A monoclonal antibody combination and an AlphaLISA detection method and a colloidal gold immunochromatographic detection method for detection of nipah virus

Abstract

This invention provides a monoclonal antibody combination for Nipah virus detection, along with an AlphaLISA and colloidal gold immunochromatographic assay for Nipah virus detection, belonging to the field of virus detection technology. This invention isolates hundreds of antigen-specific B cells from immunized mice, obtaining five stably secreted mAbs. All antibody heavy and light chains specifically recognize the Nipah virus G protein. The AlphaLISA assay using the NiV-22 / NiV-12 antibody combination has a detection limit as low as 0.024 ng / mL, approximately 100 times lower than traditional ELISA, and shows no cross-reactivity in the tests of five heterologous viral antigens. The colloidal gold immunochromatographic assay using the NiV-22 / NiV-12 antibody combination achieves a sensitivity of 1 ng / mL for G protein detection and a sensitivity of 945 TU / mL for Nipah virus pseudovirus detection, showing no cross-reactivity in the tests of five heterologous viral antigens.

Description

Technical Field

[0001] This invention belongs to the field of virus detection technology, and particularly relates to a monoclonal antibody combination for Nipah virus detection and an AlphaLISA and colloidal gold immunochromatographic detection method for Nipah virus. Background Technology

[0002] Nipah virus (NiV) is a highly pathogenic zoonotic virus, first identified in Malaysia in 1998-1999. Clinical manifestations of infection include asymptomatic infection, acute respiratory symptoms, and fatal encephalitis. Currently, there are no specific drugs or vaccines against this virus, and treatment mainly relies on supportive care. The virus binds to receptors on the surface of host cells via its G protein. After entering the cell, it replicates and transcribes to produce new viral particles, leading to impaired cell function and ultimately inducing clinical symptoms.

[0003] Accurate and rapid diagnostic techniques are crucial for Nipah virus. Currently, detection methods for Nipah virus mainly include nucleic acid testing, antibody testing, and virus isolation and neutralization assays. Nucleic acid testing, specifically reverse transcription polymerase chain reaction (RT-PCR), is widely used for the early diagnosis of Nipah virus. However, RT-PCR has limitations: it requires sophisticated equipment and advanced laboratory facilities, is expensive and complex, and is highly dependent on the skill level of the personnel. It can also lead to false positives, especially in the presence of laboratory contamination. Serological testing methods, including enzyme-linked immunosorbent assay (ELISA) and immunofluorescence assay (IFA), while simple and inexpensive, also have a window period limitation. In the early stages of infection, the host may not have produced sufficient specific antibodies, leading to false negatives in acute-phase diagnosis. Furthermore, serological testing faces the risk of cross-reactivity, especially in regions with complex epidemiological backgrounds, where antibodies between different viruses can cause misdiagnosis and affect the accuracy of test results. Virus isolation and neutralization tests have extremely strict biosafety requirements for laboratories, are complex to operate, and are costly, so they are not widely used in routine clinical diagnosis. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a monoclonal antibody combination for Nipah virus detection and an AlphaLISA and colloidal gold immunochromatographic detection method for Nipah virus.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] This invention provides a monoclonal antibody combination for Nipah virus detection, comprising NiV-12 and NiV-22.

[0007] The amino acid sequence of the heavy chain variable region of NiV-12 is shown in SEQ ID No: 1, specifically EVQLQESGPGLVAPSQSLSITCTVSGFSIIGYGVNWVRQPPGKGLEWLGMIWGDGSTDYNSALKSRLSISKDNSKSQVFLKMNSLQTDDTARYYCARDPHYYGYYGMDYWGQGTSVTVSS. The amino acid sequence of the light chain variable region of NiV-12 is shown in SEQ ID No: 2, specifically DIVMTQSPLSLPVSLGDQASISCRSSQSLVHSHGETYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPWTFGGGTKLEIK.

[0008] The amino acid sequence of the heavy chain variable region of NiV-22 is shown in SEQ ID No: 3, specifically EVQLQESGAELVRPGSSVKISCKASGYVFSIYWMNWVKQRPGQGPEWIGQIYPGQGDTNYNGKFKGKATVTVDKSSSTAYMQLSSLTSEDSAVYFCVRGSFYAMDYWGQGTSVTVSS. The amino acid sequence of the light chain variable region of NiV-22 is shown in SEQ ID No: 4, specifically DIVVTQSPAILSASPGEKVTMTCRASSSVSYMHWYQQKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSSNPLTFGAGTKLELK.

[0009] Preferably, the heavy chain variable region of NiV-12 has three complementarity-determining regions CDR1, CDR2 and CDR3. The amino acid sequence of the heavy chain CDR1 is shown in SEQ ID No: 5, specifically GYGVN; the amino acid sequence of the heavy chain CDR2 is shown in SEQ ID No: 6, specifically MIWGDGSTDYNSALKS; and the amino acid sequence of the heavy chain CDR3 is shown in SEQ ID No: 7, specifically DPHYYGYYGMDY.

[0010] The light chain variable region of NiV-12 has three complementarity-determining regions, CDR1, CDR2, and CDR3. The amino acid sequence of the light chain CDR1 is shown in SEQ ID No: 8, specifically RSSQSLVHSHGETYLH. The amino acid sequence of the light chain CDR2 is shown in SEQ ID No: 9, specifically KVSNRFS. The amino acid sequence of the light chain CDR3 is shown in SEQ ID No: 10, specifically SQSTHVPWT.

[0011] Preferably, the heavy chain variable region of NiV-22 has three complementarity-determining regions CDR1, CDR2 and CDR3. The amino acid sequence of the heavy chain CDR1 is shown in SEQ ID No: 11, specifically IYWMN; the amino acid sequence of the heavy chain CDR2 is shown in SEQ ID No: 12, specifically QIYPGQGDTNYNGKFKG; and the amino acid sequence of the heavy chain CDR3 is shown in SEQ ID No: 13, specifically GSFYAMDY.

[0012] The light chain variable region of NiV-22 has three complementarity-determining regions, CDR1, CDR2, and CDR3. The amino acid sequence of the light chain CDR1 is shown in SEQ ID No: 14, specifically RASSSVSYMH. The amino acid sequence of the light chain CDR2 is shown in SEQ ID No: 15, specifically ATSNLAS. The amino acid sequence of the light chain CDR3 is shown in SEQ ID No: 16, specifically QQWSSNPLT.

[0013] The present invention also provides a polynucleotide encoding the monoclonal antibody combination for Nipah virus detection, wherein the nucleotide sequence encoding the amino acid of the heavy chain variable region of NiV-12 is as shown in SEQ ID. As shown in No. 17, specifically GAAGTGCAGCTCCAAGAGAGCGGGCCCGGCCTCGTGGCCCCTAGCCAAAGCCTGAGCATCACCTGCACCGTGAGCGGCTTCAGCATCATCGGCTACGGCGTGAACTGGGTGAGACAGCCCCCCGGCAAGGGCCTGGAGTGGCTGGGCATGATCTGGGGCGACGGCAGCACCGACTACAACAGCGCCCTGAAGAGCAGACTGAGCATCAGCAAGGACAACAGCAAGAGCCAAGTGTTCCTGAAGATGAACAGCCTGCAGACCGACGACACCGCTAGATACTACTGCGCTAGAGACCCCCACTACTACGGCTACTACGGCATGGACTACTGGGGCCAAGGCACAAGCGTGACCGTGAGCAGC, the nucleotide sequence encoding the amino acid of the light chain variable region of NiV-12 is as shown in SEQ ID. No: 18 is shown, specifically GACATCGTGATGACACAGAGCCCCCTGAGCCTGCCCGTGAGCCTGGGCGACCAAGCTAGCATCAGCTGCAGAAGCAGCCAAAGCCTGGTGCACAGCCACGGCGAGACCTACCTGCACTGGTACCTGCAGAAGCCCGGGCAGAGCCCCAAGCTGCTGATCTACA AGGTGAGCAACAGATTCTCCGGCGTGCCCGACAGATTCAGCGGCAGCGGCAGCGGGACCGACTTCACCCTGAAGATCAGCAGAGTGGAGGCCGAGGACCTGGGCGTGTACTTCTGCAGCCAAAGCACCCACGTGCCCTGGACCTTCGGCGGGGGCACCAAGCTGGAGATCAAG;

[0014] The nucleotide sequence encoding the amino acids of the heavy chain variable region of NiV-22 is shown in SEQ ID No: 19, specifically: GAGGTGCAGCTGCAAGAGAGCGGCGCCGAGCTGGTGAGACCCGGCAGCAGCGTGAAGATCAGCTGCAAGGCTAGCGGCTACGTGTTCAGCATCTACTGGATGAACTGGGTGAAGCAGAGACCCGGCCAAGGCCCCGAGTGGATCGGGCAGATCTACCCCGGCCAAGGCGATACCAACTACAACGGCAAGTTCAAGGGCAAGGCCACCGTGACCGTGGACAAGAGCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGACAAGCGAGGACAGCGCCGTGTACTTCTGCGTGAGAGGCAGCTTCTACGCCATGGACTACTGGGGCCAAGGCACAAGCGTGACCGTGAGCAGC. The nucleotide sequence encoding the amino acids of the light chain variable region of NiV-22 is shown in SEQ ID No: 19. No: 20 is shown, specifically GACATCGTGGTGACAGAGCCCCGCCATCCTGAGCGCTAGCCCCGGCGAGAAGGTGACCATGACCTGCAGAGCTAGCAGCAGCGTGAGCTACATGCACTGGTATCAGCAGAAGCCCGGCAGCAGCCCCAAGCCCTGGATTTACGCCACAAGCA ACCTGGCTAGCGGCGTGCCCGCTAGATTCAGCGGCAGCGGCAGCGGCACAAGCTACAGCCTGACCATCAGCAGTGGAGGCCGAGGACGCCGCCACCTACTACTGTCAGCAGTGGAGCAGCAACCCCCTGACCTTCGGCGCCGGCACCAAGCTGGAGCTGAAG.

[0015] Preferably, the nucleotide sequence encoding the NiV-12 heavy chain CDR1 is as shown in SEQ ID No: 21, specifically GGCTACGGCGTGAAC; the nucleotide sequence encoding the NiV-12 heavy chain CDR2 is as shown in SEQ ID No: 22, specifically ATGATCTGGGGCGACGGCAGCACCGACTACAACAGCGCCCTGAAGAGC; and the nucleotide sequence encoding the NiV-12 heavy chain CDR3 is as shown in SEQ ID No: 23, specifically GACCCCCACTACTACGGCTACTACGGCATGGACTAC.

[0016] The nucleotide sequence encoding the NiV-12 light chain CDR1 is shown in SEQ ID No: 24, specifically AGAGCAGCCAAAGCCTGGTGCACAGCCACGGCGAGACCTACCTGCAC; the nucleotide sequence encoding the NiV-12 light chain CDR2 is shown in SEQ ID No: 25, specifically AAGGTGAGCAACAGATTCTCC; and the nucleotide sequence encoding the NiV-12 light chain CDR3 is shown in SEQ ID No: 26, specifically AGCCAAAGCACCCACGTGCCCTGGACC.

[0017] Preferably, the nucleotide sequence encoding the NiV-22 heavy chain CDR1 is as shown in SEQ ID No: 27, specifically ATCTACTGGATGAAC; the nucleotide sequence encoding the NiV-22 heavy chain CDR2 is as shown in SEQ ID No: 28, specifically CAGATCTACCCCGGCCAAGGCGATACCAACTACAACGGCAAGTTCAAGGGC; and the nucleotide sequence encoding the NiV-22 heavy chain CDR3 is as shown in SEQ ID No: 29, specifically GGCAGCTTCTACGCCATGGACTAC.

[0018] The nucleotide sequence encoding the NiV-22 light chain CDR1 is shown in SEQ ID No: 30, specifically AGAGCTAGCAGCAGCGTGAGCTACATGCAC; the nucleotide sequence encoding the NiV-22 light chain CDR2 is shown in SEQ ID No: 31, specifically GCCACAAGCAACCTGGCTAGC; and the nucleotide sequence encoding the NiV-22 light chain CDR3 is shown in SEQ ID No: 32, specifically CAGCAGTGGAGCAGCAACCCCCTGACC.

[0019] The present invention also provides the application of the monoclonal antibody combination described above for Nipah virus detection in the detection of Nipah virus.

[0020] Preferably, the method for detecting Nipah virus includes an immunoassay method, which includes AlphaLISA, ELISA, colloidal gold / latex chromatography, and immunofluorescence assay.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] This invention successfully isolated hundreds of antigen-specific B cells from immunized mice, and after gene cloning and expression, obtained five stably secreted mAbs (named NiV-6, NiV-8, NiV-12, NiV-22, and NiV-63). SDS-PAGE and ELISA confirmed that the heavy and light chain sizes of all antibodies were consistent with expectations and that they specifically recognized the native conformation of the Nipah virus G protein. The standard curve of the NiV-22 / NiV-12 antibody combination showed linearity in the AlphaLISA assay range of 0.012-100 ng / mL (R0.012). 2 =0.9989), with a detection limit as low as 0.024 ng / mL, approximately 100 times higher than traditional ELISA. Intra-assay repeatability CV was 6.4%-8.0% (low, medium, and high concentration samples), and inter-assay CV was 8.1%-9.5%; no cross-reactivity was observed in the tests for five heterologous viral antigens. The NiV-22 / NiV-12 antibody combination achieved a sensitivity of 1 ng / mL for G proteins and 945 TU / mL for Nipah virus pseudovirus in colloidal gold immunochromatography, with no cross-reactivity observed in the tests for five heterologous viral antigens.

[0023] This invention integrates single-B cell technology with various immunoassay platforms to achieve an innovative combination of efficient preparation of Nipah virus monoclonal antibodies and ultrasensitive detection / rapid on-site detection technologies, constructing a complete process system from the antigen to the mature immunoassay protocol. The "antibody discovery-detection development" dual-track system established by this invention not only provides an integrated solution for the precise diagnosis and therapeutic antibody development of Nipah virus, but also offers a modular development path for the rapid development of tools for the prevention and control of emerging infectious diseases, demonstrating significant universal value. Attached Figure Description

[0024] Figure 1 It is the IgG titer of mouse serum against Nipah virus G protein;

[0025] Figure 2 It is a flow cytometry sorting method for NiV-G protein-specific memory B cells;

[0026] Figure 3This is the electrophoresis result of single-cell PCR amplification of the antibody variable region gene (Maker: DNA molecular standard mass; Haven chain: antibody heavy chain; Light chain: antibody light chain).

[0027] Figure 4 It is an indirect ELISA assay to detect the binding activity of the antibody to the NiV-G protein;

[0028] Figure 5 It is the determination of the antibody affinity constant;

[0029] Figure 6 It is an indirect ELISA assay to detect the binding activity of the antibody to the NiV-G protein;

[0030] Figure 7 It is a NiV monoclonal antibody SDS-PAGE (Marker: standard mass of DNA molecule, H: antibody heavy chain, L: antibody light chain).

[0031] Figure 8 It is the screening of AlphaLISA antibody pairs;

[0032] Figure 9 The optimization of the dilution ratio of biotinylated mouse anti-NiV monoclonal antibody conjugated with mouse anti-NiV monoclonal antibody receptor microspheres and donor microspheres in the AlphaLISA detection system (where A is the optimization of the donor microsphere dilution ratio, B is the optimization of the biotinylated Nipah virus antibody dilution ratio, and C is the optimization of the dilution ratio of the Nipah virus antibody and receptor microsphere conjugate).

[0033] Figure 10 This is the standard curve for AlphaLISA detection of NiV antigen;

[0034] Figure 11 This is the detection limit for NiV antigen;

[0035] Figure 12 This is the standard curve for AlphaLISA detection of NiV pseudovirus;

[0036] Figure 13 This is the detection limit for NiV pseudovirus;

[0037] Figure 14 This is the standard curve for detecting NiV antigen using AlphaLISA porcine serum simulated samples;

[0038] Figure 15 This is the detection limit for NiV antigen in swine serum simulated samples;

[0039] Figure 16 This is the standard curve of NiV pseudovirus from AlphaLISA porcine serum simulated samples;

[0040] Figure 17This is the detection limit for NiV pseudovirus in swine serum simulated samples;

[0041] Figure 18 This is the optimal dilution ratio of human serum to PBS;

[0042] Figure 19 This is the standard curve of NiV antigen from human serum simulated samples for AlphaLISA;

[0043] Figure 20 This is the detection limit for NiV antigen in human serum simulated samples;

[0044] Figure 21 This is the standard curve of NiV pseudovirus from human serum simulated samples obtained by AlphaLISA;

[0045] Figure 22 This is the detection limit for NiV pseudovirus in human serum simulated samples;

[0046] Figure 23 This is a specific study of AlphaLISA for detecting Nipah virus antigens (IAV: Influenza A virus, IBV: Influenza B virus, JEV19: Japanese encephalitis virus, CA73: Coxsackie virus, ZiKa: Zika virus).

[0047] Figure 24 This is a diagram showing the specificity results of the Nipah virus immunochromatographic test strip.

[0048] Figure 25 This is a graph showing the sensitivity results of the immunochromatographic test strip for detecting Nipah virus G protein;

[0049] Figure 26 This is a graph showing the sensitivity results of the immunochromatographic test strip for detecting Nipah virus pseudovirus. Detailed Implementation

[0050] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0051] Example 1

[0052] Sorting and antibody sequence determination of single B cells from mice secreting NiV antibodies

[0053] Expi293 cells were provided by the Institute of Microbiology and Epidemiology, Academy of Military Medical Sciences, and six-week-old female BALB / c mice were purchased from Spiford (Beijing) Biotechnology Co., Ltd. NiV-G protein was synthesized by Beijing Yiqiao Shenzhou Technology Co., Ltd.

[0054] I. Evaluation of Immune and Humoral Immune Response Levels in Experimental Mice

[0055] 1. Immunization of laboratory animals and collection of blood samples

[0056] Blood was collected from newly purchased mice via tail vein sampling before immunization. Immunization was performed every 21 days for a total of four immunizations. For the first immunization, 150 μL of antigen (50 μg / mouse) was administered intraperitoneally after mixing Freund's complete adjuvant with the antigen. For the subsequent three immunizations, 150 μL of antigen (50 μg / mouse) was administered intraperitoneally after mixing Freund's incomplete adjuvant with the antigen. Tail vein blood was collected one week after the third and fourth immunizations. The collected blood was incubated at 37°C for 1 hour, then placed in a 4°C refrigerator for 3 hours, followed by centrifugation at 8000 rpm for 10 minutes. The supernatant was collected and stored at -20°C.

[0057] 2. Indirect ELISA method for determining the titer of mouse serum IgG anti-NiV-G protein

[0058] The antigen was coated with NiV-G protein, and the antibody serum binding titer was detected by indirect ELISA at different time points after immunization. The experimental steps are as follows:

[0059] ① Coating: Dilute the 10× coating solution 10 times with ultrapure water, use the diluted coating solution to dilute the NiV-G protein to 1 μg / mL, add 100 μL of the diluted NiV-G protein to each well of the enzyme strip using a pipette, and incubate at 4℃ for 12 h.

[0060] ② Washing the plate: Pat the plate dry on kitchen paper, wash the plate with PBST, add 200μL to each well, use a multi-channel pipette to wash the plate, after adding PBST, place the plate on a shaker to shake the plate, pat dry again, repeat the washing process three times in total;

[0061] ③ Blocking: Pat the plate dry, add 280 μL of 3% BSA dissolved in PBST to each well using a pipette, and block overnight (16-24 h) in a 4°C refrigerator.

[0062] ④ Washing the plate: Pat the plate dry on kitchen paper, wash the plate with PBST, add 300μL to each well, use a multi-channel pipette to wash the plate, after adding PBST, place it on a plate shaker to shake the plate, pat dry again, repeat five times in total, and it can be stored at -20℃ for 3 months.

[0063] ⑤ Incubate primary antibody: Dilute the immunized mouse serum with PBST, dilute it in an enzyme-linked strip, and dilute the antigen by 100 times, 1000 times, 10000 times, 100000 times, etc. Add the diluted immunized mouse serum to the coated plate using a multi-channel pipette, 100 μL / well, and incubate in a 37℃ incubator for 30 min.

[0064] ⑥ Washing the plate: Pat the plate dry on kitchen paper, wash the plate with PBST, add 200μL to each well, use a combustor to wash the plate, after adding PBST, place the plate on a shaker to shake the plate, pat dry again, repeat for a total of five times;

[0065] ⑦ Incubate with secondary antibody: Dilute goat anti-mouse HRP antibody 10,000 times with PBST, vortex to mix, and add it to the enzyme strip using a multi-channel pipette, 100 μL / well, and incubate at 37°C for 30 min.

[0066] ⑧ Washing the plate: Pat the plate dry on kitchen paper, wash the plate with PBST, add 200μL to each well, use a combustor to wash the plate, after adding PBST, place the plate on a shaker to shake the plate, pat dry again, repeat for a total of five times;

[0067] ⑨ Color development: Add 100 μL of TMB color development solution to each well and react at room temperature in the dark for 5 min (determine the color development time according to the color development situation). Add 50 μL of stop solution to each well to stop the reaction and use an ELISA reader to measure the absorbance at 450 nm.

[0068] Experimental results: such as Figure 1 As shown, after the third immunization, the immunogenicity of all four mice reached 1:10. 5 (log10[EC50]=5.0±0.3); After the fourth immunization, the immune titers of the four mice were significantly increased. Among them, Mouse-A and Mouse-B mice showed a high level of immune response to NiV-G protein, with Mouse-B mice showing the highest level and an immune titer of up to 1:1. (log10=7.2). The ELISA results showed that after immunization with Nipah virus G protein, BALB / c mice produced anti-Nipah virus antibodies in their serum. The serum antibody titer increased after each immunization with statistical differences, which was consistent with the expected immunogenicity.

[0069] II. Flow cytometry sorting of NiV-G protein-specific memory B cells

[0070] 1. Separate mouse spleen and obtain spleen cells

[0071] ① First, the mouse was euthanized by dislocation and then immersed in a 500 mL beaker containing 75% alcohol. The entire procedure was performed in a biosafety cabinet. The mouse was fixed belly-up on the dissection table, and the outer layer of skin was cut open to remove the spleen.

[0072] ② Add 5 mL of culture medium (1640 plus 5% fetal bovine serum) to a clean culture dish in advance. Place the mouse spleen in a 40 μm filter and grind it with the rubber tip of a 5 mL syringe. After grinding, rinse the filter screen with cell culture medium. Aspirate all the cell fluid through the filter and put it into a 15 mL centrifuge tube. Centrifuge at 1500 rpm for 15 min at 4 °C. Keep the experiment on ice throughout the experiment.

[0073] ③ After centrifugation, discard the upper culture medium and add 3 mL of red blood cell lysis buffer. Lyse at room temperature for 2 min, then fill with culture medium and centrifuge at 1500 rpm for 15 min at 4℃.

[0074] ④ After centrifugation, discard the liquid, resuspend in PBS, centrifuge at 4℃ and 1500 rpm for 15 min, discard the supernatant, and finally resuspend in 1 mL of flow cytometry buffer.

[0075] Experimental Results: After the fourth immunization of mice, the serum antibody titer was measured. Compared with the third immunization, the antibody titer increased, but not significantly. Therefore, it was decided not to perform a follow-up immunization after the fourth immunization and directly proceed to the sorting of memory B cells. Splenic cells were obtained from the spleens of the mice for sorting. A total of three mice were selected for spleen cell collection. The spleens of the three mice were significantly larger than those of normal mice. A total of 2 × 10⁶ cells were isolated. 8 The number of spleen cells showed a viability of greater than 98% in the cell counter.

[0076] 2. Flow cytometry sorting of NiV-G antigen-specific single B cells

[0077] ① The concentration of the counted spleen cell suspension was adjusted to 1×1 using flow cytometry staining buffer. live cells / mL;

[0078] ② Prepare five 1.5 mL test tubes, and transfer 20 μL of cell suspension (containing 2 × 10⁻⁶ cells) into each tube. 6 (200 live cells) were used as a single-label control; another test tube was filled with 200 μL of suspension (containing 2 × 10⁻⁶ live cells). 7 (10 live cells) were used as the master sample for sorting;

[0079] ③ Add 10 μg of mouse isoglobulin to each single label tube and 20 μg of isoglobulin to the master sample tube. Incubate in an ice bath in the dark for 10 min to block Fc receptors and reduce non-specific binding.

[0080] ④ Referring to the preset fluorescent labeling scheme (Table 1), add specific antibodies to each detection tube, set up unlabeled samples as blank controls, and incubate at 4℃ in the dark for 30 min;

[0081] ⑤ Add 3 mL of pre-cooled buffer to each tube and gently suspend. Use a gradient centrifugation program (parameter settings: heating and cooling rate level 3, 1000×g, 4℃, 5 min), and wash twice to remove free antibodies. Discard the supernatant and retain the precipitate.

[0082] ⑥ In the single-label group, NiV-G treated tubes and co-stained tubes were supplemented with 0.5 μL of PE-Cy7 labeled streptavidin secondary antibody, and the remaining samples were mixed with 1 mL of buffer and temporarily stored in the dark at 4℃.

[0083] ⑦ After the antigen-labeled group was stained twice, it was washed three times by centrifugation (1500×g, 4℃), and finally resuspended in 1 mL of buffer. All operations were performed under light-protected conditions.

[0084] ⑧ After filtering the cells in each tube through a 200-mesh cell sieve, the NiV-specific memory B cells were sorted using a BD FACSAria III flow cytometer. After starting the machine, the flow rate was adjusted to a stable state, and then naked cells and single-stained tubes were loaded sequentially with compensation adjustments. The sorting tubes were loaded onto the machine, and lymphocytes were circled using FSC and SSC, designated as gate 1. In gate 1, dead cells and adhesions were excluded using 7AAD, and single cells were circled using PerCP Cy5.5, designated as gate 2. In gate 2, single memory B cells were circled using FITC, designated as gate 3. In gate 3, CD19+ memory B cells were circled using BV510, designated as gate 4. In gate 4, IgG+ antigen-specific memory B cells were circled using PE-Cy7 and APC-Cy7, designated as gate 5. In gate 5, the desired cells were circled using PE and APC. The cell population in gate 6 (CD19+ / IgG+ / PE+ / APC+) consisted of antigen-specific memory B lymphocytes.

[0085] ⑨ Configure a 100μm nozzle, select the single-cell sorting mode, sort the target cell population (CD19+ / IgG+ / PE+ / APC+) into a 96-well PCR plate pre-filled with 5μL of lysis buffer, and immediately transfer to an ultra-low temperature freezer at -80℃ for storage after sorting.

[0086] Table 1. Fluorescently labeled antibodies and antigen dosages for sorting antigen-specific B lymphocytes

[0087]

[0088] Experimental results: such as Figure 2As shown, TruStain FcX reagent blocked non-specific Fc receptors and de-adhesion lymphocytes (Gate 1). 7AAD was selected to remove dead cells (Gate 2). FITC-negative cells (excluding T cells) were selected to select B cells (Gate 3). BV510-positive cells were selected. CD19+ is a marker molecule for memory B cells. IgG is expressed on the surface of memory B cells. Memory B cells expressing IgG were co-selected using CD19+ and IgG fluorescent monoclonal antibodies (Gate 4). PE-Cy7-negative cells and APC-Cy7-negative cells were then selected sequentially (Gate 5). Finally, a cell population double-positive for PE and APC-labeled antigens was selected (Gate 6). Ultimately, this cell population accounted for less than 0.01%, resulting in 288 memory B cells with phenotypic titers (CD19+ / IgG+ / PE+ / APC+) in a 96-well plate.

[0089] III. Amplification of Antibody Variable Region Genes

[0090] 1. Preparation of single B cell cDNA molecules

[0091] cDNA synthesis was performed on lysed single B cells using the Aomei Bio premixed reverse transcription kit. The reaction conditions were set as follows: 50°C for 15 min to complete the reverse transcription reaction, followed by 90°C for 1 min to inactivate the reverse transcriptase, and finally storage at 4°C to terminate the reaction. The resulting cDNA product was immediately transferred to a -80°C cryogenic freezer for subsequent nested PCR amplification experiments. The reverse transcription PCR reaction system is shown in Table 2.

[0092] Table 2 PCR Reverse Transcription System

[0093]

[0094] 2. Primer design for nested amplification of antibody variable regions

[0095] For each cell, cDNA was generated using a two-step PCR with different primers. The first round of amplification employed a mixed primer system, using nested PCR to amplify the variable heavy and light chain domains. The forward primer specifically binds to the V(D)J gene leader sequence, while the reverse primer targets the conserved 3' region of the immunoglobulin heavy chain constant domain (CH1 region). The second round utilized a nested amplification strategy, with the forward primer designed for the FR1 region of the 5' frame sequence in the variable region, and the reverse primer using nested complementary oligonucleotides targeting conserved sites within the CH1 region. Primer design considered all functional genes from the BALB / c mouse strain. The forward primer targeted the conserved region of the V gene family (covering functional genes in BALB / c, 129 / Sv, and C57BL / 6 strains), while the reverse primer was designed for the constant region (IgG-CH1 or Igκ / Igλ-C region). The PCR products were then purified and sequenced.

[0096] Table 3 Primer sequences and product sizes for antibody variable region amplification

[0097]

[0098] 3. Single-cell PCR amplification of antibody light and heavy chain variable region genes

[0099] Antibody heavy chains can be classified into five types—μ (IgM), α (IgA), δ (IgD), ε (IgE), and γ (IgG)—based on the amino acid composition and sequence differences in their constant region (C region). The structural characteristics of different antibody classes are further determined by the number and spatial distribution of disulfide bonds in the C region of their heavy chains, thus classifying them into different subclasses. Given that the B cells isolated in this invention specifically express IgG-type antibodies, it is necessary to amplify the variable region (VH) gene of its γ chain heavy chain to analyze the diversity of the antibody complementarity-determining region (CDR). Antibody light chains are divided into two subtypes: λ and κ. The λ light chain accounts for only 5% in mice (λ:κ ≈ 1:19), therefore, the variable region (VL) gene of the κ light chain was amplified for subsequent analysis. Two rounds of PCR are required to amplify the variable region genes of the antibody heavy and light chains, as follows:

[0100] Using the reverse transcription product as a template, multiple pairs of first-round PCR primers designed targeting the variable regions of the H and L chains of the murine antibody were used to amplify the antibody variable region gene. PCR reactions were performed using a 2×M5 Hiper plus Taq HiFi PCR mix (hereinafter referred to as MIX) according to the reaction system in Table 4. The first-round PCR reaction system is shown in Table 4.

[0101] Table 4 First-round PCR reaction system

[0102]

[0103] PCR conditions: 95°C, 3 min; 94°C, 25 s; 7°C, 25 s; and 72°C, 25 s for a total of 35 cycles; 72°C, 5 min.

[0104] The copy number of the antibody gene in a single B cell is low, and the target band cannot be seen in the first round of PCR products. A second PCR step is required to amplify the variable region sequence of the antibody. The reaction system for the second round of PCR is shown in Table 5.

[0105] Table 5 Second round PCR reaction system

[0106]

[0107] PCR conditions: 95°C, 3 min; 35 cycles of 94°C, 25 s, 57°C, 20 s and 72°C, 25 s, followed by 2°C, 5 min.

[0108] After the second round of PCR, 10 μL of the amplification product was taken out using a pipette and verified by 1% agarose gel electrophoresis. The target band was recovered by following the instructions of the gel recovery kit.

[0109] PCR product sequencing:

[0110] ① Ligation: The fragments of antibody variable region recovered by gel were ligated to the pMD-19T vector. The ligated system was placed in a water bath at 16°C and heated for 15 min. The ligation system is shown in Table 6.

[0111] Table 6 Connection System

[0112]

[0113] ② Transformation: Using a pipette, take 10 μL of the ligation product and mix it with 100 μL of Top 10 competent cells into a 1.5 mL EP tube. Incubate on ice for 30 min, then transfer to a 42℃ water bath for heat shock for 50 s. Immediately place on ice for 2 min to terminate the reaction. Under aseptic conditions, add 1 mL of antibiotic-free LB liquid medium and culture in a shaker at 37℃ and 200 rpm for 1 h. Then, centrifuge the bacteria at 4000 xg for 5 min, discard 500 μL of supernatant, and retain about 100 μL of bacterial culture. Spread it evenly on the surface of LB solid medium containing ampicillin (100 μg / mL) and incubate upside down in a 37℃ incubator for 16-18 h.

[0114] ③ Identification: Single colonies from overnight culture were resuspended in 10 μL of ultrapure water, vortexed to mix, and 2 μL was used as PCR template. The remaining 8 μL was transferred to LB liquid medium containing 1 mL of ampicillin (100 μg / mL) and amplified at 37℃ and 220 rpm for 6 h. Amplification was performed using universal M13 primers (forward: Primer M13F; reverse: Primer M13R). The bacterial PCR system is shown in Table 7.

[0115] Table 7 Bacterial PCR Reaction System

[0116]

[0117] PCR conditions: 35 cycles of 94℃ for 3 min; 94℃ for 30 s; 55℃ for 30 s; 72℃ for 1 min and 72℃ for 5 min; 25℃ for 1 min.

[0118] 4. Sequencing: PCR amplification products were analyzed for quality control using 1.5% agarose gel electrophoresis (120 V, 30 min). Positive clones with band sizes matching expectations were screened and sequenced using the Sanger dideoxy chain termination method by Tianyi Huiyuan Biotechnology Co., Ltd. The raw sequencing peaks were assembled and their quality assessed using DNASTAR software. Germline alignment was further performed using the NCBI IgBLAST database (https: / / www.ncbi.nlm.nih.gov / igblast / ) to determine the antibody variable region (V / D / J) gene composition and complementarity-determining region (CDR) location.

[0119] Experimental Results: Through systematic primer screening and reaction condition optimization, this invention established a stable antibody gene amplification system. Initially, through parallel comparison of four candidate primers, combined with annealing gradient experiments (temperature range 55-65℃), template concentration gradient tests, and evaluation of different commercial enzyme systems, the M5 HiPer Plus Taq HiFi pcrMiX was ultimately selected, as it significantly improves amplification efficiency and reduces non-specific products. First, the lysates of 288 sorted single cells were reverse transcribed to synthesize cDNA. Then, the optimized two-stage nested PCR strategy was used to amplify the variable regions of the immunoglobulin heavy chain (IgH) and light chain (IgL) genes, respectively. Agarose gel electrophoresis analysis showed (e.g.) Figure 3 As shown in the figure, the target fragments of the heavy chain are concentrated around 500 bp, and the target fragments of the light chain are concentrated around 300 bp, which is consistent with the theoretical expectation. Gene amplification efficiency statistics show that 116 samples successfully amplified the IgH variable region (positive rate 40.2%), and 158 clones (54.8%) effectively amplified the IgL variable region, of which 23.9% (69 / 288) achieved paired expression of the light and heavy chains. These data indicate that the established single-cell PCR system has high amplification specificity, laying a technical foundation for the subsequent construction of antibody gene libraries.

[0120] Based on the good light and heavy chain sequencing results, the gene sequences of 69 antibody light and heavy chains were finally obtained. The DNAstar software was used for analysis and comparison to screen out antibody light and heavy chain variable region amino acid sequences that were the same, and those with certain differences were selected for subsequent plasmid synthesis.

[0121] Example 2

[0122] Expression and identification of murine antibodies

[0123] Twenty-three pairs of antibodies were selected from the variable region gene sequences of the heavy and light chains of 69 cell antibodies and entrusted Guangzhou Chengyuan Bio-Immunology Technology Co., Ltd. to construct eukaryotic expression plasmids for the heavy and light chains. These plasmids were then co-transfected into HEK293 cells to express and purify Nipah virus monoclonal antibodies. The biological activity of the antibodies was further verified by indirect ELISA and SDS-PAGE experiments, and monoclonal antibodies were screened.

[0124] The Expi293F cells used were provided and preserved by the Institute of Microbiology and Epidemiology, Academy of Military Medical Sciences, and the antibody expression plasmid was synthesized by Guangzhou Chengyuan Bioimmunotechnology Co., Ltd.

[0125] I. Methods

[0126] 1. Construction of antibody expression plasmids

[0127] TransT1 competent cells were used in the transformation experiment. The specific procedures were as follows: Under aseptic conditions, 100 μL of TransT1 competent cell suspension was placed in a pre-chilled 1.5 mL EP tube and aliquoted into 50 μL tubes. 2 μL of recombinant plasmid was added to each tube, gently mixed, and incubated on ice for 30 min. After the binding reaction was complete, the cells were quickly transferred to a 42℃ water bath for heat shock (30 s), and then immediately placed on ice for 2 min. The tubes should be kept stable during the process, avoiding vigorous shaking. For the recovery phase, 500 μL of sterile LB liquid medium (antibiotic-free) was injected into each tube to resuspend the cells, and the tubes were incubated at 37℃ and 200 rpm for 60 min. 100 μL of the bacterial suspension was evenly spread on the surface of LB solid medium containing the appropriate antibiotic, and the plates were inverted and incubated at 37℃ for 12-16 h. The following day, morphologically intact single colonies were selected and inoculated into 5 mL of LB liquid medium (containing antibiotics) for primary amplification (37℃, 200 rpm, 6-7 h). Once the bacterial concentration reached the logarithmic growth phase, the bacterial culture was transferred at a 1% inoculum volume to an Erlenmeyer flask containing 100 mL of LB liquid medium (containing ampicillin) and cultured with shaking for another 14-16 h. Finally, plasmid purification was performed using an endotoxin-free plasmid extraction kit to ensure the product met the requirements for downstream experiments. The steps are as follows:

[0128] ① Take the bacterial culture that has been cultured overnight for 14-16 hours and put it into a 500 mL centrifuge bucket. Centrifuge at 8000 rpm for 3 minutes, discard the supernatant, and place the centrifuge bucket upside down on kitchen paper for 5 minutes to completely absorb the supernatant.

[0129] ② Add 10 mL of colorless solution RB (be sure to add RNase A before use), shake thoroughly to suspend the bacteria, and mix the bacterial cells completely, leaving no small bacterial clumps;

[0130] ③ Add 10 mL of Buffer RB (containing premixed RNase A) to the bacterial pellet, vortex until the bacterial pellet is completely resuspended, and avoid leaving any small clumps;

[0131] ④ Add NB1 to the clear blue solution, gently stir and turn 6-10 times. Note that the supernatant will become colorless and there will be a pale yellow flocculent precipitate at the bottom. Then place it on an ice bath for 3 minutes.

[0132] ⑤ Centrifuge the above solution at 8000 rpm for 20 min, pour the supernatant into a push filter and push it into a brand new 50 mL centrifuge tube;

[0133] ⑥ Column activation is required during the centrifugation process in the previous step. Add 5 mL of solution AB to the Maxi-Plasmid Spin Column CollectionTube centrifuge column to activate the column, and centrifuge at 8000 rpm for 1 min.

[0134] ⑦ Add 1.5 mL of orange solution ER to the liquid in step ⑤, and mix by inverting until a clear orange solution is obtained;

[0135] ⑧ Add 9 mL of isopropanol to the above solution, invert and mix well, transfer the liquid into the centrifuge in several equal portions, centrifuge at 8000 rpm for 1 min, and discard the liquid flowing out from the bottom layer.

[0136] ⑨ Add 5 mL of WB solution to the centrifuge column, centrifuge at 8000 rpm for 1 min, and repeat this step twice to clean the column;

[0137] ⑩ Centrifuge the column at 8000 rpm for 3 min to completely remove residual WB. Transfer the column to a new 50 mL Collection Tube and leave it at room temperature with the lid off for 5 min to allow the ethanol to evaporate completely.

[0138] Add 1.5 mL of preheated Elution Buffer to the center of the column membrane twice (incubate at room temperature for 5 min each time), centrifuge at 8000 rpm for 2 min to elute plasmid DNA, and combine the eluents;

[0139] Plasmid concentration was tested using nanodrop2000. After sterilizing the plasmids by filtering them with a disposable filter, they were stored at -20°C.

[0140] 2. Small-scale expression and identification of monoclonal antibodies

[0141] To identify NiV antibody expression, plasmids of each antibody heavy and light chain were first expressed in small quantities into Expi293 cells according to the transfection instructions. The cell supernatant was then used for indirect ELISA identification. The specific steps are as follows:

[0142] ① The cell concentration was adjusted to 1×10⁻⁶ the day before the transfer. 6 The cells / mL were replaced with fresh, antibiotic-free culture medium. The cells were counted the next day, stained with trypan blue, and cell viability was checked using a cell counter. Cell viability should be above 90%. 23 plasmids were required for transfection, requiring 1.2-1.5 × 10⁻⁶ cells / mL. 6 40 mL of cells at a concentration of cells / mL;

[0143] ② Dilute the light and heavy chain plasmids to a total volume of 50 μL with OPTI-MEM and mix gently by pipetting.

[0144] ③ Add 1410 μL of OPTI-MEM medium to a 1.5 mL sterile EP tube, then add 90 μL of suspension-sensitive transfection reagent, gently pipette to mix, and incubate for 5 min.

[0145] ④ After incubation for 5 min, add 50 μL of the diluted transfection reagent to the EP tube of the diluted plasmid, mix by pipetting, and incubate for 20-30 min.

[0146] ⑤ Using a 3 mL 24-well plate, add 1.4 mL or more of the adjusted concentration of Expi293 cells to each well, and add DNA and transfection reagent complex after 25 min;

[0147] ⑥ Incubate at 37℃ in a shaker with 8% CO2 for 7 days. After 7 days, collect the liquid into a 1.5 mL EP tube, centrifuge at 10000 rpm for 10 min, and transfer the supernatant into another new EP tube. Store at -20℃ for long-term storage.

[0148] 3. Indirect ELISA screening for antigen-binding antibodies

[0149] To screen for monoclonal antibodies with NiV-G protein binding activity, NiV-G protein was selected as the screening antigen. The supernatant of transfected cells was identified using an indirect ELISA method. Cells identified as having binding activity were then transfected in large quantities to express antibodies. Specific steps are detailed in Example 1.

[0150] Experimental results: such as Figure 4 As shown in the figure. The results showed that 16 antibodies could bind to NiV-G protein. Antibodies with a binding titer of 1:100 or higher were selected for large-scale purification, and a total of 11 antibodies were selected (6, 7, 8, 9, 12, 14, 22, 42, 50, 61, 63).

[0151] 4. Determination of monoclonal antibody affinity

[0152] Affinity assays were performed on 23 antibodies from cell supernatants collected in small-scale transfections using the Gator label-free biomolecular interaction analysis system. The specific steps are as follows:

[0153] ① Dilute the antigen to a concentration of 100 nmol, take 1700 μL of Q buffer solution and add 17 μL of NiV-G protein. The cell supernatant does not need to be diluted.

[0154] ② Add 250 μL of Q buffer to each well in the first column of the probe plate. Carefully add the dried and stored Protein G probe to the wells with Q buffer using tweezers, being careful not to let the probe touch the well wall.

[0155] ③ Add 200 μL of diluted antigen to each well in the first column of the sample plate; add 200 μL of buffer 1 (Expi 293 medium) to each well in the second column; add 200 μL of cell supernatant to each well in the third column, noting that each well should contain cell supernatant corresponding to a different cell number; add 200 μL of Q buffer to each well in the fourth column; add 1200 μL of buffer to each well in the fifth column; add 200 μL of cell supernatant to each well in the sixth column; add 200 μL of Q buffer to each well in the seventh column; add 1200 μL of buffer to each well in the eighth column; add 200 μL of cell supernatant to each well in the ninth column; add 200 μL of Q buffer to each well in the tenth column, for a total of 23 cell supernatants. Add one negative well, and add 200 μL of medium to the negative well.

[0156] ④ Set up the computer, click K assay, Plate Set Up, which will display the position of the sample plate and probe plate buffer or sample in the 96-well plate. Click the number above the column, and click the button corresponding to the sample (Buffer, Sample, Probe, Load) to define the sample column attributes. Set it according to the classic five-step kinetic method, set Buffer1-cell supernatant-Buffer1-antigen-Qbuffer, and then enter the cell number corresponding to the sample.

[0157] ⑤ Under Assay Steps, define the Position, time, and speed for each step. Note that the speed should be reduced by 5% during dissociation, and the dissociation time should be manually extended as appropriate.

[0158] Experimental results: such as Figure 5As shown in Table 8, ten antibodies with high affinity were selected, namely 6, 7, 8, 9, 12, 14, 22, 42, 61, and 63, which were basically consistent with the ELISA results. Antibodies with high affinity and high ELISA titers were selected for large-scale purification. The affinity constants of the selected antibodies are shown in Table 8.

[0159] Table 8 Combined with dynamic parameters

[0160]

[0161] 5. The Expi293 expression system expresses antibodies in large quantities.

[0162] ① Eight days before transfection, expand the culture of Expi293 cells. This requires transfection with 11 plasmids, totaling 6 × 10⁶ cells. 8 One cell;

[0163] ② Prepare a 50mL disposable sterile centrifuge tube, add 180μg of light chain plasmid and 160μg of heavy chain plasmid, and dilute it to a total volume of 8mL using OPTI-MEM medium;

[0164] ③ Transfecting 11 plasmids requires 12 transfection reaction systems. Add 2.4 mL of suspension-sensitive transfection reagent and 37.6 mL of OPTI-MEM medium to a 50 mL disposable sterile centrifuge tube. This requires two 40 mL systems of diluted transfection reagent and one system consisting of 1.2 mL of suspension-sensitive transfection reagent and 18.8 mL of OPTI-MEM medium. After dilution, gently invert the centrifuge tube to mix and incubate for 5 min.

[0165] ④ After incubation for 5 min, use a pipette to transfer 8 mL of diluted transfection reagent into the diluted plasmid, gently invert to mix, and then incubate for 25 min.

[0166] ⑤ Use a disposable sterile 50 mL centrifuge tube to measure 50 mL of a concentration of 1×10⁻⁶. 6 Add Expi293F cells / mL to a 1000 mL cell shake flask, then add 174 mL of Wayne293 medium;

[0167] ⑥ Add the incubated DNA and transfection reagent complex to the 1000 mL cell shake flask from step ⑤, slowly adding the DNA dropwise while shaking the flask, and label the different antibodies accordingly.

[0168] ⑦ Place the cell shake flask in a shaker at 37°C and 8% CO2 for 7 days.

[0169] 6. Antibody purification

[0170] After seven days of culture, the transfection solution was centrifuged at 8000 rpm for 10 min. The supernatant was collected and filtered through a 0.45 μm filter membrane. Purification was then performed using a Protein A affinity column, as detailed below:

[0171] ① Install the Protein A column according to the instructions. Turn on the computer first, then turn on the instrument. Rinse the AB pump of the instrument and flush it with 25 mL of equilibration buffer to equilibrate the column. Set the flow rate to 3 mL / min.

[0172] ② Load the sample at a flow rate of 1 mL / min and set the UV to 0. Avoid generating bubbles during the loading process.

[0173] ③ After loading the sample, continue to rinse with 60 mL of equilibration buffer to remove any unbound contaminating proteins;

[0174] ④ Prepare a 10 mL centrifuge tube, add 900 μL of Tris-HCl in advance, start rinsing to remove the buffer, and start collecting the sample when the UV value is higher than the equilibration buffer value by 50. The sample is collected into a centrifuge tube containing 900 μL of Tris-HCl in advance for 10 min. The resulting liquid is the purified monoclonal antibody.

[0175] ⑤ Add the monoclonal antibody to a 3.5 kDa dialysis bag that has been boiled and sterilized, dialyze in 1×PBS solution, and incubate overnight at 4°C;

[0176] ⑥ The concentration of the dialysis antibody was determined using Nano Drop 2000. Antibodies with a concentration of less than 1 mg / mL were ultrafiltered. All purified antibodies were aliquoted and stored at -20°C.

[0177] 7. Indirect ELISA assay for antibody binding activity

[0178] To identify the expression effect of the antibodies, an indirect ELISA method was used for detection. Antibodies with strong binding ability to antigens were screened. The harvested antibodies were first diluted to 1 mg / mL, and then diluted 100 times to detect their binding activity.

[0179] Experimental results: such as Figure 6 As shown, the five antibodies, NiV-6, NiV-8, NiV-12, NiV-22 and NiV-63, exhibited significant antigen-binding activity. Among them, NiV-6 had a half-maximum binding titer (EC50) as high as 1:107, indicating that it has a super antigen affinity.

[0180] 8. SDS-PAGE identification of antibody purification results

[0181] The purified antibody was diluted 1:1 with PBS. 40 μL of the diluted antibody was added to a 1.5 mL EP tube, along with 10 μL of 5×SDS-PAGE Loading Buffer. The tube was boiled in a water bath for 10 min. Using a 5%-20% pre-prepared gel, 10 μL of sample was added to each well. The gel was set to 220 V and timed for 50 min. Observe for bubble formation; the presence of bubbles indicates normal operation. After electrophoresis, the gel was removed and placed on a disposable square culture dish containing Coomassie Brilliant Blue staining solution. The dish was placed on a shaker and stained overnight. After staining, destaining solution was added for destaining. After one day of destaining on a shaker, the antibody purification results were observed using a gel imaging system.

[0182] Experimental results: such as Figure 7 As shown, five high-purity monoclonal antibodies were successfully obtained by reduced SDS-PAGE electrophoresis. The molecular weights of the heavy and light chains were approximately 55 kDa and 25 kDa, respectively, which are consistent with the typical structural characteristics of IgG antibodies, proving that the purification process can effectively maintain the structural integrity and functional activity of the antibodies.

[0183] Example 3

[0184] Establishment of an AlphaLISA detection method for Nipah virus

[0185] EZ-Link SµLfo-NHS-LC-Biotinylation Kit (Thermor Science, Rockford, USA) Tris-HCl (Sinopharm Chemical Reagent Co., Ltd.) Proclin-300 (Sinopharm Chemical Reagent Co., Ltd.) HEPES (Sinopharm Chemical Reagent Co., Ltd.) Tween-20 (Bailingwei Chemical Reagent Co., Ltd.) Acceptor beads (PE Company) Donorbeads (PE Company) Europium ion acceptor microspheres, streptavidin-modified donor microspheres, 1 / 2 AreaPlate™-96-well plates (PerkinElmer, USA).

[0186] Antibody-coupled receptor microsphere buffer: 100 mM HEPES pH=7.4; 100 mM Tris-HCl pH=8.0 (store at 4℃); 800 mM NaOH; 400 mM NaBH3CN (prepare fresh); 1×PBS pH=7.4. AlphaLISA buffer (Buffer Y): 25 mM HEPES, pH 7.4, 0.1% casein, 1 mg / mL dextran-500, 0.5% Triton X-100 and 0.05% Proclin-300.

[0187] I. Biotin-labeled antibodies

[0188] Biotinylation was performed on five purified NiV monoclonal antibodies (NiV-6, NiV-8, NiV-12, NiV-22, NiV-63) according to the EZ-Link® Sulfo-NHS-LC-Biotinylation Kit instructions. The specific method is as follows:

[0189] ① Dilute the antibody to 1 mg / mL with PBS;

[0190] ② Weigh 3 mg of biotin into a 1.5 ml EP tube, add 525 μL of dimethylformamide, vortex to mix, dispense 10 μL into 5 tubes in total, and store at -20℃.

[0191] ③ Add 100 μL of diluted antibody to a 1.5 mL EP tube, then add 3 μL of biotin solution, and mix by rotating at room temperature for 1 h.

[0192] ④ Add 300 μL PBS to the desalting column and wash it. Centrifuge at 1500 xg for 1 min. Repeat the washing process three times. Set aside for later use.

[0193] ⑤ After the antibody and biotin mixture has been incubated at room temperature, use a pipette to add it to the desalting column, centrifuge at 1500 xg for 2 min to remove excess biotin, and store the obtained biotin-labeled antibody at 4℃.

[0194] 2. Receptor microsphere conjugate antibody

[0195] The conjugation of antibodies to receptor microspheres was performed according to the experimental instructions provided by PE Company. NiV-6, NiV-8, NiV-12, NiV-22, and NiV-63 antibodies were conjugated to receptor microspheres respectively. The specific experimental steps are as follows:

[0196] ① Activation: Take 25 μL of receptor microspheres (10 mg / mL) and add them to 200 μL of 0.05 M pH=6.0 MES buffer in a 1.5 mL EP tube. Then add 1 μL of 10 mg / mL EDC and NHS solutions to activate the microspheres. Vortex to mix and let stand at room temperature for 30 min. Centrifuge at 16000 xg for 15 min at 4℃. Use a pipette to slowly aspirate the supernatant.

[0197] ② Labeling: Take 200 μL of 0.05 M pH 7.0 MES buffer, resuspend and mix well, add 25 μg of NiV monoclonal antibody, and incubate at 37℃ using a vortex mixer for 2 h.

[0198] ③ Blocking: Add 25 μL of 2% BSA blocking solution and mix in an incubator at 37°C using a vortex mixer for 2 h;

[0199] ④ Washing: After inverting the solution for 2 hours, centrifuge at 16000 xg for 15 min at 4℃, collect the supernatant, resuspend it in 100 μL of 0.05M pH=8.0 Tris buffer, and sonicate (10s, 1 pulse, 1s interval, power=20%). Repeat the above operation twice.

[0200] ⑤ Preservation: Resuspend the washed receptor microsphere-conjugated antibody in 100 μL of microsphere preservation solution and mix well. Store at 4°C.

[0201] II. Screening for the optimal antibody pair for AlphaLISA

[0202] 1. AlphaLISA Preliminary Experiment

[0203] The entire AlphaLISA reaction can be completed within 30 minutes. The experiment used 1 / 2 AreaPlate™-96 well plates, and the final assay was performed using a SpectraMax™ I3 microplate reader. The specific operating steps are as follows:

[0204] ① Use Buffer Y to dilute the receptor microsphere-conjugated antibody and biotinylated antibody at a certain ratio, mix them, and add 20 μL to the well plate;

[0205] ② Dilute the NiV-G protein with Buffer Y, add 10 μL to each well and incubate at 37°C for 15 min;

[0206] ③ During incubation, the donor microspheres of streptavidin were diluted in proportion under light-protected conditions with Buffer Y under green light, wrapped in aluminum foil to protect from light, and mixed by vortexing.

[0207] ④ After incubation for 15 min, add 10 μL of diluted donor microspheres to each well, cover with a film to protect from light, and incubate at 37°C for 10 min;

[0208] ⑤ Perform the assay using the AlphaScreen plate in the microplate reader and save the data.

[0209] 2. AlphaLISA for screening optimal antibody pairs

[0210] Five antibodies with the highest titers selected by indirect ELISA were paired, and five to six antibodies were tested in a single assay, for a total of four groups. The receptor-labeled antibody at a 1:200 dilution and another biotinylated monoclonal antibody at a 1:4000 dilution were diluted with Buffer Y and vortexed. 20 µL of each diluted antibody was added to each well. The antigen was diluted to 100 ng / mL and 10 µL was added to each well. Three sub-wells were prepared for each sample according to the gradient concentration. After thorough shaking, the samples were transferred to an incubator at 37°C for 15 min. Subsequently, in a dark environment, 20 µL of donor fluorescent microsphere suspension diluted 1:250 (v / v) was precisely added to each well, and the samples were incubated for another 10 min in the dark at the same temperature. Finally, the absorbance signal of each sample was recorded at an excitation wavelength of 650 nm using the chemiluminescence detection mode of the SpectraMax™ I3 multifunctional detection system. The cutoff value was defined as the average absorbance value of the negative control plus three standard deviations. The antibody pair with the highest signal-to-noise ratio (the ratio of the absorbance of 100 ng / mL Nipah virus antigen to the critical value) was selected. The best antibody pair among the four groups was selected and compared in one experiment. The antibody pair with the highest detection signal-to-noise ratio was the best antibody pair for AlphaLISA detection.

[0211] Experimental Results: Six antibodies with high affinity and strong binding ability—NiV-6, NiV-8, NiV-12, NiV-22, and NiV-63—were selected and paired with different antibodies, respectively, as biotinylated antibodies and receptor-microsphere-labeled antibodies. For example, in the figure, numbers 8-12 represent receptor-microsphere-labeled antibody 8 and biotinylated antibody 12. The results are as follows: Figure 8 As shown in the diagram, the antibody pair with the highest signal-to-noise ratio (SNR) from the four experimental groups was selected and the AlphaLISA experiment was repeated. The pair with the highest SNR among the four antibody pairs was then selected to demonstrate that this antibody pair achieves the optimal AlphaLISA detection sensitivity. The results showed that the system exhibited a high SNR (S / N) when using antibody-conjugate microspheres (No. 22) and biotinylated antibody (No. 12), and antibody-conjugate microspheres (No. 6) and biotinylated antibody (No. 8). Therefore, antibody-conjugate microspheres (No. 22) and biotinylated antibody (No. 12) were subsequently selected as the optimal antibody pair for the AlphaLISA detection system.

[0212] III. Optimization of the AlphaLISA Detection System

[0213] Since the dilution ratios of receptor microspheres, biotinylated antibodies, and donor microspheres used in the antibody pair screening were for preliminary experiments and were not the optimal detection system, the AlphaLISA detection system was optimized to ensure more accurate subsequent detection values.

[0214] To optimize the detection system, the dilution ratios of biotinylated antibody and donor microspheres were varied at 1:4000 and 1:250, with the recipient microsphere dilution ratios changed to 1:100, 1:200, 1:400, 1:800, 1:1600, and 1:3200. At a Nipah virus antigen concentration of 10 ng / mL, the signal-to-noise ratio (SNR) of each dilution ratio was compared. With the optimal recipient and donor microsphere dilution ratio at 1:250, the biotinylated antibody dilution ratio was varied to 1:1000, 1:2000, 1:4000, 1:8000, and 1:16000. At a Nipah virus antigen concentration of 10 ng / mL, the SNR of each dilution ratio was compared to select the optimal biotinylated antibody dilution ratio. Having confirmed the optimal dilution ratios of the receptor microsphere-labeled antibody and the biotinylated antibody, the dilution ratios of the donor microspheres were changed to 1:31.25, 1:62.5, 1:125, and 1:250. At a Nipah virus antigen concentration of 10 ng / mL, the signal-to-noise ratios of the different donor microsphere dilution ratios were compared, and the highest value was confirmed as the optimal dilution ratio.

[0215] Experimental results: such as Figure 9 As shown in the figure, the signal-to-noise ratio (SNR) of the Nipah virus monoclonal antibody receptor microspheres was highest at a dilution of 1:400, showing a significant difference from the other two groups. The biotinylated Nipah virus monoclonal antibody had the highest SNR at a dilution of 1:8000, also showing a significant difference from the other two groups. The donor microspheres had the highest SNR at a dilution of 1:62.5, showing a significant difference from one of the other groups. The optimal conditions for the ALphaLISA assay were determined to be: receptor microspheres 1:400, biotinylated Nipah virus monoclonal antibody 1:8000, and donor microspheres 1:62.5.

[0216] IV. Sensitivity of AlphaLISA method for detecting Nipah virus antigen

[0217] First, the Nipah virus antigen was serially diluted 2-fold (100 ng / mL - 0.012 ng / mL) using 100 ng / mL. AlphaLISA was then performed using the selected optimal antibody pair and reaction system. Each concentration was repeated three times, and a standard curve model for Nipah virus antigen detection was established. A standard curve was plotted with NiV-G protein concentration (Log10) on the x-axis and signal-to-noise ratio on the y-axis (e.g., ...). Figure 10 As shown), the four-parameter fitting equation is Y=0.1803+(309.7-0.1803) / (1+(X / 1.368)^(-0.9908), and the correlation coefficient R0 is 0.1803+(309.7-0.1803) / (1+(X / 1.368)^(-0.9908)). 2 =0.9989. The cutoff value is defined as the average absorbance of the negative control group plus three times the standard deviation. The signal-to-noise ratio is the ratio of the absorbance of each concentration to the cutoff value. The detection limit is set to 1, and the lowest detection limit is the smallest concentration with a signal-to-noise ratio greater than 1.

[0218] Experimental results: such as Figure 11 As shown, the sensitivity of AlphaLISA in the buffer solution for detecting NiV antigen is 0.024 ng / mL.

[0219] V. Sensitivity of AlphaLISA for Detecting Nipah Spurious Virus

[0220] The pseudovirus was purchased from Beijing Yiqiao Shenzhou Company. The pseudovirus titer was 9.45E+06 TU / mL. The Nipah virus and the Nipah pseudovirus were simultaneously diluted 2-fold from 9450 TU / mL to 18.5 TU / mL and detected by AlphaLISA. A standard curve model for the detection of Nipah virus pseudovirus was established. A standard curve was plotted with pseudovirus concentration (Log10) on the x-axis and signal-to-noise ratio (S / N) on the y-axis (e.g., [image of curve]). Figure 12 As shown), the four-parameter fitting equation is Y=1.614+(323.8-1.614) / (1+(X / 6.781)^(-1.176), and the correlation coefficient R0 is 1.614+(323.8-1.614) / (1+(X / 6.781)^(-1.176)). 2 =0.9934. Using the optimal antibody pair and optimal reaction system, three replicates were set for each concentration, and the average absorbance values ​​were calculated. The cutoff value was defined as the average absorbance value of the negative control group plus three times the standard deviation. The signal-to-noise ratio (S / N) was the ratio of the absorbance value of each concentration to the cutoff value. The detection limit was set to 1, and a positive result was defined as an S / N greater than 1.

[0221] Experimental results: such as Figure 13 As shown, the limit of detection (LOD) for AlphaLISA in detecting Nipah pseudovirus is 36.9 TU / mL.

[0222] VI. AlphaLISA Repeatability Experiment

[0223] To verify the intra-batch reproducibility of the method, Nipah virus antigen was diluted 10-fold starting at 10 ng / mL, resulting in three concentration gradients. Each concentration was run in 12 replicates to calculate the coefficient of variation. To verify the inter-batch reproducibility of the method, three replicates were run at each concentration (10 ng / mL, 1 ng / mL, 0.1 ng / mL) at different time points, and the coefficient of variation was calculated.

[0224] Experimental results are shown in Table 9. The intra-assay coefficient of variation (CV = 6.42-8.03) and inter-assay coefficient of variation (CV = 6.14-9.51) for the three concentration gradients indicate that AlphaLISA has good reproducibility.

[0225] Table 9. Differences between and within batches of NiV antigen

[0226]

[0227] VII. AlphaLISA Simulated Sample Detection

[0228] Because humans and pigs are susceptible to Nipah virus infection, porcine serum and human serum were selected as background samples for testing. Different concentrations of NiV-G protein or NiV pseudovirus were added to solutions diluted with serum in a certain ratio using PBS as simulated samples. Antigen concentrations were serially diluted twofold (100 ng / mL - 0.012 ng / mL), and Nipah pseudovirus concentrations were serially diluted twofold (9450 TU / mL - 18.5 TU / mL). The experimental method was optimized as follows: three parallel detection wells were set up for each concentration gradient sample. A blank control system was established using a solution diluted with Buffer Y buffer and porcine serum (1:1). Buffer Y buffer was diluted with human serum at 1:19 and 1:49 to determine the optimal dilution ratio. Through statistical analysis, the mean absorbance of the negative control group plus three standard deviations was defined as the cutoff value. The signal-to-noise ratio (S / N) of the test sample was obtained by calculating the ratio of each concentration detection value to the threshold. A positive detection was defined when the S / N ratio was greater than 1, and the minimum detection limit standard of the system was established accordingly.

[0229] Experimental results: such as Figures 14-17 As shown, the detection sensitivity of NiV antigen in simulated samples can reach 0.048 ng / mL. The four-parameter fitting equation is Y = -0.884 + (211.6 + 0.884) / (1 + (X / 1.799)^(-0.7292)), and the correlation coefficient R is... 2 =0.9910, the detection sensitivity of NiV pseudovirus in simulated samples can be diluted up to 51200 times, the lowest detectable viral titer is 184.5 TU / mL, the four-parameter fitting equation is Y=1.273+(79.84-1.273) / (1+(X / 4.608)^(-1.275), and the correlation coefficient R 2 =0.9916. The swine serum simulated sample had no significant effect on the detection sensitivity of NiV antigen. However, when the spurious virus concentration was diluted 1000 times, the swine serum simulated sample had a significant impact on the detection of its luminescent signal, and the detection sensitivity decreased. Virus particles above 184.5 TU / ml were required for detection.

[0230] like Figures 18-22As shown. Human serum simulants significantly affect the detection signal values ​​of antigens and pseudoviruses. Even when human serum is diluted 20-fold, it still noticeably affects the detection signal values. Therefore, human serum simulants were used in experiments with a serum:PBS dilution ratio of 1:49. The detection sensitivity of NiV antigen in human serum simulants reached 0.028 ng / mL. The four-parameter fitting equation was Y = -0.2.310 + (432.3 + 2.310) / (1 + (X / 1.847)^(-0.7317)), and the correlation coefficient R... 2 =0.9928, the detection sensitivity of NiV pseudovirus in simulated samples can be diluted up to 51200 times, and the lowest detectable viral titer is 184.5 TU / mL. The four-parameter fitting equation is Y=1.022+(249-1.022) / (1+(X / 5.503)^(-1.035), and the correlation coefficient R is 0.9928. 2 =0.9948.

[0231] VIII. AlphaLISA Specificity Assay

[0232] The specificity of the AlphaLISA detection method was evaluated using several RNA viruses similar to Nipah virus, viruses with similar pathogenicity to Nipah virus, and viruses from pigs equally susceptible to Nipah virus. Five viruses were selected: Zika virus, influenza A, influenza B, Japanese encephalitis virus, and Coxsackie virus. NiV antigen and the other viruses were diluted with Buffer Y to three concentration gradients (100 ng / mL, 10 ng / mL, and 1 ng / mL) for AlphaLISA experiments. The negative control group consisted of Buffer Y. Three replicates were set for each virus and the negative control. The cutoff value was defined as the average absorbance value of the negative control group plus three times the standard deviation. The signal-to-noise ratio (SNR) was the ratio of the absorbance value at each concentration to the cutoff value. The limit of detection (LOD) was set to 1; a positive result was considered to occur when the S / N ratio was greater than 1.

[0233] Experimental results: such as Figure 23 As shown, no positive results were found for Zika virus, Japanese encephalitis virus, influenza A virus, or influenza B virus in the buffer solution, indicating that the Nipah virus AlphaLISA monitoring system has high specificity.

[0234] Example 4

[0235] Establishment of a colloidal gold immunochromatographic method for the detection of Nipah virus

[0236] I. Preparation of Immunogold Colloidal Test Strips

[0237] 1. Preparation of colloidal gold

[0238] 40 nm colloidal gold was prepared by the trisodium citrate reduction method (refer to Li Haiyan, Xin Xiaoguang, Tian Guobin, et al. Optimal working conditions for detecting avian influenza antibodies by indirect enzyme-linked immunosorbent assay [J]. Chinese Journal of Animal Infectious Diseases, 1998, 20(4).).

[0239] 2. Labeling of colloidal gold and antibodies

[0240] Following the method described in the references (Shyu RH, Shyu HF, LIU HW. Colloidal gold-based immunochromatographic assay for detection of ricin[J]. Toxicon, 2002, 40:255-258. Wang BL, Scopsi L, Martvig M, et al. Simplified purification and testing of colloidal gold probes[J]. Histochemistry, 1985, 83:109-115.), 100 mL of colloidal gold solution was measured, and the pH of the colloidal gold solution was adjusted to 8.5 with 0.1 M K2CO3. The Nipah virus monoclonal antibody NiV-12, with high titer and good affinity, was added to the colloidal gold solution at the optimal protein dosage, and the mixture was stirred for 30 min. Then, 5% BSA was added to a final concentration of 1%, and the mixture was stirred for 30 min. Centrifuge at 1200 r / min for 20 min, discard the precipitate, centrifuge at 12000 r / min for 30 min, discard the supernatant, wash the precipitate twice with 1% BSA (prepared with Tris-HCl buffer), dissolve the precipitate with 1% BSA and concentrate it to 1 / 10 of the original volume, then add 0.05% Proclin 300 for preservation and store at 4℃.

[0241] 3. Preparation of gold-labeled pads

[0242] The glass fiber membrane was first rinsed in Tris-HCl buffer to remove surface impurities, and then vacuum dried at 37°C for 30 min. The gold-labeled antibody prepared above was sprayed onto the glass fiber membrane, lyophilized using a freeze dryer, and then dried, sealed and stored for later use.

[0243] 4. Preparation of nitrocellulose membranes

[0244] The purified Nipah virus monoclonal antibody NiV-22 and goat anti-mouse IgG were diluted to 2 mg / mL using 0.01 M pH 7.6 PBS and sprayed onto nitrocellulose membranes (NC) to form parallel test and control lines spaced 0.5 cm apart. The membranes were then dried in a humidified chamber at 37°C for 1 hour, and subsequently sealed for storage.

[0245] 5. Assembly of colloidal gold immunochromatographic test strips

[0246] First, fix the NC membrane (14 mm) onto the PVC base plate, with the control line and the test line 5 mm apart. On one side of the NC membrane test line are the gold label pad (8 mm) and the sample pad (20 mm), and on the other side of the control line is the absorbent paper (32 mm). When assembling, overlap each other by 2 mm. Use a paper cutter to cut the assembled test paper into 4 mm wide test strips, which is the finished product of the Nipah virus colloidal gold immunochromatographic test strip.

[0247] II. Performance Testing of Colloidal Gold Test Strips

[0248] 1. Specificity test

[0249] The specificity of a self-made test strip was tested using Nipah virus G protein against inactivated Zika virus, influenza A, influenza B, Japanese encephalitis virus, and Coxsackie virus.

[0250] Experimental results: such as Figure 24 As shown, the detection band was observed only in the Nipah virus G protein sample, and no cross-reactivity was observed in the tests of the five heterologous inactivated viral antigens, demonstrating excellent specificity.

[0251] 2. Sensitivity test

[0252] The Nipah virus G protein was serially diluted (10 ng / mL, 1 ng / mL, 0.1 ng / mL), and the Nipah virus pseudovirus was serially diluted (9450 TU / mL, 945 TU / mL, 94.50 TU / mL), and the results were detected using the prepared test strips.

[0253] Experimental results: such as Figure 25 and 26 As shown, the NiV-22 / NiV-12 antibody combination was found to achieve a sensitivity of 1 ng / mL for G protein and a sensitivity of 945 TU / mL for Nipah virus pseudovirus detection in colloidal gold immunochromatography.

[0254] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A monoclonal antibody combination for Nipah virus detection, characterized in that, Including Nipah virus G protein antibodies NiV-12 and NiV-22; The amino acid sequence of the heavy chain variable region of NiV-12 is shown in SEQ ID No: 1, and the amino acid sequence of the light chain variable region of NiV-12 is shown in SEQ ID No: 2; The amino acid sequence of the heavy chain variable region of NiV-22 is shown in SEQ ID No: 3, and the amino acid sequence of the light chain variable region of NiV-22 is shown in SEQ ID No:

4.

2. The monoclonal antibody combination for Nipah virus detection according to claim 1, characterized in that, The heavy chain variable region of NiV-12 has three complementarity-determining regions CDR1, CDR2 and CDR3. The amino acid sequence of the heavy chain CDR1 is shown in SEQ ID No: 5, the amino acid sequence of the heavy chain CDR2 is shown in SEQ ID No: 6, and the amino acid sequence of the heavy chain CDR3 is shown in SEQ ID No:

7. The light chain variable region of NiV-12 has three complementarity-determining regions, CDR1, CDR2 and CDR3. The amino acid sequence of the light chain CDR1 is shown in SEQ ID No: 8, the amino acid sequence of the light chain CDR2 is shown in SEQ ID No: 9, and the amino acid sequence of the light chain CDR3 is shown in SEQ ID No:

10.

3. The monoclonal antibody combination for Nipah virus detection according to claim 1, characterized in that, The heavy chain variable region of NiV-22 has three complementarity-determining regions CDR1, CDR2 and CDR3. The amino acid sequence of the heavy chain CDR1 is shown in SEQ ID No: 11, the amino acid sequence of the heavy chain CDR2 is shown in SEQ ID No: 12 and the amino acid sequence of the heavy chain CDR3 is shown in SEQ ID No:

13. The light chain variable region of NiV-22 has three complementarity-determining regions, CDR1, CDR2 and CDR3. The amino acid sequence of the light chain CDR1 is shown in SEQ ID No: 14, the amino acid sequence of the light chain CDR2 is shown in SEQ ID No: 15, and the amino acid sequence of the light chain CDR3 is shown in SEQ ID No:

16.

4. A polynucleotide encoding a monoclonal antibody combination for Nipah virus detection as described in any one of claims 1 to 3, characterized in that, The nucleotide sequence encoding the amino acid of the heavy chain variable region of NiV-12 is shown in SEQ ID No: 17, and the nucleotide sequence encoding the amino acid of the light chain variable region of NiV-12 is shown in SEQ ID No:

18. The nucleotide sequence encoding the amino acid of the heavy chain variable region of NiV-22 is shown in SEQ ID No: 19, and the nucleotide sequence encoding the amino acid of the light chain variable region of NiV-22 is shown in SEQ ID No:

20.

5. The polynucleotide according to claim 4, characterized in that, The nucleotide sequence encoding the NiV-12 heavy chain CDR1 is shown in SEQ ID No: 21, the nucleotide sequence encoding the NiV-12 heavy chain CDR2 is shown in SEQ ID No: 22, and the nucleotide sequence encoding the NiV-12 heavy chain CDR3 is shown in SEQ ID No:

23. The nucleotide sequence encoding the NiV-12 light chain CDR1 is shown in SEQ ID No: 24, the nucleotide sequence encoding the NiV-12 light chain CDR2 is shown in SEQ ID No: 25, and the nucleotide sequence encoding the NiV-12 light chain CDR3 is shown in SEQ ID No:

26.

6. The polynucleotide according to claim 4, characterized in that, The nucleotide sequence encoding the NiV-22 heavy chain CDR1 is shown in SEQ ID No: 27, the nucleotide sequence encoding the NiV-22 heavy chain CDR2 is shown in SEQ ID No: 28, and the nucleotide sequence encoding the NiV-22 heavy chain CDR3 is shown in SEQ ID No:

29. The nucleotide sequence encoding the NiV-22 light chain CDR1 is shown in SEQ ID No: 30, the nucleotide sequence encoding the NiV-22 light chain CDR2 is shown in SEQ ID No: 31, and the nucleotide sequence encoding the NiV-22 light chain CDR3 is shown in SEQ ID No:

32.

7. The use of the monoclonal antibody combination for Nipah virus detection as described in any one of claims 1 to 3 in the preparation of products for detecting Nipah virus.

8. The application according to claim 7, characterized in that, The method for detecting Nipah virus is an immunoassay.

9. The application according to claim 8, characterized in that, The immunoassay method is selected from AlphaLISA.

10. The application according to claim 8, characterized in that, The immunoassay method is selected from ELISA.

11. The application according to claim 8, characterized in that, The immunoassay method is selected from colloidal gold / latex chromatography.

12. The application according to claim 8, characterized in that, The immunoassay method is selected from immunofluorescence assay.

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