Monoclonal antibody for resisting rabies virus N protein, detection reagent and application of monoclonal antibody

Abstract

The invention discloses an anti-rabies virus N protein monoclonal antibody, a detection reagent and application of the anti-rabies virus N protein monoclonal antibody. The monoclonal antibody for resisting the rabies virus N protein comprises a heavy chain variable region and a light chain variable region, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 1; the amino acid sequence of the light chain variable region is as shown in SEQ ID No.2. The monoclonal antibody with relatively high affinity and detection sensitivity for resisting rabies virus N protein lays a foundation for research, development and popularization of colloidal gold test strips and fluorescence immunoassay test strips.

Description

Technical Field

[0001] The present invention relates to the technical field of rapid biological detection of animal diseases, and in particular to a monoclonal antibody against rabies virus N protein, a detection reagent and applications thereof. Background Art

[0002] Rabies virus (RV) is a member of the Rhabdoviridae family and the genus Lyssavirus. It has a bullet-shaped appearance, a helically symmetrical nucleocapsid, an envelope, and contains single-stranded RNA. It is the causative agent of rabies. The RV encodes five proteins: glycoprotein (G), nucleoprotein (N), polymerase (L), phosphoprotein (P), and matrix protein (M). The N protein encapsidates the viral genome and interacts with the P and L proteins to form the viral nucleocapsid. The nucleoprotein is highly conserved, with amino acid identity between different strains ranging from 98% to 99.6%.

[0003] The rabies virus is primarily present in the brain tissue of infected animals, with large amounts of virus also often found in the salivary glands and saliva. Bites, scratches, or mucosal infection from a rabid animal can cause rabies. Under certain conditions, it can also be transmitted through respiratory aerosols. The incubation period of rabies varies, depending on factors such as the site of viral entry and the viral load. It is typically two to three months, but can be as short as less than a week or as long as a year or even multiple years.

[0004] After entering the body, the rabies virus first replicates in the bitten muscle tissue. It then invades the peripheral nervous system through the end plates and axons of motor neurons, moving centripetally toward the central nervous system. Once there, it rapidly multiplies and spreads through nerves to the salivary glands. Saliva from the infected animal then contaminates wounds or mucous membranes of other animals, transmitting the virus to the next host. Once symptoms begin, the rabies virus is almost 100% fatal to humans or animals. The only effective way to prevent rabies is timely vaccination and enhanced monitoring of vaccine efficacy. Therefore, it is necessary to develop highly specific and sensitive rabies virus monoclonal antibodies for the specific identification and detection of rabies virus. Summary of the Invention

[0005] To this end, the present invention provides a monoclonal antibody against rabies virus N protein, a detection reagent and applications thereof.

[0006] In order to achieve the above object, the present invention provides the following technical solutions: According to a first aspect of an embodiment of the present invention, there is provided a monoclonal antibody against rabies virus N protein, wherein the monoclonal antibody against rabies virus N protein comprises a heavy chain variable region and a light chain variable region; The amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID No. 1; The amino acid sequence of the light chain variable region of the monoclonal antibody is shown in SEQ ID No. 2; The heavy chain variable region and the light chain variable region are both composed of complementarity determining regions and framework regions, and the complementarity determining regions are both composed of CDR1, CDR2 and CDR3; The amino acid sequence of CDR1 of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID No. 5; The amino acid sequence of CDR2 of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID No. 6; The amino acid sequence of CDR3 of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID No. 7; The amino acid sequence of CDR1 of the light chain variable region of the monoclonal antibody is shown in SEQ ID No. 8; The amino acid sequence of CDR2 of the light chain variable region of the monoclonal antibody is shown in SEQ ID No. 9; The amino acid sequence of CDR3 of the light chain variable region of the monoclonal antibody is shown in SEQ ID No. 10.

[0007] Furthermore, the monoclonal antibody is a mouse monoclonal antibody.

[0008] According to a second aspect of an embodiment of the present invention, a nucleic acid molecule is provided, comprising a nucleotide sequence encoding any one of the above monoclonal antibodies.

[0009] Furthermore, the nucleotide sequence encoding the heavy chain variable region is shown in SEQ ID No. 3; The nucleotide sequence encoding the light chain variable region is shown in SEQ ID No.4.

[0010] According to a third aspect of an embodiment of the present invention, an expression vector is provided, comprising any one of the nucleotide sequences described above.

[0011] According to a fourth aspect of the embodiments of the present invention, a host cell is provided, comprising any one of the nucleotide sequences described above, or the expression vector described above.

[0012] According to a fifth aspect of the embodiments of the present invention, a test strip is provided, comprising any one of the monoclonal antibodies described above.

[0013] According to a sixth aspect of an embodiment of the present invention, there is provided a use of the above-mentioned detection reagent in detecting a biological sample containing rabies virus antibodies.

[0014] The present invention has the following advantages: The present invention uses genetic engineering technology and hybridoma cell technology to screen a monoclonal antibody with high affinity and detection sensitivity to rabies virus N protein. The antibody has the characteristics of strong specificity and high sensitivity, laying the foundation for the research, development and promotion of colloidal gold test strips and fluorescent immunoassay strips. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] To more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for the embodiments or the description of the prior art. Obviously, the drawings described below are merely exemplary, and those skilled in the art can derive other implementation drawings based on the provided drawings without inventive effort.

[0016] Figure 1 This is an SDS-PAGE identification diagram of the purified rabies virus N protein provided in an embodiment of the present invention; Figure 2 This is the SDS-PAGE electrophoresis diagram of the anti-rabies virus N protein monoclonal antibody provided in the embodiment of the present invention. DETAILED DESCRIPTION

[0017] The following describes the implementation of the present invention using specific embodiments. Those skilled in the art will readily understand the other advantages and benefits of the present invention from the disclosure herein. Obviously, the embodiments described are only a portion of the present invention, not all of it. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without inventive effort are intended to fall within the scope of protection of the present invention.

[0018] Example 1 Preparation of rabies virus N protein 1. Expression process of rabies virus N protein According to the published nucleotide sequence of the rabies virus N protein gene, the gene sequence was codon-optimized and then submitted to Wuhan Jinkairui Bioengineering Co., Ltd. for gene synthesis. The correctly identified positive recombinant plasmid was transformed into BL21 (DE3) competent cells and cultured in a constant temperature incubator at 37°C overnight. A single positive clone was picked and inoculated into LB liquid medium containing ampicillin for culture at 37°C with shaking at 220 rpm. According to the bacterial growth curve, 2 ml of the bacterial suspension was taken for bacterial preservation after about 8 hours. The bacterial liquid was inoculated into 300 mL of Amp + LB liquid medium at a ratio of 1:100 and cultured at 37°C for about 90 min. It was activated by shaking at 220 rpm to an OD value of approximately 0.6, and then IPTG was added to a final concentration of 0.8 mM for induction for about 24 hours. The cells were collected by centrifugation at 10,000 rpm for 15 min at 4°C, the supernatant was discarded, and the cells were resuspended and washed in 20 ml of PBS and centrifuged again to discard the supernatant. Resuspend the cells in 20 ml of 1× Binding buffer without urea, 5 ml of PMSF, and bacterial lysis buffer per 100 ml of the original bacterial liquid. Sonicate and lyse the cells under ice-bath conditions. Centrifuge at 10,000 rpm for 15 min at 4°C, separate the supernatant and precipitate, and analyze the protein expression using SDS-PAGE. Figure 1 .

[0019] 2. Protein purification The supernatant after ultrasonic lysis was filtered through a 0.45 μm filter and purified on a column, and the flow-through was collected; ① Add 10 vol 1× Binding Buffer; ②Add 6 vol 1× Wash Buffer; ③ Add 6 vol 1× Elute Buffer and collect 1 tube for every 1 ml of effluent, for a total of 6 tubes, labeled E1 to E6; ④ Add 6 vol 1× Strip Buffer for destaining and collect 1 tube of effluent labeled S1; ⑤ Stop fractionation after the resin purification column is completely decolorized and store at 4°C. Measure the protein concentrations in protein fraction samples E1 to E6 and label them.

[0020] The recombinant N protein was affinity purified by nickel column and then identified by SDS-PAGE. Figure 1 As shown, an obvious target band can be seen at the position of about 55 kDa.

[0021] 3. Protein Verification The purified rabies virus N protein was verified by ELISA. Use 1 μg / ml purified rabies virus N protein antigen, 100 μl / well, coat overnight at 4℃, add 100 μl 0.1% BSA to each well, block at 37℃ for 2 h, wash with PBST washing solution (0.1%), 300 μl / well, wash the plate three times, add 100 μl / well of diluted clinical positive / negative serum, incubate at room temperature for 30 min (the positive control group is rabies virus antibody positive serum, and the negative control group is rabies virus antibody negative serum, and each group is repeated three times), wash the plate three times, pat dry, add 100 μl / well of HRP-labeled rabbit anti-canine secondary antibody (diluted 1:10000 in PBS), react at room temperature for 30 min, wash the plate again three times, pat dry, add 100 μl / well of TMB color development solution (commercial), develop at room temperature for 10 min, finally add 50 μl / well of 0.5 M sulfuric acid to stop the reaction, and measure the OD with a microplate reader. 450 The results are shown in Table 1. The rabies virus N protein reacted with the positive control and had good antigenic activity.

[0022] Table 1 Antigen activity verification results

[0023] Example 2 Preparation of monoclonal antibodies against rabies virus N protein 1. Mouse immunization Four female Balb / c mice aged 6-8 weeks were taken, and each was immunized with 50 μg of rabies virus N protein prepared in Example 1. During the first immunization, the protein was emulsified with equal volumes of Freund's complete adjuvant, and the mice were immunized by multiple injections subcutaneously. A total of three immunizations were performed, one every two weeks. The second and third immunizations were both emulsified with Freund's incomplete adjuvant for antigen emulsification, and the immunization dose and method remained unchanged. 7-10 days after the three immunizations, blood was collected from the tail vein of the mice, and the serum was collected after centrifugation and its titer was measured by conventional indirect ELISA method. Blood was collected from the tail vein of the mice, and after the serum was precipitated, the serum was taken and diluted in multiples with PBS. At the same time, the serum of blank mice was used as a control. The OD value of the experimental group and the control group was 0.04477 / 0.0337. 450 The maximum serum dilution factor at which the nm ratio exceeds 2.1 is the mouse serum titer. Select mice with high titers and perform a booster immunization with 50 μg of antigen intraperitoneally. Cell fusion can be performed three days later.

[0024] 2. Culture of SP2 / 0 Myeloma Cells Remove a tube of SP2 / 0 myeloma cells frozen in liquid nitrogen and immediately transfer it to a 37°C water bath. Gently shake the tube occasionally and remove the cells when they thaw to a semi-crystal state. Working in a sterile environment, transfer the SP2 / 0 cells from the tube to a 50ml sterile centrifuge tube. Slowly add 10ml of preheated 1640 complete medium dropwise to the tube. Centrifuge at 1000 rpm for 5 minutes and discard the supernatant. Gently break up the cell clumps, resuspend the cells in 5ml of medium, and transfer them to a T75 cell culture flask. Add an additional 5ml of medium, shake the flask in a "cross" motion, and place in a CO2 incubator at 37°C. Observe the cells under a microscope and subculture the SP2 / 0 cells when the density reaches approximately 80%.

[0025] 3. Cell fusion (1) Take 100 μg of protein and dilute it to 200 μl with 1× PBS. Intraperitoneally inject the mouse for shock immunization. Cell fusion can be performed three days later. Take the orbital blood of the experimental mouse after shock and collect it in an EP tube. After standing at 37℃ for 2 hours, centrifuge it at 4000 rpm for 10 minutes. Collect the serum for subsequent screening of monoclonal antibodies as a positive control. The mouse is killed by dislocating the neck and soaked in 75% alcohol for disinfection.

[0026] (2) Preparation of spleen cells: In a biosafety cabinet, use sterile scissors and forceps to cut open the mouse skin. Use a new set of sterile scissors and forceps to cut open the mouse abdominal cavity. Use another set of sterile scissors and forceps to carefully remove the spleen and trim away excess fat. Prepare a sterile 15ml centrifuge tube, add 10ml of DMEM medium, and place the spleen into the centrifuge tube. After the spleen is moistened, carefully discard the excess medium. Then aspirate 10 ml of DMEM culture medium and place it in a sterile dish. Grind the spleen with a frosted glass slide to make a single-cell suspension and filter it through a 200-mesh nylon mesh into a sterile centrifuge tube. Add 30 ml of DMEM to a 50 ml sterile centrifuge tube, rinse the nylon mesh with a pipette, centrifuge the centrifuge tube containing the spleen cell suspension at 1500 rpm for 5 min, discard the supernatant, gently break up the cell clumps by hand, add 30 ml of DMEM culture medium to resuspend, and centrifuge again. Then discard the supernatant, gently break up the cell clumps by hand, and add 10 ml of DMEM culture medium to resuspend.

[0027] (3) Cell fusion: Collect well-grown SP2 / 0 cells by centrifugation at 1000 rpm for 5 min and place them in a 50 ml centrifuge tube. Gently break up the SP2 / 0 cell clumps, add 30 ml of DMEM medium to resuspend, centrifuge once more, add 10 ml of DMEM medium to resuspend, then mix the spleen cell suspension with the SP2 / 0 cell suspension, centrifuge at 1000 rpm for 5 min, discard the supernatant, gently break up the cell clumps, place in a 37°C water bath, draw 1 ml of PEG fusion agent and drop it into the centrifuge tube. Add 1 ml within 1 min. At this time, the cell clumps are red homogeneous quicksand, and the tube wall feels like frosted glass when rotated.

[0028] (4) Termination of fusion: 9 ml of pre-warmed DMEM medium was pipetted to terminate fusion in three stages. The first stage was to add 1 ml dropwise within 1 minute, the second stage was to add 1 ml dropwise within 1 minute, and the third stage was to add the remaining 7 ml of medium dropwise within 3 minutes. The cells were then allowed to stand in a 37°C water bath for 5 minutes and then centrifuged at 800 rpm for 5 minutes.

[0029] (5) Plating: Discard the supernatant, gently break up the cell clumps, add HAT medium (for example, if you want to plate 5 96-well plates, 200 μl / well, excluding the 100 μl / well feeder layer cells that have been plated in advance, then you need to add 50 ml of HAT medium). After mixing the cells, evenly plate the fused cell suspension in the 96-well cell plate that has been added with the feeder layer cells, 100 μl / well, and culture in a CO2 cell culture incubator at 37°C.

[0030] 4. Screening of positive hybridoma cells Seven days after cell fusion, when the cell clusters were observed to be relatively large, the cell supernatant was tested by indirect ELISA. 1 μg / mL of inactivated rabies virus antigen and inactivated canine parvovirus antigen were used as the antigens for detection, respectively. The positive control was the serum of the fused mice, and the negative control was the serum of the mice immunized with PBS. The hybridoma cell wells with the strongest color reaction and no reaction with the inactivated canine parvovirus antigen were selected as positive wells. The positive hybridoma cells screened were subjected to limiting dilution. The hybridoma cell lines that could stably secrete monoclonal antibodies obtained after subcloning were expanded and transferred to T75 cell flasks. When the cell count was about 80%, they could be collected for preparation of ascites.

[0031] 5. Ascites Preparation Add 10 ml of sterile 1× PBS to the cell flask, blow off the cell layer, resuspend, and transfer to a 15 ml centrifuge tube. Centrifuge at 1000 rpm for 10 minutes. Discard the supernatant, resuspend the pellet in another 1 ml of sterile 1× PBS, and aspirate using a 1 ml syringe. Inject approximately 500 μl of the cell suspension per mouse. Pay attention to the growth of the mouse. After one week, wait until the mouse abdomen is enlarged before collecting ascites. Collect the ascites in a centrifuge tube, centrifuge at 8000 rpm for 20 minutes, and aspirate the middle layer.

[0032] 6. Antibody Purification The collected ascites was first subjected to crude purification using the caprylic acid-ammonium sulfate method. Ascites fluid (ml) was collected, recorded in name, centrifuged, and the volume recorded. Add 3 ml of 0.06 M sodium acetate buffer (pH 4.0), mix for 5 minutes, add 10 μl of caprylic acid, and stir at 4°C for 15 minutes. Filter once through cotton wool using a syringe. Centrifuge at 12,000 rpm for 15 minutes at 4°C. Collect the supernatant and add saturated ammonium sulfate to an equal volume of the supernatant to a final concentration of 50%. Stir while adding, and incubate at 4°C for 3 hours. Centrifuge at 12,000 rpm for 15 minutes at 4°C. Discard the supernatant and add 1.8 ml of 0.01 M PBS (pH 7.2) to the centrifuge tube until the precipitate is completely dissolved. Use a pipette to determine the total volume. Add 1 / 2 volume of saturated ammonium sulfate to a concentration of 33%. Stir while adding, and incubate at 4°C overnight. Centrifuge at 12,000 rpm for 15 minutes at 4°C. Discard the supernatant and add 0.45ml 0.01M PBS to dissolve the precipitate. Treat the dialysis bag (boil for 5 minutes, rinse with pure water to remove leaks). Add the dissolved solution to the dialysis bag and dialyze it overnight in 0.01M PBS. Then use Protein G pre-packed column to purify the crude monoclonal antibody for the second time. SDS-PAGE is used to identify the purity of the purified monoclonal antibody. The results are shown in Figure 2 , purity is about 96%.

[0033] Example 3 Identification of monoclonal antibodies against rabies virus N protein 1. Concentration determination The concentration of the monoclonal antibody prepared in Example 2 was 2.36 mg / mL.

[0034] 2. Specific identification An indirect ELISA method was used to test the specificity of monoclonal antibodies against rabies virus N protein. Inactivated rabies virus antigen, canine C-reactive protein, inactivated canine parvovirus antigen, inactivated canine distemper virus antigen, and inactivated canine adenovirus type II antigen were coated at 1 μg / mL. The 1 mg / mL monoclonal antibody was diluted 1:5000 to verify the specificity of the monoclonal antibody. The results are shown in Table 2. The prepared rabies virus N protein monoclonal antibody reacted with inactivated rabies virus antigen but did not react with canine C-reactive protein, inactivated canine parvovirus antigen, or inactivated canine distemper virus antigen, indicating good specificity.

[0035] Table 2 Monoclonal antibody specificity identification results

[0036] Example 4 Cloning of Heavy and Light Chain Variable Region Genes of Monoclonal Antibodies Against Rabies Virus N Protein 1. Hybridoma cell culture and total RNA extraction Hybridoma cells were cultured in RPMI 1640 complete medium in a 37°C, 5% CO2 incubator until the cell count reached 1×10 7 Total RNA was extracted from cells using a total RNA extraction kit (purchased from Tiangen).

[0037] 2. Synthesis of the first strand of cDNA The first-strand cDNA was synthesized using a reverse transcription kit (purchased from TAKARA) with the extracted total RNA as the amplification template.

[0038] 3. Gene amplification Design downstream primers for lambda chain, kappa chain, heavy chain and upstream universal primers.

[0039] Primer: F (SEQ ID No. 11): AAGCAGTGGTATCAACGCAGA, R κ (SEQ ID No.12): AAATTGATGTCTTTGGGGTAGAA, R λ (SEQ ID No.13): AATCGTACACACCAGTGTGTGGG, R H (SEQ ID No. 14): AGGGATCCAGAGTTCCAGGT.

[0040] PCR was performed using the first strand of cDNA as a template in a 50 μl reaction system.

[0041] Touchdown PCR reaction system: 3 μl template, 2.5 μl upstream primer and downstream primer (10 μM) each, 25 μl 2× pfu PCR Master Mix, 17 μl ddH2O.

[0042] Touchdown PCR reaction conditions were as follows: 98°C for 30 s; 98°C for 15 s, 64°C-58°C for 30 s, decreasing by 0.5°C each time until 58°C, for 10 cycles; 72°C for 30 s; 98°C for 15 s, 56°C for 30 s, 72°C for 30 s, for 15 cycles; 72°C for 7 min.

[0043] 4. Cloning and screening of PCR amplification products The PCR products were subjected to 1% agarose gel electrophoresis, and the antibody kappa chain, lambda chain, and heavy chain fragments were recovered using a PCR product recovery kit (Tiangen). The fragments were inserted into the pLB vector using the pLB Zero Background Rapid Cloning Kit (Tiangen), and transformed into DH5α competent cells (ampicillin resistance). The recombinant positive clones were screened and sequenced.

[0044] Nucleotide sequence encoding the heavy chain variable region such as SEQ ID Shown in No.3: GACGTGAAACTGGTGGAGAGCGGAGGAGGACTGGTGAAACCCGGAGGCTCTCTGAAGCTGTCTTGCGCCGCCAGCGGCTTTACATTCAGCCGGTACACCATGTCTTGGGTCCGGCAGACACCAGAGAAGCGCCTCGAGTGGGTGTCCCTTATCAACAACGACGGCAGC TTCACCTACTACAGCGACAGCGTGCGGGAAGATTCACCATCAGCCGGGACAACGCCAAGAACACCCTGTACCTGCAGATGAGCAGCCTGAAGAGCGAGGACACCGCCATGTTCTATTGCACCAGGCCAGGCGGAGCCTATTGGGGACAGGGAACACTGGTGACCGTGTCTAGC.

[0045] The amino acid sequence of the heavy chain variable region is shown in SEQ ID No. 1: DVKLVESGGGLVKPGGSLKLSCAASGFTFSRYTMSWVRQTPEKRLEWVSLINNDGSFTYYSDSVRGRFTISRDNAKNTLYLQMSSLKSEDTAMFYCTRPGGAYWGQGTLVTVSS.

[0046] The nucleotide sequence encoding the light chain variable region is shown in SEQ ID No. 4: GACGTCGTGATGACACAGACCCCTCTGACCCTGAGCGTGACAATCGGACAGCCAGCCAGCATCAGCTGCAAGAGCAGCCAGAGCCTGCTGGATAGCGACGGCAAGACCTACCTGAATTGGCTGCTGCAGAGACCAGGACAGAGCCCTAAGCGGCTGATCTACCTGGTG TCCAAGCTGGACAGCGGCGTGCCAGATAGATTCACCGGAAGCGGAAGCGGCACCGACTTCACCCTGAAGATCTCTAGAGTGGAGGCCGAGGATCTGGGCATCTACTACTGTTGGCAGGGCACCCACTTCCCTCAGACATTTGGCGGAGGCACCAAGCTGGAGATCAAG.

[0047] The amino acid sequence of the light chain variable region is shown in SEQ ID No. 2: DVVMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGIYYCWQGTHFPQTFGGGTKLEIK.

[0048] 5. Variable region amino acid sequence and homology analysis Comparison of the heavy and light chain gene sequences in the NCBI database revealed that the monoclonal antibody heavy chain amino acid sequence shared the highest homology with a synthetic immunoglobulin heavy chain amino acid sequence (Sequence ID: XUS51565.1), with 102 / 119 sequences sharing a homology ratio of 102 / 119 and an 86% identity. The monoclonal antibody heavy chain nucleotide sequence shared the highest homology with the mammalian expression vector pEXPR50 (Sequence ID: MH107787.1), with 243 / 295 sequences sharing a homology ratio of 82%. The monoclonal antibody light chain amino acid sequence shared the highest homology with a mouse immunoglobulin kappa chain amino acid sequence (Sequence ID: pirC32513), with 110 / 112 sequences sharing a 98% identity ratio. The monoclonal antibody light chain nucleotide sequence showed the highest homology with the synthetic anti-Kv1.2 scFv sequence (Sequence ID: PP840917.1), with a homology ratio of 282 / 332 and an 85% identity. Homology analysis of the nucleotide and amino acid sequences encoding the heavy and light chains of the monoclonal antibody revealed no sequences identical to those of the present invention.

[0049] 6. CDR region analysis The amino acid sequences of the heavy and light chain variable regions of the monoclonal antibody were analyzed at https: / / www.novopro.cn / tools / cdr.html to obtain their CDR regions.

[0050] Antibody heavy chain CDR region: CDR-H1 (SEQ ID No. 5): GFTFSRYT CDR-H2 (SEQ ID No. 6): INNDGSFT CDR-H3 (SEQ ID No.7): TRPGGAY Antibody light chain CDR region: CDR-L1 (SEQ ID No.8): QSLLDSDGKTY CDR-L2 (SEQ ID No. 9): LVS CDR-L3 (SEQ ID No. 10): WQGTHFPQT.

[0051] Example 5 Preparation of Rabies Virus Fluorescence Quantitative Detection Test Strips 1. Preparation of Rabies Virus N Protein Monoclonal Antibody Markers (1) Dilution of time-resolved fluorescent microspheres: Ultrasonicate 200 nm time-resolved fluorescent microspheres for 5 minutes. Take 100 μL of microspheres and add 900 μL of MES (50 mmol / L, pH 6.0). Centrifuge at 16,000 rpm for 10 minutes, remove the supernatant, and resuspend the microspheres in 1 mL of MES. Centrifuge again at 16,000 rpm for 10 minutes, remove the supernatant, and resuspend the microspheres in MES. (2) Activation of microspheres: Weigh 20 mg of NHS and EDC respectively, dissolve them in labeling buffer, and prepare them immediately before use, i.e., 20 mg / mL NHS and EDC; take 10 μL of NHS and add it to the washed microspheres, and mix quickly; then take 5 μL of EDC and add it to the microspheres, mix quickly, and incubate at room temperature for 20 min; (3) Wash and remove residual EDC: Centrifuge the activated microspheres at 16,000 r / min for 10 min, remove the supernatant, and add 1 mL of MES to resuspend the microspheres; Centrifuge at 16,000 r / min for 10 min, discard the supernatant, and add 1 mL of MES to resuspend the microspheres for later use; (4) Conjugation of time-resolved fluorescent microspheres and antibodies: Add 0.02 mg of rabies virus N protein monoclonal antibody, add activated microspheres, mix quickly, and incubate at room temperature for 2 h; (5) Blocking: Add the same volume of blocking solution (10% BSA) as the measured microspheres and incubate at room temperature for 1 h; (6) Remove unbound antibodies: Centrifuge at 16,000 rpm for 10 min, discard the supernatant, and add 1 mL of MES to resuspend the microspheres. Repeat twice to remove unbound antibodies. (7) Resuspend and re-dissolve: Finally, resuspend the microspheres with 1 mL (0.02 M Tris-HCl + 20% sucrose + 20% trehalose, pH 8.0) to obtain the antibody-microsphere labeled complex. Place at 4°C for later use.

[0052] 2. Preparation of Chicken IgY Marker (1) Dilution of time-resolved fluorescent microspheres: Take 200 nm time-resolved fluorescent microspheres and sonicate for 5 minutes. Take 100 μL of microspheres and add 900 μL MES (50 mmol / L, pH 6.0). Centrifuge at 16,000 r / min for 10 minutes, remove the supernatant, add 1 mL MES to resuspend the microspheres, centrifuge again at 16,000 r / min for 10 minutes, remove the supernatant, and add MES to resuspend the microspheres. (2) Activation of microspheres: Weigh 20 mg of NHS and EDC respectively, dissolve them in labeling buffer, and prepare them immediately before use, i.e., 20 mg / mL NHS and EDC; take 10 μL of NHS and add it to the washed microspheres, and mix quickly; then take 5 μL of EDC and add it to the microspheres, mix quickly, and incubate at room temperature for 20 min; (3) Wash and remove residual EDC: Centrifuge the activated microspheres at 16,000 r / min for 10 min, remove the supernatant, and add 1 mL of MES to resuspend the microspheres; Centrifuge at 16,000 r / min for 10 min, discard the supernatant, and add 1 mL of MES to resuspend the microspheres for later use; (4) Time-resolved fluorescent microspheres coupled with antibodies: Add 0.01 mg chicken IgY, add activated microspheres, mix quickly, and incubate at room temperature for 2 h; (5) Blocking: Add the same volume of blocking solution (10% BSA) as the measured microspheres and incubate at room temperature for 1 h; (6) Remove unbound antibodies: Centrifuge at 16,000 rpm for 10 min, discard the supernatant, and add 1 mL of MES to resuspend the microspheres. Repeat twice to remove unbound antibodies. (7) Resuspend and re-dissolve: Finally, resuspend the microspheres with 1 mL of re-dissolving solution to obtain the antibody-microsphere labeled complex. Place the solution at 4°C for later use.

[0053] Reconstitution solution: 0.02 mol / L Tris-HCl buffer containing 20% ​​trehalose, 20% sucrose (mass fraction), pH 8.0.

[0054] Step 2: Preparation of conjugate release pad Pretreatment of the conjugate release pad: Prepare a conjugate pad treatment solution containing 5% sucrose, 0.5% Tween 20, 0.5% BSA, and 0.02M Tris-HCl, pH 8.0. Place the glass fiber in the conjugate pad treatment solution until the liquid covers the paper. Shake on an orbital shaker for 30 minutes, dehydrate, and dry in a 40°C oven overnight.

[0055] Use a gold label gold sprayer to evenly spray the prepared time-resolved fluorescent microsphere-labeled rabies virus N protein monoclonal antibody and chicken IgY on the conjugate release pad. Spray 2.5 μL of the conjugate of time-resolved fluorescent microsphere-labeled rabies virus N protein monoclonal antibody and chicken IgY per 1 cm of the conjugate release pad, then place it in a 40°C environment for 16 hours, take it out, and store it in a dry environment for later use.

[0056] Step 3: Sample pad processing Sample pad treatment solution: Prepare a 0.1M Tris-HCl sample pad treatment solution containing 0.5% BSA (IgG-free), 0.6% Triton-100, and 0.03% PC-300, pH 8.0. Place the glass fiber in the sample pad treatment solution until the liquid covers the paper. Shake on an orbital shaker for 30 minutes, dehydrate, and dry in a 40°C oven overnight.

[0057] Step 4: Preparation of nitrocellulose membrane The rabies virus N protein polyclonal antibody was coated on the nitrocellulose membrane to form the detection line, and the rabbit anti-chicken IgY was coated on the nitrocellulose membrane to form the quality control line.

[0058] Coating procedure: Dilute rabies virus N protein polyclonal antibody to 0.8 mg / mL in 0.05 mol / L, pH 7.2 phosphate buffer and coat the test line (T) on a nitrocellulose membrane using a gold-labeled streaker at a coating volume of 1.0 μL / cm. Dilute rabbit anti-chicken IgY to 20 μg / mL in 0.01 mol / L, pH 7.2 phosphate buffer and coat the control line (C) on a nitrocellulose membrane using a gold-labeled streaker at a coating volume of 1.0 μL / cm. Dry the coated nitrocellulose membrane at 40°C for 16 hours before use.

[0059] Step 5: Assembling the components A sample absorption pad, conjugate release pad, nitrocellulose membrane, and absorbent paper are sequentially attached to a PVC substrate. The conjugate release pad is covered with the sample absorption pad from its starting point, and the nitrocellulose membrane is covered with the conjugate release pad from its starting point. The end of the nitrocellulose membrane is connected to the starting point of the absorbent paper. The starting point of the sample absorption pad is aligned with the starting point of the PVC substrate, and the end of the absorbent paper is aligned with the end of the PVC substrate. The nitrocellulose membrane is marked with a test line (T) and a control line (C), both of which are strips perpendicular to the length of the test strip. The test line is located near the end of the conjugate release pad; the control line is located away from the end of the conjugate release pad. The test strip is cut into 4.05 mm wide strips by machine, placed in a special plastic card holder, and sealed in an aluminum foil bag. It can be stored at 2-30°C for 12 months.

[0060] Example 6 Characterization of Rabies Virus N Protein Fluorescence Quantitative Detection Test Strips 1. Sensitivity identification Using inactivated rabies virus antigen as a sample and following the instructions of each method, the detection sensitivity of the rabies virus fluorescence quantitative detection test strip was compared with that of the national standard for rabies diagnostic technology (GB / T 18639-2023). The results are shown in Table 3.

[0061] Table 3 Comparison of sensitivity of rabies virus fluorescence quantitative test strips and fluorescent RT-PCR for detecting inactivated rabies virus antigen

[0062] The national standard rabies fluorescent RT-PCT result judgment: when the sample to be tested has a Ct ≤ 33, it is judged to be positive for rabies virus nucleic acid; when there is no Ct or Ct> 37, it is judged to be negative for rabies virus nucleic acid.

[0063] The judgment criteria of Nabai Bio test strips: value ≤10IU, judged as negative; value >10IU and ≤30IU, judged as weak positive; value >30IU, judged as positive.

[0064] As shown in Table 3, when samples 1 and 2 were diluted 10,000-fold or less, both the rabies virus fluorescent quantitative test strip and the fluorescent RT-PCR test results were positive. When samples 1 and 2 were diluted 100,000-fold, the rabies virus fluorescent quantitative test strip test results were negative, and the fluorescent RT-PCR results were suspicious. When samples 1 and 2 were diluted 1,000,000-fold, both methods tested negative. Although the sensitivity of the rabies virus fluorescent test strip is one degree lower than that of the fluorescent RT-PCR, considering its ease of use, the rabies virus fluorescent quantitative test strip still has good application prospects.

[0065] 2. Specific identification Canine parvovirus, feline calicivirus, feline herpesvirus, feline panleukopenia virus, and the commercially available canine distemper-canine parvovirus combination vaccine and feline calicivirus-feline herpesvirus-feline panleukopenia virus combination vaccine were selected as specific test samples. The test strips were operated and the results were determined according to the test strip instructions. As shown in Table 4, the rabies virus fluorescence quantitative test strips tested all of the above specific samples negative, indicating good specificity.

[0066] The judgment criteria of Nabai Bio test strips: value ≤10IU, judged as negative; value >10IU and ≤30IU, judged as weak positive; value >30IU, judged as positive.

[0067] Table 4 Specificity test of rabies virus fluorescence quantitative detection test strip

[0068] Although the present invention has been described in detail above using general descriptions and specific embodiments, it will be apparent to those skilled in the art that modifications and improvements may be made thereto. Therefore, such modifications and improvements, without departing from the spirit of the present invention, are intended to be within the scope of protection claimed herein.

Claims

1. A monoclonal antibody against rabies virus N protein, characterized in that: The monoclonal antibody against rabies virus N protein comprises a heavy chain variable region and a light chain variable region; The amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID No. 1; The amino acid sequence of the light chain variable region of the monoclonal antibody is shown in SEQ ID No. 2; The heavy chain variable region and the light chain variable region are both composed of complementarity determining regions and framework regions, and the complementarity determining regions are both composed of CDR1, CDR2 and CDR3; The amino acid sequence of CDR1 of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID No. 5; The amino acid sequence of CDR2 of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID No. 6; The amino acid sequence of CDR3 of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID No. 7; The amino acid sequence of CDR1 of the light chain variable region of the monoclonal antibody is shown in SEQ ID No. 8; The amino acid sequence of CDR2 of the light chain variable region of the monoclonal antibody is shown in SEQ ID No. 9; The amino acid sequence of CDR3 of the light chain variable region of the monoclonal antibody is shown in SEQ ID No.

10.

2. The monoclonal antibody against rabies virus N protein according to claim 1, characterized in that: The nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody is shown in SEQ ID No. 3; The nucleotide sequence encoding the light chain variable region of the monoclonal antibody is shown in SEQ ID No.

4.

3. An expression vector, characterized in that It comprises the nucleotide sequence according to claim 2.

4. A host cell, characterized in that It comprises the nucleotide sequence according to claim 2 or the expression vector according to claim 3.

5. A test strip, characterized in that: The monoclonal antibody according to claim 1.

6. Use of the test strip according to claim 5 in detecting biological samples containing rabies virus antibodies.

Images