density gradient centrifugation, and antigen-specific B cells were obtained by flow cytometry sorting (FACS).

[0066] (2) Antibody gene cloning and expression:

[0067] First, individual B cells were lysed. Pujian Biotechnology Co., Ltd. used a high-throughput sequencing platform to construct a single B cell library and perform sequencing to obtain the naturally paired heavy and light chain variable region (VH and VL) sequences corresponding to the single B cell. Primers designed for the antibody variable region were used to amplify the heavy and light chain variable region genes of the rabbit antibody.

[0068] (3) Antibody expression and screening:

[0069] The VH and VL genes were cloned into the mammalian expression vector PTT5, which contains the constant region of rabbit antibodies. Heavy and light chain expression plasmids were co-transfected into CHO cells, specifically by simultaneous transfection of paired light and heavy chain plasmids into each well of a deep-well plate. Cell culture supernatants were collected and purified by IgG affinity chromatography. Antibodies capable of binding the NP protein with high affinity were screened using indirect ELISA. The purified prokaryotic and eukaryotic expressed NP proteins were used as coating antigens to detect the effects of the culture supernatants from 12 monoclonal cells.

[0070] The results are as follows Figure 3 As shown: When using prokaryotic NP protein expression coating, 8 antibodies (5D11-2, 9A12, 9F5-1, 10G5, 10H10, 11G12, 16A9, 16B12) could simultaneously recognize the NP proteins of HTNV and SEVO; 3 antibodies (3C3, 9F11, 14H7) bound only the HTNV NP protein; and 1 antibody (10G11-3) showed weak binding to the SEVO NP protein. When using eukaryotic NP protein expression coating, the binding profile changed: 9 antibodies (5D11-2, 9A12, 9F5-1, 10G5, 10H10, 11G12, 16A9, 16B12, and 10G11-3) showed good binding activity to both viral NP proteins; 2 antibodies (3C3, 9F11) still only recognized the HTNV NP protein; while 14H7 showed no significant binding to either NP protein.

[0071] (4) Screening of monoclonal cell lines and identification of antibodies

[0072] Single-cell subcloning of positive-well cells was performed using a limiting dilution method. Following expansion culture and stability screening, stable antibody-secreting monoclonal CHO cell lines were obtained. Through multiple rounds of screening, 11 high-affinity rabbit monoclonal antibodies were finally obtained (5D11-2, 9A12, 9F5-1, 10H10, 11G12, 16A9, 16B12, 3C3, 9F11, 14H7, 10G11-3). The purified antibodies were then subjected to ELISA titer determination and half-maximal effective concentration (EC50) determination using indirect ELISA. 50 The measurement results are as follows: Figure 4 As shown.

[0073] Example 3: Development of a Hantavirus DAS-ELISA (Double Antibody Sandwich Enzyme-Linked Immunosorbent Assay) Detection Kit

[0074] 1. Screening for anti-Hantavirus NP protein antibodies

[0075] The 11 monoclonal antibodies obtained in Example 2 were diluted to an optimal working concentration of 1000 μg / mL with coating buffer (pH 9.6, 0.05 M carbonate buffer), and 100 μL was added to each well of an ELISA plate and incubated overnight at 4°C. Using a horseradish peroxidase labeling kit (Wuhan Sanying Biotechnology Co., Ltd.), the 11 monoclonal antibodies were conjugated with HRP to prepare the corresponding detection antibodies.

[0076] Eleven unlabeled antibodies were used as capture antibodies, each coated onto an ELISA plate. The corresponding eleven HRP-labeled antibodies were used as detection antibodies. An 11×11 antibody pairing matrix was constructed for checkerboard assay. By comparing the signal-to-noise ratio (SNR) of each combination, the pairing scheme of 9A12 as the capture antibody and HRP-labeled 16B12 as the detection antibody was determined to be the best, producing the highest SNR (S / N value) among all test combinations, demonstrating optimal detection performance. Figure 5 ).

[0077] 2. The sequence information of rabbit monoclonal antibody 9A12 is as follows:

[0078]

[0079]

[0080] The sequence information of rabbit monoclonal antibody 16B12 is as follows:

[0081]

[0082]

[0083] 3. DAS-ELISA detection of antibody pairs based on anti-hantavirus NP protein ( Figure 6 )

[0084] (1) Coating: Dilute the capture antibody 9A12 to 0.125 μg / mL with coating buffer (pH 9.6, 0.05M carbonate buffer), add it to the ELISA plate, and incubate at 37°C for 60 min.

[0085] (2) Washing and blocking: The next day, discard the coating solution and wash the plate 3 times with PBST (PBS containing 0.05% Tween-20). Add blocking solution PBST + 5% FBS and block at 37℃ for 120 min.

[0086] (3) Sample addition: Discard the blocking solution and wash the plate 3 times. Add the sample to be tested and incubate at 37°C for 90 min.

[0087] (4) Add detection antibody: Discard the sample and wash the plate 5 times. Add HRP (horseradish peroxidase) labeled 16B12 diluted at a ratio of 1:1000 and incubate at 37°C for 60 min.

[0088] (5) Color development and termination: Discard the detection antibody and wash the plate 5 times. Add 100 μL of TMB substrate solution and develop the color at 37°C in the dark for 15 minutes. Add 50 μL of 2M H2SO4 to terminate the reaction.

[0089] (6) Plate reading: Read the absorbance (OD) at 450 nm on the microplate reader. 450 ).

[0090] Example 4 Performance validation of Hantavirus NP protein DAS-ELISA detection

[0091] This embodiment systematically evaluates and validates the performance of the DAS-ELISA method for Hantavirus NP antigen established in Example 3.

[0092] 1. Standard Curve

[0093] To evaluate the detection performance of the established DAS-ELISA method for different types of hantavirus NP proteins, prokaryotically expressed HTNV-NP, SERV-NP, and DBSV-NP proteins (DBSV XN36 / 2025 NP, NCBI database Accession No: PX852834) were used as standards, following the same procedure as the previous preparation of HTNV and SERV NP prokaryotic proteins. Each antigen was serially diluted within the concentration range of 1-10,000 ng / mL, and parallel detection was performed using the DAS-ELISA method. A four-parameter logistic regression (4PL) model was used in GraphPadPrism 9.0 software to perform curve fitting between standard concentrations and corresponding OD values. Three replicates were set for each concentration point, and the coefficient of variation (CV%) was calculated for each concentration point.

[0094] The results showed that the DAS-ELISA method exhibited good linearity in the detection of HTNV-NP, SEEV-NP, and DBSV-NP proteins within a concentration range of 1.953–250,000 ng / mL. The coefficients of determination (R²) of the three standard curves were 0.998, 0.992, and 0.998, respectively, indicating a significant dose-dependent relationship between OD values ​​and antigen concentration. Furthermore, the coefficients of variation (CV) for all concentration points within this range were between 1.03% and 9.58% (all below 10%), demonstrating the method's excellent reproducibility. The EC50 of DBSV-NP was also measured. 50 The lowest (15.4 ng / mL) EC of SEEV-NP 50 The highest (89.5 ng / mL) EC of HTNV-NP 50 The value was in the middle (76.9 ng / mL).

[0095] 2. Determining the Cut-off Value

[0096] Background values ​​from 32 healthy serum samples were analyzed. The cut-off value was calculated using the formula "mean OD value of blank sample + 3 × standard deviation (Mean_blank + 3SD)". This value provides a positive threshold of ≥0.07. Sensitivity experiments were conducted using serially diluted prokaryotically expressed HTNV-NP recombinant protein (concentration range 1.0 μg / mL-0.1 ng / mL). The results showed that the limit of detection (LOD) for HTNV-NP using the DAS-ELISA method of this application was 1 ng / mL.

[0097] 3. Specificity

[0098] Cross-reactivity tests were conducted using various unrelated viral proteins prepared in the laboratory previously as antigens, including HTNV-Gn, LCMV-N, WENV-N, Nai-N, Romv-P, HEV-ORF2, HEV-ORF3, and HTNV-NP, SEOV-NP, and DBSV-NP antigens from the Hantavirus genus. The explanations of the relevant abbreviations are as follows:

[0099]

[0100]

[0101] The results showed that only HTNV-NP, SERV-NP, and DBSV-NP antigens within the Hantavirus genus showed strong positive reactions, while the OD values ​​of all other non-NP antigens, including HTNV-Gn, LCMV-N, and WENV-N, and irrelevant viral antigens, were close to or lower than the cut-off value. This indicates that the established method has high specificity for Hantavirus NP proteins and can broadly detect different types of Hantavirus, including HTNV, SERV, and DBSV.

[0102] 4. Clinical Applications

[0103] The above-described DAS-ELISA method was applied to detect the culture supernatant of Vero E6 cells infected with Hantavirus (HTNV 76-118 strain). Samples were collected for 11 consecutive days. At the same time, the NP antigen in the supernatant was detected by the DAS-ELISA method, and the copy number of the genomic RNA (vRNA) carried by the virus was detected in parallel by qPCR.

[0104] The results showed that from 1 to 5 days post-infection, the NP protein level detected by DAS-ELISA and the viral genomic RNA (vRNA) copy number detected by qPCR showed a synchronous upward trend and were highly correlated. This result confirms that the established DAS-ELISA method can effectively detect hantavirus particles in real infected samples and is suitable for dynamic monitoring of viral replication.

[0105] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An antibody pair against Hantavirus NP protein, characterized in that, The antibody pair includes a first monoclonal antibody and a second monoclonal antibody; each monoclonal antibody includes a light chain variable region and a heavy chain variable region; the light chain variable region includes light chain CDR1, light chain CDR2 and light chain CDR3; the heavy chain variable region includes heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3. The sequence information of the first monoclonal antibody is as follows: The amino acid sequence of the light chain CDR1 is shown in SEQ ID NO: 3; The amino acid sequence of the light chain CDR2 is shown in SEQ ID NO: 4; The amino acid sequence of the light chain CDR3 is shown in SEQ ID NO: 5; The amino acid sequence of heavy chain CDR1 is shown in SEQ ID NO: 8; The amino acid sequence of the heavy chain CDR2 is shown in SEQ ID NO: 9; The amino acid sequence of the heavy chain CDR3 is shown in SEQ ID NO: 10; The sequence information of the second monoclonal antibody is as follows: The amino acid sequence of the light chain CDR1 is shown in SEQ ID NO: 17; The amino acid sequence of the light chain CDR2 is shown in SEQ ID NO: 18; The amino acid sequence of the light chain CDR3 is shown in SEQ ID NO: 19; The amino acid sequence of heavy chain CDR1 is shown in SEQ ID NO: 22; The amino acid sequence of the heavy chain CDR2 is shown in SEQ ID NO: 23; The amino acid sequence of the heavy chain CDR3 is shown in SEQ ID NO:

24.

2. The antibody pair according to claim 1, characterized in that, The amino acid sequence of the light chain variable region of the first monoclonal antibody is shown in SEQ ID NO: 2, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 7; The amino acid sequence of the light chain variable region of the second monoclonal antibody is shown in SEQ ID NO: 16, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:

21.

3. The antibody pair according to claim 2, characterized in that, The amino acid sequence of the light chain of the first monoclonal antibody is shown in SEQ ID NO: 1, and the amino acid sequence of the heavy chain is shown in SEQ ID NO: 6; The amino acid sequence of the light chain of the second monoclonal antibody is shown in SEQ ID NO: 15, and the amino acid sequence of the heavy chain is shown in SEQ ID NO:

20.

4. Use of the antibody pair according to any one of claims 1-3 in the preparation of a kit for detecting Hantavirus.

5. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the amino acid sequence of the first or second monoclonal antibody of any one of claims 1-3.

6. The nucleic acid molecule according to claim 5, characterized in that, The nucleic acid molecule encoding the variable region sequence of the light chain of the first monoclonal antibody is shown in SEQ ID NO: 12; The nucleic acid molecule encoding the heavy chain variable region sequence of the first monoclonal antibody is shown in SEQ ID NO: 14; The nucleic acid molecule encoding the variable region sequence of the light chain of the second monoclonal antibody is shown in SEQ ID NO: 26; The nucleic acid molecule encoding the heavy chain variable region sequence of the second monoclonal antibody is shown in SEQ ID NO:

28.

7. The nucleic acid molecule according to claim 6, characterized in that, The nucleic acid molecule encoding the light chain sequence of the first monoclonal antibody is shown in SEQ ID NO: 11; The nucleic acid molecule encoding the heavy chain sequence of the first monoclonal antibody is shown in SEQ ID NO: 13; The nucleic acid molecule encoding the light chain sequence of the second monoclonal antibody is shown in SEQ ID NO: 25; The nucleic acid molecule encoding the heavy chain sequence of the second monoclonal antibody is shown in SEQ ID NO:

27.

8. A kit for detecting Hantavirus, characterized in that, Includes the antibody pair according to any one of claims 1-3.

9. The kit according to claim 8, characterized in that, The detection kit is an enzyme-linked immunosorbent assay (ELISA) kit, an ELISA kit, an immunohistochemistry kit, an immunofluorescence kit, an immunoblotting kit, or a flow cytometry kit.

10. The kit according to claim 9, characterized in that, The kit is a double-antibody sandwich enzyme-linked immunosorbent assay kit, wherein the first monoclonal antibody is a capture antibody, and the second monoclonal antibody is a detection antibody with a detection marker attached.

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