A monoclonal antibody against rabies virus N protein, a detection reagent and its application

Abstract

This invention discloses a monoclonal antibody against rabies virus N protein, a detection reagent, and their applications. The monoclonal antibody against rabies virus N protein includes a heavy chain variable region and a light chain variable region; the amino acid sequence of the heavy chain variable region is shown in SEQ ID No. 1; the amino acid sequence of the light chain variable region is shown in SEQ ID No. 2. This invention provides a monoclonal antibody with high affinity and detection sensitivity against rabies virus N protein, laying the foundation for the research and development and promotion of colloidal gold test strips and fluorescent immunoassay strips.

Description

Technical Field

[0001] This invention relates to the field of rapid biological detection technology for animal diseases, specifically to a monoclonal antibody against rabies virus N protein, a detection reagent, and their applications. Background Technology

[0002] Rabies virus (RV) is an important member of the family Rhabdoviridae and the genus Lyssavirus. Rabies virus is bullet-shaped with a helical nucleocapsid and an envelope containing single-stranded RNA. It is the pathogen that causes rabies. Rabies virus encodes five proteins: a glycoprotein (G), a nucleoprotein (N), a polymerase (L), a phosphoprotein (P), and a matrix protein (M). The N protein encapsulates the viral genome and interacts with the P and L proteins to form the viral nucleocapsid. The nucleoprotein is highly conserved, with amino acid homology between different strains ranging from 98% to 99.6%.

[0003] Rabies virus is mainly found in the brain tissue of infected animals, and its salivary glands and saliva often contain large amounts of the virus. Rabies can be caused by bites, scratches, or infection through mucous membranes from animals with rabies. Under certain conditions, it can also be transmitted through respiratory aerosols. The incubation period of rabies is variable, depending on factors such as the location of viral invasion and the viral load. It is usually 2 to 3 months, but can be as short as less than a week or as long as a year or even more.

[0004] After the rabies virus invades the body, it first replicates in the muscle tissue of the bite wound. Then, it invades the peripheral nervous system through the endplates and axons of motor neurons, subsequently migrating centripetally towards the central nervous system. Once in the central nervous system, it rapidly multiplies and spreads through nerves to the salivary glands. The saliva of infected animals then contaminates the wounds or mucous membranes of other animals, transmitting the virus to the next host. Once symptoms appear, rabies is almost 100% fatal to humans or animals. The only effective way to prevent rabies is timely vaccination and continuous monitoring of vaccine efficacy. Therefore, it is necessary to develop highly specific and sensitive monoclonal antibodies against the rabies virus for specific recognition and detection. Summary of the Invention

[0005] Therefore, the present invention provides a monoclonal antibody against rabies virus N protein, a detection reagent, and its application.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] According to a first aspect of the present invention, a monoclonal antibody against rabies virus N protein is provided, the monoclonal antibody against rabies virus N protein comprising a heavy chain variable region and a light chain variable region;

[0008] The amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID No. 1;

[0009] The amino acid sequence of the light chain variable region of the monoclonal antibody is shown in SEQ ID No. 2;

[0010] Both the heavy chain variable region and the light chain variable region are composed of complementary determination regions and framework regions, and the complementary determination regions are composed of CDR1, CDR2 and CDR3.

[0011] The amino acid sequence of CDR1 in the heavy chain variable region of the monoclonal antibody is shown in SEQ ID No. 5;

[0012] The amino acid sequence of CDR2 in the heavy chain variable region of the monoclonal antibody is shown in SEQ ID No. 6;

[0013] The amino acid sequence of CDR3 in the heavy chain variable region of the monoclonal antibody is shown in SEQ ID No. 7;

[0014] The amino acid sequence of CDR1 in the light chain variable region of the monoclonal antibody is shown in SEQ ID No. 8;

[0015] The amino acid sequence of CDR2 in the light chain variable region of the monoclonal antibody is shown in SEQ ID No. 9;

[0016] The amino acid sequence of CDR3 in the light chain variable region of the monoclonal antibody is shown in SEQ ID No. 10.

[0017] Furthermore, the monoclonal antibody is a murine monoclonal antibody.

[0018] According to a second aspect of the present invention, a nucleic acid molecule is provided that comprises a nucleotide sequence encoding a monoclonal antibody as described above.

[0019] Furthermore, the nucleotide sequence encoding the heavy chain variable region is shown in SEQ ID No. 3;

[0020] The nucleotide sequence encoding the variable region of the light chain is shown in SEQ ID No. 4.

[0021] According to a third aspect of the present invention, an expression vector is provided that comprises a nucleic acid molecule as described above.

[0022] According to a fourth aspect of the present invention, a host cell is provided comprising any of the nucleic acid molecules described above, or the expression vector described above.

[0023] According to a fifth aspect of the present invention, a test strip is provided, comprising a monoclonal antibody as described above.

[0024] The present invention has the following advantages:

[0025] This invention utilizes genetic engineering and hybridoma cell technology to screen a monoclonal antibody with high affinity and detection sensitivity for the N protein of rabies virus. This antibody exhibits high specificity and sensitivity, laying the foundation for the research and development and promotion of colloidal gold test strips and fluorescent immunoassay strips. Attached Figure Description

[0026] To more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are merely exemplary, and those skilled in the art can derive other embodiments based on the provided drawings without creative effort.

[0027] Figure 1 This is an SDS-PAGE identification image of the purified rabies virus N protein provided in an embodiment of the present invention.

[0028] Figure 2 This is an SDS-PAGE electrophoresis image of the anti-rabies virus N protein monoclonal antibody provided in an embodiment of the present invention. Detailed Implementation

[0029] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0030] Example 1: Preparation of Rabies Virus N Protein

[0031] 1. Expression process of rabies virus N protein

[0032] Based on the published nucleotide sequence of the rabies virus N protein gene, the gene sequence was codon-optimized and then sent to Wuhan Jinkairui Biotechnology Co., Ltd. for gene synthesis. The correctly identified positive recombinant plasmid was transformed into BL21(DE3) competent cells and cultured overnight at 37°C. Single positive colonies were picked and inoculated into LB liquid medium containing ampicillin and cultured at 37°C with shaking at 220 rpm. After about 8 hours according to the bacterial growth curve, 2 ml of the bacterial culture was collected for bacterial preservation. The bacterial culture 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. The culture was activated by shaking at 220 rpm until the OD value was about 0.6. 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 after centrifugation, and the cells were resuspended in 20 ml of PBS and washed. The supernatant was discarded after centrifugation again. Resuspend the bacterial culture in 20 ml of urea-free 1× Binding buffer, 5 ml of PMSF, and bacterial lysis buffer per 100 ml of original volume. The cells are then sonicated under ice bath conditions to lyse the bacterial cells. After centrifugation at 10,000 rpm for 15 min at 4°C, the supernatant and precipitate are separated and analyzed by SDS-PAGE protein electrophoresis to determine the expression pattern of the protein. Results are shown below. Figure 1 .

[0033] 2. Protein purification

[0034] The supernatant after ultrasonic lysis was filtered through a 0.45 μm filter, purified by column loading, and the flow-through was collected.

[0035] ① Add 10 vol 1× Binding Buffer;

[0036] ② Add 6 vol 1×Wash Buffer;

[0037] ③ Add 6 vol 1×Elute Buffer, collect 1 tube for every 1 ml of eluent, collect a total of 6 tubes, and label them E1 to E6;

[0038] ④ Add 6 vol 1×Strip Buffer for decolorization, and collect one tube of eluent labeled S1;

[0039] ⑤ After the resin purification column is completely decolorized, stop the fractionation and store at 4℃. Determine and label the protein concentrations in protein fractionation samples E1–E6.

[0040] The recombinant N protein was purified by nickel affinity column chromatography and then identified by SDS-PAGE. Figure 1 As shown, a distinct target band can be seen at around 55 kDa.

[0041] 3. Protein validation

[0042] The purified rabies virus N protein was verified using the ELISA method. Use 1 μg / ml purified rabies virus N protein antigen, 100 μl / well, and coat overnight at 4℃. Add 100 μl of 0.1% BSA to each well and block at 37℃ for 2 h. Wash with 300 μl / well of PBST (0.1%), 300 μl / well, for 3 washes. Add 100 μl / well of diluted clinical positive / negative serum and incubate at room temperature for 30 min (positive control group: rabies virus antibody positive serum; negative control group: rabies virus antibody negative serum; each group is repeated three times). Wash 3 times, blot dry, add 100 μl / well of HRP-labeled rabbit anti-canine secondary antibody (diluted 1:10000 with PBS), react at room temperature for 30 min, wash 3 times again, blot dry, add 100 μl / well of TMB chromogenic solution (commercial), incubate at room temperature for 10 min, and finally add 50 μl / well of 0.5 M sulfuric acid to stop the reaction. Measure OD using a microplate reader. 450 nm value. The results are shown in Table 1. The rabies virus N protein reacted with the positive control and had good antigenic activity.

[0043] Table 1. Results of antigen activity verification

[0044]

[0045] Example 2: Preparation of monoclonal antibodies against rabies virus N protein

[0046] 1. Mouse immunization

[0047] Four 6-8 week old female Balb / c mice were used, and each mouse was immunized with 50 μg of the rabies virus N protein prepared in Example 1. For the first immunization, the protein was emulsified with an equal volume of Freund's complete adjuvant and injected subcutaneously at multiple sites. A total of three immunizations were performed, one every two weeks. For the second and third immunizations, the antigen was emulsified with Freund's incomplete adjuvant, and the immunization dosage and method remained unchanged. Seven to ten days after the three immunizations, blood was collected from the tail vein of the mice. After centrifugation, the serum was collected and its titer was determined using a conventional indirect ELISA method. Blood was collected from the tail vein, and after the serum was separated, it was serially diluted with PBS. Blank mouse serum served as a control. OD values ​​were measured in the experimental group and the control group. 450 The maximum serum dilution factor, i.e., the mouse serum titer, is defined as the nm ratio greater than 2.1. Mice with high titers are selected and boosted with 50 μg of antigen via intraperitoneal injection. Cell fusion can be performed 3 days later.

[0048] 2. Culture of SP2 / 0 myeloma cells

[0049] One vial of SP2 / 0 myeloma cells, frozen in liquid nitrogen, was immediately transferred to a 37°C water bath. The cryovial was gently agitated periodically until the cells reached a semi-ice crystal state. Under sterile conditions, the SP2 / 0 cells were transferred to a 50ml sterile centrifuge tube. 10ml of preheated 1640 complete culture medium was slowly added dropwise to the centrifuge tube. The tube was centrifuged at 1000rpm for 5 minutes, and the supernatant was discarded. The cell clumps were gently dispersed, and the cells were resuspended in 5ml of culture medium and transferred to a T75 cell culture flask. An additional 5ml of culture medium was added, and the flask was agitated in a "cross" motion before being placed in a CO2 cell culture incubator at 37°C. The cell condition was observed under a microscope. When the cell density reached approximately 80%, the SP2 / 0 cells were passaged.

[0050] 3. Cell fusion

[0051] (1) Take 100 μg of protein, dilute it with 1×PBS to 200 μl, and inject it intraperitoneally into mice for shock immunization. Cell fusion can be performed three days later. Blood was collected from the orbital rim of the mice after the shock immunization and placed in EP tubes. After standing at 37°C for 2 h, the tubes were centrifuged at 4000 rpm for 10 min. The serum was collected as a positive control for subsequent screening of monoclonal antibodies. The mice were euthanized by cervical dislocation and disinfected by immersion in 75% alcohol.

[0052] (2) Preparation of spleen cells: In a biosafety cabinet, use sterilized scissors and forceps to cut open the mouse skin. Replace with a new set of sterilized scissors and forceps to cut open the mouse abdominal cavity. Then, carefully remove the spleen using a set of sterilized scissors and forceps, and trim away excess fat. Prepare a sterile 15ml centrifuge tube, add 10ml of DMEM culture medium, place the spleen into the centrifuge tube, moisten the spleen, and carefully discard the excess culture medium. Take another 10 ml of DMEM culture medium and place it in a sterile Petri dish. Grind the spleen with a ground glass slide to prepare a single-cell suspension. Filter the suspension 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. Resuspend the cells in 30 ml of DMEM culture medium and centrifuge again. Discard the supernatant, gently break up the cell clumps by hand, and resuspend the cells in 10 ml of DMEM culture medium.

[0053] (3) Cell fusion: Centrifuge at 1000 rpm for 5 min to collect well-grown SP2 / 0 cells into a 50 ml centrifuge tube, gently break up the SP2 / 0 cell clusters, add 30 ml of DMEM medium to resuspend, centrifuge again, 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 clusters, place in a 37℃ water bath, and add 1 ml of PEG fusion agent to the centrifuge tube within 1 min. At this time, the cell clusters are red, homogeneous, and quicksand-like, and rotating the tube wall feels like frosted glass.

[0054] (4) Termination of fusion: Take 9 ml of preheated DMEM medium to terminate the fusion, which is divided into three stages. The first stage is to add 1 ml in the first 1 min, the second stage is to add 1 ml in the first 1 min, and the third stage is to add the remaining 7 ml of medium in the first 3 min. Then, after standing in a 37℃ water bath for 5 min to stabilize, centrifuge at 800 rpm for 5 min.

[0055] (5) Plating: Discard the supernatant, gently break up the cell clumps, add HAT medium (for example, to plate 5 96-well plates, 200 μl / well, remove the feeder layer cells that have been pre-plated at 100 μl / well, then add 50 ml of HAT medium), mix the cells, and then evenly spread the fused cell suspension into the 96-well cell plate with the feeder layer cells added, 100 μl / well, and incubate in a CO2 cell incubator at 37°C.

[0056] 4. Screening of positive hybridoma cells

[0057] Seven days after cell fusion, when the cell clusters were relatively large, the cell supernatant was analyzed using an indirect ELISA method. Rabies virus inactivated antigen (1 μg / mL) and canine parvovirus inactivated antigen (1 μg / mL) were used as the detection antigens, respectively. Serum from fused mice was used as the positive control, and serum from mice immunized with PBS was used as the negative control. Hybridoma cell wells showing the strongest colorimetric reaction and not reacting with canine parvovirus inactivated antigen were selected as positive wells. The selected positive hybridoma cells were then subjected to limiting dilution. The hybridoma cell lines that stably secreted monoclonal antibodies, identified after subcloning, were expanded and cultured in T75 cell flasks. When the cell count reached approximately 80%, the cells were collected for ascites preparation.

[0058] 5. Preparation of ascites

[0059] Add 10 ml of sterile 1×PBS to the cell culture flask, blow off the cell layer, resuspend it, and transfer it to a 15 ml centrifuge tube. Centrifuge at 1000 rpm for 10 min. Discard the supernatant, resuspend the precipitate in 1 ml of sterile 1×PBS, mix well, and aspirate using a 1 ml syringe. Inject approximately 500 μl of the cell suspension into each mouse, observing their growth. Collect ascites fluid one week later, when the mouse's abdomen has swelled. Collect the ascites fluid into a centrifuge tube, centrifuge at 8000 rpm for 20 min, and aspirate the middle ascites layer.

[0060] 6. Antibody purification

[0061] The collected ascites fluid was first crudely purified using the caprylic acid-ammonium sulfate method. Ascites fluid (ml) was collected and its name recorded. It was centrifuged, and the volume recorded. 3 ml of pH 4.0 0.06M sodium acetate buffer was added, and the mixture was stirred for 5 min. 10 μl of caprylic acid was added, and the mixture was stirred at 4°C for 15 min. The mixture was filtered once through absorbent cotton using a syringe. Centrifugation was performed at 12000 rpm for 15 min at 4°C. The supernatant was collected, and an equal volume of saturated ammonium sulfate was added to a final concentration of 50%, stirring continuously. The mixture was incubated at 4°C for 3 h. Centrifugation was performed at 12000 rpm for 15 min at 4°C. The supernatant was discarded, and 1.8 ml of 0.01M PBS (pH 7.2) was added to the centrifuge tube until the precipitate was completely dissolved. The total volume was determined using a pipette, and half a volume of saturated ammonium sulfate was added to a concentration of 33%, stirring continuously. The mixture was incubated overnight at 4°C. Centrifugation was performed at 12000 rpm for 15 min at 4°C. Discard the supernatant and dissolve the precipitate in 0.45 ml of 0.01 M PBS. Treat the dialysis bag (boil for 5 min, rinse with pure water and check for leaks). Add the dissolved solution to the dialysis bag and dialyze overnight in 0.01 M PBS. Then, perform secondary purification of the crudely purified monoclonal antibody using a Protein G pre-packed column. SDS-PAGE was used to identify the purity of the purified monoclonal antibody. Results are shown below. Figure 2 The purity is approximately 96%.

[0062] Example 3: Identification of monoclonal antibodies against rabies virus N protein

[0063] 1. Concentration determination

[0064] The concentration of monoclonal antibodies was detected using a nucleic acid protein concentration analyzer. The concentration of the monoclonal antibody prepared in Example 2 was 2.36 mg / mL.

[0065] 2. Specificity identification

[0066] The specificity of monoclonal antibodies against rabies virus N protein was detected using an indirect ELISA method. Rabies virus inactivated antigen, canine C-reactive protein, canine parvovirus inactivated antigen, canine distemper virus inactivated antigen, and canine adenovirus type II inactivated antigen were coated at 1 μg / mL, respectively. The monoclonal antibody was then diluted 1 mg / mL at a 1:5000 ratio to verify its specificity. The results are shown in Table 2. The prepared rabies virus N protein monoclonal antibody reacted with rabies virus inactivated antigen but did not specifically react with canine C-reactive protein, canine parvovirus inactivated antigen, or canine distemper virus inactivated antigen, indicating good specificity.

[0067] Table 2 Results of Monoclonal Antibody Specificity Identification

[0068]

[0069] Example 4: Cloning of heavy and light chain variable region genes of monoclonal antibodies against rabies virus N protein

[0070] 1. Hybridoma cell culture and total RNA extraction

[0071] Hybridoma cells were cultured in RPMI 1640 complete medium at 37°C in a 5% CO2 incubator until the cell count reached 1×10⁻⁶. 7 Total RNA was extracted from cells using a total RNA extraction kit (purchased from Tiangen).

[0072] 2. Synthesis of the first strand of cDNA

[0073] The first strand of cDNA was synthesized using a reverse transcription kit (purchased from TAKARA) with extracted total RNA as the amplification template.

[0074] 3. Gene amplification

[0075] Design downstream primers and upstream universal primers for lambda, kappa, and heavy chains.

[0076] Primer: F (SEQ ID No. 11): AAGCGTGGTATCAACGCAGA,

[0077] R κ (SEQ ID No.12): AAATTGATGTCTTTGGGGTAGAA,

[0078] R λ (SEQ ID No.13): AATCGTACACACCAGTGTGTGGG,

[0079] R H (SEQ ID No. 14): AGGGATCCAGAGTTCCAGGT.

[0080] PCR was performed using the first strand of cDNA as a template in a 50 μl reaction volume.

[0081] Landing PCR reaction system: 3 μl template, 2.5 μl each of upstream and downstream primers (10 μM), 25 μl 2×pfu PCRMasterMix, and 17 μl ddH2O.

[0082] The landing PCR reaction conditions were as follows: 98℃ for 30s; 98℃ for 15s, 64℃-58℃ for 30s, decreasing by 0.5℃ each time until reaching 58℃, for 10 cycles; 72℃ for 30s; 98℃ for 15s, 56℃ for 30s, 72℃ for 30s, for 15 cycles; 72℃ for 7min.

[0083] 4. Cloning and screening of PCR amplification products

[0084] The PCR products were subjected to 1% agarose gel electrophoresis. 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 a pLB zero-background rapid cloning kit (Tiangen), transformed into DH5α competent cells (ampicillin-resistant), and recombinant positive clones were screened and sequenced.

[0085] The nucleotide sequence encoding the variable region of the heavy chain is shown in SEQ ID No. 3:

[0086] GACGTGAAACTGGTGGAGAGCGGAGGAGGACTGGTGAAACCCGGAGGCTCTCTGAAGCTGTCTTGCGCCGCCAGCGGCTTTACATTCAGCCGGTACACCATGTCTTGGGTCCGGCAGACACCAGAGAAGCGCCTCGAGTGGGTGTCCCTTATCAACAACGACGGCAGCTTC ACCTACTACAGCGACAGCGTGCGGGGAAGATTCACCATCAGCCGGGACAACGCCAAGAACACCCTGTACCTGCAGATGAGCAGCCTGAAGAGCGAGGACACCGCCATGTTCTATTGCACCAGGCCAGGCGGAGCCTATTGGGGACAGGGAACACTGGTGACCGTGTCTAGC.

[0087] The amino acid sequence of the heavy chain variable region is shown in SEQ ID No. 1:

[0088] DVKLVESGGGLVKPGGSLKLSCAASGFTFSRYTMSWVRQTPEKRLEWVSLINNDGSFTYYSDSVRGRFTISRDNAKNTLYLQMSSLKSEDTAMFYCTRPGGAYWGQGTLVTVSS.

[0089] The nucleotide sequence encoding the variable region of the light chain is shown in SEQ ID No. 4:

[0090] GACGTCGTGATGACACAGACCCCTCTGACCCTGAGCGTGACAATCGGACAGCCAGCCAGCATCAGCTGCAAGAGCAGCCAGAGCCTGCTGGATAGCGACGGCAAGACCTACCTGAATTGGCTGCTGCAGAGACCAGGACAGAGCCCTAAGCGGCTGATCTACCTGGTG TCCAAGCTGGACAGCGGCGTGCCAGATAGATTCACCGGAAGCGGAAGCGGCACCGACTTCACCCTGAAGATCTCTAGAGTGGAGGCCGAGGATCTGGGCATCTACTACTGTTGGCAGGGCACCCACTTCCCTCAGACATTTGGCGGAGGCACCAAGCTGGAGATCAAG.

[0091] The amino acid sequence of the light chain variable region is shown in SEQ ID No. 2:

[0092] DVVMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGIYYCWQGTHFPQTFGGGTKLEIK.

[0093] 5. Variable region amino acid sequence and homology analysis

[0094] The heavy and light chain gene sequences were compared and analyzed in the NCBI database. The results showed that the monoclonal antibody heavy chain amino acid sequence had the highest homology with the synthetic immunoglobulin heavy chain amino acid sequence (Sequence ID: XUS51565.1), with a homology of 102 / 119, or 86%. The monoclonal antibody heavy chain nucleotide sequence had the highest homology with the mammalian expression vector pEXPR50 (Sequence ID: MH107787.1), with a homology of 243 / 295, or 82%. The monoclonal antibody light chain amino acid sequence had the highest homology with the mouse immunoglobulin Kappa chain amino acid sequence (Sequence ID: pirC32513), with a homology of 110 / 112, or 98%. The light chain nucleotide sequence of the monoclonal antibody showed the highest homology with the artificially synthesized anti-Kv1.2 scFv sequence (Sequence ID: PP840917.1), with a homology of 282 / 332, representing 85% homology. Homology analysis of the heavy and light chain nucleotide and amino acid sequences encoding the monoclonal antibody revealed no sequences identical to those of this invention.

[0095] 6. CDR Area Analysis

[0096] The amino acid sequences of the heavy chain variable region and light chain variable region of the monoclonal antibody were analyzed at https: / / www.novopro.cn / tools / cdr.html to obtain its CDR region.

[0097] Antibody heavy chain CDR region:

[0098] CDR-H1 (SEQ ID No.5): GFTFSRYT

[0099] CDR-H2 (SEQ ID No. 6): INNDGSFT

[0100] CDR-H3 (SEQ ID No.7): TRPGGAY

[0101] Antibody light chain CDR region:

[0102] CDR-L1 (SEQ ID No.8): QSLLDSDGKTY

[0103] CDR-L2 (SEQ ID No. 9): LVS

[0104] CDR-L3 (SEQ ID No. 10): WQGTHFPQT.

[0105] Example 5: Preparation of rabies virus fluorescent quantitative detection test strip

[0106] 1. Preparation of monoclonal antibody markers for rabies virus N protein

[0107] (1) Dilution of time-resolved fluorescent microspheres: Take time-resolved fluorescent microspheres with a particle size of 200 nm, sonicate for 5 min, take 100 μL of microspheres and add 900 μL of MES (50 mmol / L, pH 6.0). Centrifuge at 16000 r / min for 10 min, remove the supernatant, add 1 mL of MES to resuspend the microspheres, centrifuge again at 16000 r / min for 10 min, remove the supernatant, add MES to resuspend the microspheres;

[0108] (2) Activation of microspheres: Weigh 20 mg NHS and EDC, dissolve them in labeling buffer, prepare fresh each time, i.e. 20 mg / mL NHS and EDC; take 10 μL NHS and add it to the washed microspheres, mix quickly; then take 5 μL EDC and add it to the microspheres, mix quickly, and incubate at room temperature for 20 min.

[0109] (3) Cleaning to remove residual EDC: Centrifuge the activated microspheres at 16000 r / min for 10 min, remove the supernatant, and add 1 mL MES to resuspend the microspheres; Centrifuge at 16000 r / min for 10 min, discard the supernatant, and add 1 mL MES to resuspend the microspheres for later use;

[0110] (4) Coupling of time-resolved fluorescent microspheres with antibodies: Add 0.02 mg of rabies virus N protein monoclonal antibody, add the activated microspheres, mix quickly, and incubate at room temperature for 2 h;

[0111] (5) Blocking: Add the same volume of blocking solution (10% BSA) as the measured microspheres and incubate at room temperature for 1 h;

[0112] (6) Removal of unbound antibodies: Centrifuge at 16000 r / min for 10 min, discard the supernatant, and resuspend the microspheres in 1 mL of MES. Repeat twice to remove unbound antibodies;

[0113] (7) Resuspension and reconstitution: Finally, resuspend the microspheres in 1 mL (0.02M Tris-HCl + 20% sucrose + 20% trehalose, pH 8.0) to obtain the antibody-microsphere labeled complex. Store at 4℃ for later use.

[0114] 2. Preparation of chicken IgY markers

[0115] (1) Dilution of time-resolved fluorescent microspheres: Take time-resolved fluorescent microspheres with a particle size of 200 nm, sonicate for 5 min, take 100 μL of microspheres and add 900 μL of MES (50 mmol / L, pH 6.0). Centrifuge at 16000 r / min for 10 min, remove the supernatant, add 1 mL of MES to resuspend the microspheres, centrifuge again at 16000 r / min for 10 min, remove the supernatant, and add the MES to resuspend the microspheres;

[0116] (2) Activation of microspheres: Weigh 20 mg NHS and EDC, dissolve them in labeling buffer, prepare fresh each time, i.e. 20 mg / mL NHS and EDC; take 10 μL NHS and add it to the washed microspheres, mix quickly; then take 5 μL EDC and add it to the microspheres, mix quickly, and incubate at room temperature for 20 min.

[0117] (3) Cleaning to remove residual EDC: Centrifuge the activated microspheres at 16000 r / min for 10 min, remove the supernatant, and add 1 mL MES to resuspend the microspheres; Centrifuge at 16000 r / min for 10 min, discard the supernatant, and add 1 mL MES to resuspend the microspheres for later use;

[0118] (4) Coupling of time-resolved fluorescent microspheres with antibodies: Add 0.01 mg chicken IgY, add activated microspheres, mix quickly, and incubate at room temperature for 2 h;

[0119] (5) Blocking: Add the same volume of blocking solution (10% BSA) as the measured microspheres and incubate at room temperature for 1 hour;

[0120] (6) Removal of unbound antibodies: Centrifuge at 16000 r / min for 10 min, discard the supernatant, and resuspend the microspheres in 1 mL of MES. Repeat twice to remove unbound antibodies;

[0121] (7) Resuspension and reconstitution: Finally, resuspend the microspheres with 1 mL of reconstitution solution to obtain the antibody-microsphere labeled complex. Store at 4°C for later use.

[0122] Reconstitution solution: 0.02 mol / L Tris-HCl buffer solution containing 20% ​​trehalose, 20% sucrose (mass fraction), pH 8.0.

[0123] Step 2: Preparation of the conjugate release pad

[0124] Pretreatment of the conjugate release pad: Prepare a 0.02M Tris-HCl conjugate pad treatment solution containing 5% sucrose, 0.5% Sinopharm Tween 20, 0.5% BSA, and pH 8.0. Place the glass fiber into the prepared conjugate pad treatment solution, ensuring the liquid completely submerges the paper. Then, shake on a track shaker for 30 minutes to dehydrate, and dry overnight in a 40℃ oven.

[0125] Using a gold sprayer, the prepared time-resolved fluorescent microsphere-labeled rabies virus N protein monoclonal antibody and chicken IgY were uniformly sprayed onto the conjugate release pad. 2.5 μL of the conjugate of time-resolved fluorescent microsphere-labeled rabies virus N protein monoclonal antibody and chicken IgY was sprayed onto each 1 cm of the conjugate release pad. The pad was then placed in a 40°C environment for 16 h and then removed and stored in a dry environment for later use.

[0126] Step 3: Sample pad preparation

[0127] Sample pad treatment solution: Prepare a 0.1M Tris-HCl sample pad treatment solution containing 0.5% BSA (IgG Free), 0.6% Triton-100, 0.03% PC-300, and pH 8.0. Place the glass fiber in the prepared sample pad treatment solution, ensuring the liquid completely submerges the paper. Shake on a track shaker for 30 minutes to dehydrate, then dry in a 40°C oven overnight.

[0128] Step 4: Preparation of nitrocellulose membrane

[0129] A detection line was constructed by coating a rabies virus N protein polyclonal antibody onto a nitrocellulose membrane, and a quality control line was constructed by coating a rabbit anti-chicken IgY onto a nitrocellulose membrane.

[0130] Coating process: Rabies virus N protein polyclonal antibody was diluted to 0.8 mg / mL with 0.05 mol / L, pH 7.2 phosphate buffer and coated onto the detection line (T) of a nitrocellulose membrane using a gold-labeled coating apparatus, with a coating volume of 1.0 μL / cm. Rabbit anti-chicken IgY was diluted to 20 μg / mL with 0.01 mol / L, pH 7.2 phosphate buffer and coated onto the control line (C) of a nitrocellulose membrane using a gold-labeled coating apparatus, with a coating volume of 1.0 μL / cm. The coated nitrocellulose membranes were then dried at 40℃ for 16 h for later use.

[0131] Step 5: Assemble the components

[0132] The sample absorption pad, conjugate release pad, nitrocellulose membrane, and absorbent paper are sequentially attached to a PVC base plate. One-quarter of the conjugate release pad's area from its starting end is covered by the sample absorption pad, and one-quarter of the nitrocellulose membrane's area from its starting end is also covered by the conjugate release pad. The end of the nitrocellulose membrane is connected to the beginning of the absorbent paper. The beginning of the sample absorption pad is aligned with the beginning of the PVC base plate, and the end of the absorbent paper is aligned with the end of the PVC base plate. The nitrocellulose membrane has 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 on the side closer to the end of the conjugate release pad, and the control line is located on the side farther from the end of the conjugate release pad. The test strip is cut into 4.05mm wide strips using a machine, packaged in a special plastic case, sealed in an aluminum foil bag, and can be stored for 12 months at 2–30℃.

[0133] Example 6 Characterization of Rabies Virus N Protein Fluorescent Quantitative Detection Test Strip

[0134] 1. Sensitivity determination

[0135] Using rabies virus inactivated antigen as a sample, and following the instructions of their respective methods, the detection sensitivity of the rabies virus fluorescent quantitative test strip and the national standard for rabies diagnostic technology (GB / T 18639-2023) were compared. The results are shown in Table 3.

[0136] Table 3. Comparison of the sensitivity of rabies virus quantitative PCR test strips and fluorescent RT-PCR for detecting rabies virus inactivated antigen.

[0137]

[0138] The national standard for interpreting rabies fluorescent RT-PCT results is as follows: if the sample has a Ct ≤ 33, it is considered positive for rabies virus nucleic acid; if there is no Ct or Ct > 37, it is considered negative for rabies virus nucleic acid.

[0139] NanoBio test strip judgment criteria: Value ≤10IU, judged as negative; value >10IU and ≤30IU, judged as weakly positive; value >30IU, judged as positive.

[0140] As shown in Table 3, for samples 1 and 2 diluted 10,000 times or less, both the quantitative rabies virus test strip and the fluorescent RT-PCR test results were positive. For samples 1 and 2 diluted 100,000 times, the quantitative rabies virus test strip result was negative, while the fluorescent RT-PCR result was suspected. For samples 1 and 2 diluted 1,000,000 times, both methods yielded negative results. Although the sensitivity of the quantitative rabies virus test strip is one gradient lower than that of fluorescent RT-PCR, considering its ease of operation, the quantitative rabies virus test strip still has good application prospects.

[0141] 2. Specificity identification

[0142] Canine parvovirus, feline calicivirus, feline herpesvirus, feline panleukopenia virus, and commercially available canine distemper-canine parvovirus bivalent vaccine and feline calicivirus-feline herpesvirus-feline panleukopenia virus trivalent vaccine were selected as specific detection samples. The test strips were operated and the results were interpreted according to the instructions. As shown in Table 4, the rabies virus quantitative fluorescence test strips showed negative results for all the above specific samples, indicating that the test strips have good specificity.

[0143] NanoBio test strip judgment criteria: Value ≤10IU, judged as negative; value >10IU and ≤30IU, judged as weakly positive; value >30IU, judged as positive.

[0144] Table 4: Specificity Tests of Rabies Virus Quantitative Detection Test Strips

[0145]

[0146] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A monoclonal antibody against rabies virus N protein, characterized in that, The monoclonal antibody against rabies virus N protein includes 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; Both the heavy chain variable region and the light chain variable region are composed of complementary determination regions and framework regions, and the complementary determination regions are composed of CDR1, CDR2 and CDR3. The amino acid sequence of CDR1 in the heavy chain variable region of the monoclonal antibody is shown in SEQ ID No. 5; The amino acid sequence of CDR2 in the heavy chain variable region of the monoclonal antibody is shown in SEQ ID No. 6; The amino acid sequence of CDR3 in the heavy chain variable region of the monoclonal antibody is shown in SEQ ID No. 7; The amino acid sequence of CDR1 in the light chain variable region of the monoclonal antibody is shown in SEQ ID No. 8; The amino acid sequence of CDR2 in the light chain variable region of the monoclonal antibody is shown in SEQ ID No. 9; The amino acid sequence of CDR3 in 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 carrier, characterized in that, It contains the nucleotide sequence as described in claim 2.

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

5. A test strip, characterized in that, Includes the monoclonal antibody as described in claim 1.

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