Monoclonal antibody for detecting A35R protein and application thereof

Abstract

The invention relates to a monoclonal antibody for detecting A35R protein and application of the monoclonal antibody. According to the invention, six strains of antibodies, including an antibody 4E7, an antibody 11G12, an antibody 6F10, an antibody 6C11, an antibody 18C3 and an antibody 23A7, are screened out; the invention specifically discloses a heavy chain variable region amino acid sequence and a light chain variable region amino acid sequence of the antibody, and specific complementary determining region amino acid sequences and framework region amino acid sequences of the heavy chain variable region and the light chain variable region of the antibody. The antibody 4E7 and the antibody 11G12 not only can be combined with A35R-MPXV protein, but also can be combined with homologous proteins A35R-CPXV, A35R-VTT8 and A35R-Variola of the A35R protein. The antibody 6F10, the antibody 6C11, the antibody 23A7 and the antibody 18C3 can specifically recognize the A35R-MPXV protein. The monoclonal antibody has high binding capacity for A35R protein, is suitable for sensitive and specific detection and prevention of monkey pox virus A35R protein, and can be used for clinical diagnosis and prevention of monkey pox virus infection related diseases.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a monoclonal antibody for detecting A35R protein and an application thereof. Background Art

[0002] Monkeypox virus (MPXV) is an orthopoxvirus belonging to the Poxviridae family of large double-stranded DNA viruses. It is closely related to the smallpox virus that has been eradicated worldwide and has replaced the smallpox virus as the most serious disease caused by orthopoxviruses (Thornhill JP, Barkati S, Walmsley S, et al. Monkeypox Virus Infection in Humans across 16 Countries-April-June 2022[J]. N Engl J Med, 2022, 387(8): 679-691.). MPXV mainly infects the body in two forms: extracellular enveloped virus (EEV) and intracellular mature virus (IMV) (Roth enburg S, Yang Z, Beard P, et al. Monkeypox emergency: Urgent questions and pers pectives[J]. Cell, 2022, 185(18): 3279-3281.). A35R is an envelope component of intracellular enveloped virions (IEV) and extracellular enveloped virions (EEV), is homologous to vaccinia virus protein A33R, and plays a crucial role in the process of cell-to-cell transmission of viral particles.

[0003] Since the first case of monkeypox virus infection was reported in 1970, more than 30,000 cases have been reported in West and Central African countries. Currently, the most effective way to prevent monkeypox virus infection is vaccination. Its diagnostic methods include nucleic acid amplification detection, enzyme-linked immunosorbent assay, immunoblotting test, immunohistochemistry and virus isolation and culture, but they all have advantages and disadvantages to varying degrees. Therefore, it is necessary to develop neutralizing antibodies against one or more epitopes of monkeypox virus to achieve a good neutralizing effect on it and provide reserves for clinical diagnosis and prevention methods. Summary of the invention

[0004] In view of the problems existing in the prior art, the present invention provides monoclonal antibodies for detecting A35R protein and their applications. The present invention prepares and screens 6 monoclonal antibodies with strong specificity, good A35R protein binding ability and easy production. In the above manner, the antibodies 4E7, 6F10, 6C11, 11G12, 23A7, and 18C3 obtained by the present invention can react specifically with the membrane protein A35R on the monkeypox virus IMV particles, and can be used in the clinical diagnosis and prevention of diseases related to monkeypox virus infection.

[0005] The technical solution of the present invention to solve the above technical problems is as follows:

[0006] The first object of the present invention is to provide a monoclonal antibody for detecting A35R protein, wherein the monoclonal antibody comprises at least one of antibody 4E7, antibody 11G12, antibody 6F10, antibody 6C11, antibody 18C3, and antibody 23A7; the monoclonal antibody comprises a heavy chain variable region and a light chain variable region;

[0007] The complementary determining regions CDR1, CDR2, and CDR3 of the heavy chain variable region of the antibody 4E7 are SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3, respectively; the complementary determining regions CDR1, CDR2, and CDR3 of the light chain variable region of the antibody 4E7 are SEQ ID NO.4, SEQ ID NO.5, and SEQ ID NO.6, respectively;

[0008] The complementary determining regions CDR1, CDR2, and CDR3 of the heavy chain variable region of the antibody 11G12 are SEQ ID NO.7, SEQ ID NO.8, and SEQ ID NO.9, respectively; the complementary determining regions CDR1, CDR2, and CDR3 of the light chain variable region of the antibody 11G12 are SEQ ID NO.10, SEQ ID NO.11, and SEQ ID NO.12, respectively;

[0009] The complementary determining regions CDR1, CDR2, and CDR3 of the heavy chain variable region of the antibody 6F10 are SEQ ID NO.13, SEQ ID NO.14, and SEQ ID NO.15, respectively; the complementary determining regions CDR1, CDR2, and CDR3 of the light chain variable region of the antibody 6F10 are SEQ ID NO.16, SEQ ID NO.17, and SEQ ID NO.18, respectively;

[0010] The complementary determining regions CDR1, CDR2, and CDR3 of the heavy chain variable region of the antibody 6C11 are SEQ ID NO.19, SEQ ID NO.20, and SEQ ID NO.21, respectively; the complementary determining regions CDR1, CDR2, and CDR3 of the light chain variable region of the antibody 6C11 are SEQ ID NO.22, SEQ ID NO.23, and SEQ ID NO.24, respectively;

[0011] The complementary determining regions CDR1, CDR2, and CDR3 of the heavy chain variable region of the antibody 18C3 are SEQ ID NO.25, SEQ ID NO.26, and SEQ ID NO.27, respectively; the complementary determining regions CDR1, CDR2, and CDR3 of the light chain variable region of the antibody 18C3 are SEQ ID NO.28, SEQ ID NO.29, and SEQ ID NO.30, respectively;

[0012] The complementary determining regions CDR1, CDR2, and CDR3 of the heavy chain variable region of the antibody 23A7 are SEQ ID NO.31, SEQ ID NO.32, and SEQ ID NO.33, respectively; the complementary determining regions CDR1, CDR2, and CDR3 of the light chain variable region of the antibody 23A7 are SEQ ID NO.34, SEQ ID NO.35, and SEQ ID NO.36, respectively.

[0013] Furthermore, the framework regions FR1, FR2, FR3, and FR4 of the heavy chain variable region of the antibody 4E7 are SEQ ID NO.37, SEQ ID NO.38, SEQ ID NO.39, and SEQ ID NO.40, respectively;

[0014] The framework regions FR1, FR2, FR3, and FR4 of the heavy chain variable region of the antibody 11G12 are SEQ ID NO.41, SEQ ID NO.42, SEQ ID NO.43, and SEQ ID NO.44, respectively;

[0015] The framework regions FR1, FR2, FR3, and FR4 of the heavy chain variable region of the antibody 6F10 are SEQ ID NO.45, SEQ ID NO.46, SEQ ID NO.47, and SEQ ID NO.48, respectively;

[0016] The framework regions FR1, FR2, FR3, and FR4 of the heavy chain variable region of the antibody 6C11 are SEQ ID NO.49, SEQ ID NO.50, SEQ ID NO.51, and SEQ ID NO.52, respectively;

[0017] The framework regions FR1, FR2, FR3, and FR4 of the heavy chain variable region of the antibody 18C3 are SEQ ID NO.53, SEQ ID NO.54, SEQ ID NO.55, and SEQ ID NO.56, respectively;

[0018] The framework regions FR1, FR2, FR3, and FR4 of the heavy chain variable region of the antibody 23A7 are SEQ ID NO.57, SEQ ID NO.58, SEQ ID NO.59, and SEQ ID NO.60, respectively.

[0019] Furthermore, the framework regions FR1, FR2, FR3, and FR4 of the light chain variable region of the antibody 4E7 are SEQ ID NO.61, SEQ ID NO.62, SEQ ID NO.63, and SEQ ID NO.64, respectively;

[0020] The framework regions FR1, FR2, FR3, and FR4 of the light chain variable region of the antibody 11G12 are SEQ ID NO.65, SEQ ID NO.66, SEQ ID NO.67, and SEQ ID NO.68, respectively;

[0021] The framework regions FR1, FR2, FR3, and FR4 of the light chain variable region of the antibody 6F10 are SEQ ID NO.69, SEQ ID NO.70, SEQ ID NO.71, and SEQ ID NO.72, respectively;

[0022] The framework regions FR1, FR2, FR3, and FR4 of the light chain variable region of the antibody 6C11 are SEQ ID NO.73, SEQ ID NO.74, SEQ ID NO.75, and SEQ ID NO.76, respectively;

[0023] The framework regions FR1, FR2, FR3, and FR4 of the light chain variable region of the antibody 18C3 are SEQ ID NO.77, SEQ ID NO.78, SEQ ID NO.79, and SEQ ID NO.80, respectively;

[0024] The framework regions FR1, FR2, FR3, and FR4 of the light chain variable region of the antibody 23A7 are SEQ ID NO.81, SEQ ID NO.82, SEQ ID NO.83, and SEQ ID NO.84, respectively.

[0025] Further, the sequence of the heavy chain variable region of the antibody 4E7 is SEQ ID NO.85; the sequence of the light chain variable region of the antibody 4E7 is SEQ ID NO.86;

[0026] The sequence of the heavy chain variable region of the antibody 11G12 is SEQ ID NO.87; the sequence of the light chain variable region of the antibody 11G12 is SEQ ID NO.88;

[0027] The sequence of the heavy chain variable region of the antibody 6F10 is SEQ ID NO.89; the sequence of the light chain variable region of the antibody 6F10 is SEQ ID NO.90;

[0028] The sequence of the heavy chain variable region of the antibody 6C11 is SEQ ID NO.91; the sequence of the light chain variable region of the antibody 6C11 is SEQ ID NO.92;

[0029] The sequence of the heavy chain variable region of the antibody 18C3 is SEQ ID NO.93; the sequence of the light chain variable region of the antibody 18C3 is SEQ ID NO.94;

[0030] The sequence of the heavy chain variable region of the antibody 23A7 is SEQ ID NO.95; the sequence of the light chain variable region of the antibody 23A7 is SEQ ID NO.96.

[0031] The beneficial effect of the present invention is that: the present invention discloses the above antibody sequence, and when the sequence is known, the above antibody can be repeatedly obtained based on gene recombination technology.

[0032] The second object of the present invention is to provide the use of a monoclonal antibody for detecting A35R protein, wherein the monoclonal antibody is used in the preparation of a detection product or a prevention product.

[0033] Furthermore, the detection product is a product for detecting monkeypox virus A35R protein.

[0034] Furthermore, the preventive product is a product for preventing infection by monkeypox virus A35R protein.

[0035] Furthermore, the antibody 6F10, the antibody 6C11, the antibody 18C3, and the antibody 23A7 specifically recognize the monkeypox virus protein A35R-MPXV.

[0036] Furthermore, the antibody 4E7 and the antibody 6F10, the antibody 4E7 and the antibody 6C11 can be used in the preparation of products for detecting monkeypox virus or preventing monkeypox virus infection by specifically recognizing the monkeypox virus protein A35R-MPXV.

[0037] Furthermore, the antibody 11G12 and the antibody 18C3, and the antibody 11G12 and the antibody 23A7 are paired and specifically recognize the monkeypox virus protein A35R-MPXV to be used in the preparation of products for detecting monkeypox virus or preventing monkeypox virus infection.

[0038] The beneficial effect of the present invention is that the 6 antibodies screened by the present invention are suitable for sensitively and specifically detecting monkeypox virus A35R-MPXV protein, and can be used for clinical diagnosis of diseases related to monkeypox virus infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 The figure is a graph showing the protein expression and purification results of Example 1 of the present invention; in the figure, M is a molecular weight indicator band, 1 is a denatured protein sample, and 2 is a non-denatured protein sample;

[0040] Figure 2 The concentration effect curve of the A35R binding antibody screening of Example 1 of the present invention; wherein (a) is antibody 4E7; (b) is antibody 11G12, (c) is antibody 6F10, (d) is antibody 6C11, (e) is antibody 18C3, and (f) is antibody 23A7;

[0041] Figure 3 The binding of the antibody of Example 2 of the present invention to the A35R-MPXV homologous protein; wherein (a) is antibody 4E7; (b) is antibody 11G12, (c) is antibody 6F10, (d) is antibody 6C11, (e) is antibody 18C3, and (f) is antibody 23A7;

[0042] Figure 4 The concentration effect curve of the A35R binding biotinylated antibody screening of Example 3 of the present invention; wherein (a) is antibody 4E7; (b) is antibody 11G12, (c) is antibody 6F10, (d) is antibody 6C11, (e) is antibody 18C3, and (f) is antibody 23A7;

[0043] Figure 5 This is a competitive ELISA bar graph of Example 3 of the present invention, wherein (a) is antibody 11G12; (b) is antibody 18C3, (c) is antibody 4E7, (d) is antibody 6C11, (e) is antibody 6F10, and (f) is antibody 23A7;

[0044] Figure 6 The concentration effect curves of the monoclonal antibodies of Example 3 of the present invention for detecting A35R-MPXV are (a) 4E7-6F10, (b) 4E7-6C11, (c) 11G12-18C3, and (d) 11G12-23A7. DETAILED DESCRIPTION

[0045] The principles and features of the present invention are described below, and the examples are only used to explain the present invention and are not used to limit the scope of the present invention. If no specific technology or conditions are specified in the embodiments, the technology or conditions described in the literature in this field or the product instructions are used. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased through regular channels.

[0046] Experimental Materials

[0047] 1. Key drugs and reagents are shown in Table 1. Other reagents are of domestic analytical grade.

[0048]

[0049]

[0050] 2. Main instruments

[0051]

[0052]

[0053] Example 1: Preparation and purification of monkeypox virus monoclonal antibodies

[0054] 1. Expression and purification of monkeypox virus A35R protein

[0055] (1) The A35R protein gene sequence of MPXV was constructed into the eukaryotic expression vector PTT5 after adding a His tag sequence at the C-terminus to prepare a recombinant expression plasmid.

[0056] (2) Expression of A35R protein: The transfection reagent PEI was added to the recombinant expression plasmid and slowly mixed to obtain a complex. The complex was incubated at room temperature for 15 minutes and then transfected into Expi293 cells to obtain recombinant cells expressing A35R-MPXV protein.

[0057] (3) The recombinant cells were placed in a 5% CO2, 37°C incubator at 120 rpm for 5 days, and then centrifuged for 30 min to obtain a supernatant, wherein the centrifugation conditions were 4°C, 3000 rpm. Subsequently, the target protein A35R-MPXV was separated and purified using Ni-NTA column affinity chromatography.

[0058] (4) Repeat the above steps (1), (2), and (3) to prepare vaccinia virus protein A35R-CPXV, smallpox virus protein A35R-Variola, and vaccinia virus Tiantan strain protein A35R-VTT8. Figure 1 shown.

[0059] 2. Acquisition of Antibodies

[0060] 2.1 Mouse immunization and antiserum detection

[0061] The A35R-MPXV antigen protein was used to immunize the experimental mice with the A35R-MPXV protein for multiple times, and the serum titer test was performed after immunization. The specific experimental steps are as follows:

[0062] (1) Five 6-week-old SPF female BalB / C mice weighing 25±2 g (purchased from Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd.) were selected as immunized animals. The mice were marked and numbered as left front, right front, left hind, right hind, and none. 30-50 μl of blood was collected from the tail vein of the five mice, and the pre-immune serum was collected by centrifugation after coagulation.

[0063] (2) First immunization: The required volume of A35R protein was calculated based on the immunization dose of 100 μg / mouse. The A35R protein was emulsified with an equal volume of Freund's complete adjuvant and then injected subcutaneously at multiple points in the mouse.

[0064] (3) Second immunization: Two weeks after the first immunization, the required volume of A35R protein was calculated based on the immunization dose of 50 μg / mouse. The A35R protein was emulsified with an equal volume of Freund's incomplete adjuvant and then injected subcutaneously at multiple points in the mice.

[0065] (4) Third immunization: One week after the second immunization, the required volume of A35R protein was calculated based on the immunization dose of 50 μg / animal, and the antigen was emulsified using an equal volume of Freund's incomplete adjuvant and injected subcutaneously at multiple points;

[0066] (5) ELISA test: One week after the third immunization, 30-50 μl of blood was collected from the tail vein of 5 mice, and the serum after three immunizations was collected by centrifugation after coagulation. The antigen (i.e., A35R protein) was diluted to a final concentration of 1 μg / ml with antigen coating solution (i.e., carbonate buffer with a pH of 7.4), and the ELISA plate was coated at 100 μL / well. After standing at 4°C overnight (12-16 h), the plate was blocked with 0.1% casein blocking solution at room temperature for 1 h and then washed. The mouse serum was diluted to the following dilutions: 1:2000, 1:4000, 1:8000, 1:16000, 1:32000, 1:64000, and 1:128000. Antibody diluent (1×PBS, 0.5% BSA) was used as a negative control. The serum titer was detected by ELISA, and the OD was measured by an enzyme reader. 450 Read the results. The results are shown in Table 1:

[0067] Table 1

[0068]

[0069] It can be seen from Table 1 that the titer of mouse serum was qualified after three immunizations, and no marked mice were fused.

[0070] 2.2 Preparation and screening of hybridoma cells

[0071] (1) One week before fusion, mouse SP2 / 0 myeloma cells were cultured and the cell state was adjusted to the logarithmic growth phase. The mice with the highest ELISA titer in Table 1, i.e., unmarked mice, were selected. Three days before fusion, the mice were boosted with A35R-MPXV recombinant protein, 50 μg / mouse, by intraperitoneal injection;

[0072] (2) Fusion: The mice were killed and the spleens were removed under sterile conditions. The mouse spleen cells were collected after mechanical disruption and large impurities were removed with a 200-mesh sieve. The cells were washed with PBS buffer for 3 times and then the cells were counted to obtain the mouse spleen cells to be fused. SP2 / 0 myeloma cells in the logarithmic phase were collected and washed with PBS for 3 times and then the cells were counted. After the cell count, the SP2 / 0 myeloma cells and the mouse spleen cells to be fused were gently mixed at a ratio of 1:2.5, centrifuged and the PBS was discarded, and the cells were fused using an electric fusion instrument to obtain fused cells. After centrifugation, complete medium containing HAT was added, and the fused cells were resuspended and mixed and then plated in a 96-well plate.

[0073] (3) Medium change: Full medium change is performed 5 days after fusion;

[0074] (4) ELISA test: Coat the test source (A35R-MPXV), aspirate the cell culture supernatant 7 days after fusion for ELISA test, record the positive cell wells (judgment standard: three times the negative result), mark them and perform half-medium change;

[0075] (5) ELISA re-examination and screening: The next day, the positive cell wells after the medium change were re-examined and the coated screening source (A35R-MPXV, A35R-CPXV, A35R-Variola, A35R-VTT8) was screened, the required clone number was recorded, and subcloning was performed.

[0076] 2.3 Establishment of stable cell lines

[0077] (1) First subcloning: Perform subclone screening in the positive cell wells of the above ELISA retest, use complete medium for limiting dilution, and plate the cells in a 96-well plate at a number of 1 cell / well; observe under a microscope after 7 days, detect the wells with clone growth, mark the single clone wells and register them, and perform ELISA test on the registered clones; discard the clone numbers that turn negative, select the positive clone wells with vigorous growth, perform half-medium change, and retest and screen the next day;

[0078] (2) Secondary subcloning: Select the positive monoclonal wells from the previous step for secondary subclone screening, perform limiting dilution using complete medium, and plate the cells in a 96-well plate at a rate of 1 cell / well; observe under a microscope 7 days later, detect the wells with clone growth, mark the monoclonal wells and register them, and perform ELISA on the registered clones;

[0079] (3) After multiple subcloning, when the ELISA test reaches 100% positive, select the monoclonal wells with vigorous growth for expansion culture and finalization, and freeze the cells for preservation, thus establishing a stable hybridoma cell line.

[0080] 3 Antibody gene sequencing

[0081] 3.1 Extraction of total RNA from hybridoma cell lines

[0082] (1) Add sufficient amount of TRIpure Reagent to the established hybridoma cell line and pipette repeatedly to obtain a homogenate.

[0083] (2) Place the homogenate at room temperature for 5 minutes to allow the nucleic acids and proteins in the homogenate to be fully dissociated. Add 0.2 mL of chloroform to every 1 mL of TRIpure Reagent, cover the tube tightly, shake vigorously by hand for 15 seconds, and then place at room temperature for 2 to 3 minutes.

[0084] (3) After centrifugation at 4°C and 12,000 g for 10 min, the upper colorless aqueous phase was added to a new test tube and the volume of the upper colorless aqueous phase in the new test tube was calculated.

[0085] (4) Add a volume of pre-cooled isopropanol equal to the colorless water in the upper layer of the new test tube, cap the tube tightly, shake gently, and let stand at room temperature for 10 minutes to allow the RNA to fully precipitate.

[0086] (5) After centrifugation at 12,000 g for 10 min at 4°C, discard the supernatant, add 1 mL of 80% ethanol to each tube for rinsing, cover the tube tightly, and gently shake the centrifuge tube to remove residual isopropanol and salt.

[0087] (6) After centrifugation at 7500g for 5 min at 4°C, discard the supernatant. Open the tube cap and dry the RNA precipitate (evaporation at room temperature or vacuum drying) to obtain the dried RNA precipitate. Note that the RNA precipitate cannot be completely dried, otherwise it will be difficult to dissolve.

[0088] (7) Dissolve the dried RNA precipitate in 50 μl RNase Free ddH2O to obtain a dissolving solution.

[0089] 3.2 Reverse transcription of RNA into cDNA

[0090] The RNA extracted in Example 3.2 was reverse transcribed into cDNA using a cDNA Synthesis Kit according to the instructions of the kit.

[0091] (1) Genomic DNA removal

[0092] ① Prepare the following mixture in an RNase free centrifuge tube:

[0093] Components volume Template RNA 8μL

[0094] Incubate at 65°C for 5 min and place on ice for 2 min.

[0095]

[0096]

[0097] ③ Use a pipette to gently pipette and mix the reaction solution from the previous step, incubate at 42°C for 2 min.

[0098] (2) Reverse transcription reaction:

[0099] Prepare the following reaction mixture in a PCR tube:

[0100]

[0101] The reverse transcription reaction conditions were set as follows: 25°C for 5 min, 37°C for 45 min, and 85°C for 5 s. After cooling to 4°C, the product (i.e., cDNA) was stored in a -80°C refrigerator.

[0102] 3.3 Antibody fragments were obtained by PCR amplification using cDNA as a template. The PCR reaction system and reaction program were set as follows:

[0103] 3.3.1 PCR reaction system

[0104] Components volume cDNA template 0.5μL GreenTaqMix 12.5μL VH-Fmix(VL-F) 0.5μL VH-Rmix(VL-R) 0.5μL <![CDATA[RNasefreeddH2O]]> up to 25 μL

[0105] PCR reaction procedure

[0106]

[0107] 3.3.2TA cloning and sequencing

[0108] The antibody fragments were TA cloned and sequenced.

[0109] ① Prepare the following reaction mixture in a PCR tube:

[0110] Components volume pMD19-TVector 1μL VH / VLPCRFragment 0.1pmol~0.3pmol <![CDATA[RNasefreeddH2O]]> up to 5 μL

[0111] ②Add 5 μL of Solution I and react at 16°C overnight.

[0112] ③ Add the ligation product to 100 μL DH5α competent cells and place on ice for 30 minutes.

[0113] ④Heat at 42℃ for 45s and ice bath for 1min.

[0114] ⑤ Add 890 μL SOC medium and resuscitate at 37°C and 200 rpm for 60 min.

[0115] ⑥Spread on LB-Amp medium and culture at 37℃ overnight to form single colonies.

[0116] ⑦Pick a single colony and perform sequencing verification.

[0117] After the above steps, 17 antibody sequences were obtained.

[0118] 4. Preparation of Antibodies

[0119] (1) Hybridoma cell recovery: Take out the hybridoma cells frozen in the liquid nitrogen tank and quickly place them in a 37°C water bath to quickly heat and thaw. After the cell suspension is fully thawed, add it to Gibico DMEM medium mixed with 10% FBS. Centrifuge at 300×g for 5 minutes and discard the supernatant. Resuspend the cell pellet with fresh medium and transfer it to a 10 cm dish and culture it in a 5% CO2, 37°C constant temperature incubator.

[0120] (2) Cell expansion culture: After the cells in the 10 cm dish have grown sufficiently, the cell suspension is collected and counted. After counting, 1.2×10 6 The cells were cultured continuously. The cell status was continuously observed during the culture process.

[0121] (3) Antibody collection and purification: After 5 days of culture, the cell supernatant was collected and the antibody was purified using Protein L agarose.

[0122] (4) Solution replacement: Ultrafilter the antibody obtained in step 3 using an ultrafiltration tube with a size of 50 KD, and replace the solution with PBS buffer to obtain an antibody solution. After replacement, the antibody concentration is measured using an ultra-micro UV spectrophotometer.

[0123] (5) Antibody purity identification: SDS-PAGE electrophoresis was used to detect whether the obtained protein was an antibody and to identify its purity.

[0124] Example 2: ELISA to detect antigen-antibody binding ability

[0125] (1) Antigen coating: A35R-Variola, A35R-VTT8, A35R-CPXV and A35R-MPXV were diluted with 1× PBS to a final concentration of 1 ng / μl. Each well of a 384-well ELISA plate was coated with 25 μl of the diluted antigen solution and incubated in a 4°C refrigerator overnight.

[0126] (2) Blocking: The next day, discard the supernatant and wash three times with 100 μl PBST (1×PBS, 0.3% TWEEN-20) per well. Then, add 100 μl 5% BSA blocking solution to each well and place in a 37°C incubator for 2 h. Discard the blocking solution and wash three times with PBST solution to obtain a blocked ELISA plate.

[0127] (3) Primary antibody incubation: Discard the supernatant and dilute the antibody three-fold with PBS solution containing 0.5% BSA, with the initial concentration of the antibody being 80 μg / mL. Dilute into 11 gradients in total and add to the blocked ELISA plate. Place the ELISA plate in a 37°C incubator and incubate for 1 h;

[0128] (4) Secondary antibody incubation: discard the supernatant, wash with PBST solution 5 times, and pat dry on absorbent paper. Add 50 μL of HRP-conjugated anti-mouse IgG-Fc antibody (1:50,000 dilution in PBS solution containing 0.5% BSA) to each well of the ELISA plate. Incubate the ELISA plate in a 37°C incubator for 1 hour;

[0129] (5) Color development: After the secondary antibody incubation is completed, discard the supernatant, wash with PBST solution 5 times, and pat dry on absorbent paper. Add 50 μL of TMB color development solution to each well, let it react at room temperature for 5 minutes, and then add 25 μL of stop solution to each well. Use an ELISA reader to read the absorbance value of each well at a wavelength of 450 nm; and draw a concentration effect curve, such as Figure 2 , 3 The antibody concentration when the binding activity was 50%, i.e., the BD50 value of the antibody-antigen affinity, was calculated using GraphPad Prism 9.5 software. The experimental results are shown in Table 2.

[0130] Table 2

[0131] Antibody Name 4E7 11G12 6F10 6C11 18C3 23A7 <![CDATA[Maximum absorbance OD 450 > 1.79 1.82 1.86 2.41 1.72 3.05 BD50(μg / mL) 0.03 0.05 0.03 0.06 0.05 0.07

[0132] From Table 2, Figure 2-3 We can get:

[0133] The experimental results show that the two antibodies 4E7 and 11G12 screened out can not only bind to the A35R-MPXV protein, but also bind to its homologous proteins A35R-CPXV, A35R-VTT8, and A35R-Variola; the results are as follows Figure 3As shown, the screened 4 antibodies 6F10, 6C11, 23A7 and 18C3 can specifically recognize A35R-MPXV protein.

[0134] Example 3: Antigen-antibody affinity test and competition experiment

[0135] 1. Antibody Biotin Labeling

[0136] (1) Prepare biotin labeling reagent (Thermo EZ-Link TM NHS-PEG4-biotin labeling reagent); calculate the required volume of biotin labeling reagent (0.67 μl) according to the amount of antibody to be labeled (100 μg), add the labeling reagent to the antibody solution to be labeled, and react at room temperature in the dark for 1 hour;

[0137] (2) After the reaction is completed, add 50mM glycine solution (100μl) of the same volume as the reaction system to remove the unreacted labeling reagent and let it stand for 10 minutes. Then use a 50KDa ultrafiltration tube to ultrafilter the labeled antibody solution to remove excess labeling reagent and glycine in the solution to obtain the labeled biotinylated antibody.

[0138] 2. Biotinylated Antibody Affinity Experiment

[0139] (1) Antigen coating: A35R-MPXV protein was diluted with coating buffer to a final concentration of 1 ng / μl. Each well of a 384-well ELISA plate was coated with 25 μl of the diluted solution and incubated in a 4°C refrigerator overnight.

[0140] (2) Blocking: The next day, discard the supernatant and wash three times with 100 μl PBST in each well. Then, add 100 μl 5% BSA blocking solution to each well and place in a 37°C incubator for 2 h. Discard the blocking solution and wash three times with PBST solution.

[0141] (3) Primary antibody incubation: Discard the supernatant and dilute the biotinylated antibody three-fold with a PBS solution containing 0.5% BSA, where the initial concentration of the antibody is 10 μg / mL. Dilute into 11 gradients in total and add to the blocked ELISA plate. Place the ELISA plate in a 37°C incubator and incubate for 1 hour.

[0142] (4) Secondary antibody incubation: Discard the supernatant, wash with PBST solution 5 times, and pat dry on absorbent paper. Add 50 μL HRP-Conjugated-Streptavidin (1:5000 dilution in PBS solution containing 0.5% BSA) to each well. Place the ELISA plate in a 37°C incubator and incubate for 1 hour.

[0143] (5) Color development: After the secondary antibody incubation is completed, discard the supernatant, wash with PBST solution 5 times, and pat dry on absorbent paper. Add 50 μL of TMB color development solution to each well, let it react at room temperature for 5 minutes, and then add 25 μL of stop solution to each well. Use an ELISA reader to read the absorbance value of each well at a wavelength of 450 nm, and draw a concentration effect curve, such as Figure 4 shown.

[0144] 3. Competitive ELISA Experiment

[0145] (1) Antigen coating: A35R-MPXV protein was diluted with coating buffer to a final concentration of 1 ng / μl. Each well of a 384-well ELISA plate was coated with 25 μl of the diluted solution and incubated in a 4°C refrigerator overnight.

[0146] (2) Blocking: The next day, discard the supernatant and wash three times with 100 μl PBST in each well. Then, add 100 μl 5% BSA blocking solution to each well and place in a 37°C incubator for 2 h. Discard the blocking solution and wash three times with PBST solution.

[0147] (3) Primary antibody incubation: Based on the above biotinylated antibody affinity test results, dilute the labeled antibody to the corresponding IC 90 Biotinylated 4E7, 11G12, 6F10, 6C11, 23A7 and 18C3 and non-biotinylated 4E7, 11G12, 6F10, 6C11, 23A7 and 18C3 antibodies were paired one by one, wherein the concentration of non-biotinylated antibodies was adjusted to 10 μg / mL, 1 μg / mL and 0.1 μg / mL, and equal volumes of unlabeled antibodies and labeled antibodies were mixed and added to the blocked ELISA plate, and incubated in a 37°C incubator for 0.5 h.

[0148] (4) Secondary antibody incubation: discard the supernatant, wash with PBST solution 5 times, and pat dry on absorbent paper. Add 50 μL HRP-Conjugated-Streptavidin (1:5000 dilution in PBS solution containing 0.5% BSA) to each well. Place the ELISA plate in a 37°C incubator and incubate for 1 hour;

[0149] (5) Color development: After the secondary antibody incubation is completed, discard the supernatant, wash with PBST solution 5 times, and pat dry on absorbent paper. Add 50 μL of TMB color development solution to each well, let it stand at room temperature for 5 minutes, and then add 25 μL of stop solution to each well. Use an ELISA reader to read the absorbance value of each well at a wavelength of 450 nm, and draw a competitive ELISA bar graph as shown below. Figure 5 shown.

[0150] 4. Monoclonal Antibody Pairing ELISA Experiment

[0151] (1) Coating antibody: Based on the results of competitive ELISA, paired antibodies were selected: 4E7-6F10 and 4E7-6C11, 11G12-23A7 and 11G12-18C3. Coating buffer was used to dilute the coating antibody to a final concentration of 1 ng / μl. 100 μl of the diluted solution was coated on each well of a 96-well ELISA plate and incubated overnight at 4°C.

[0152] (2) Blocking: The next day, discard the supernatant and wash three times with 200 μl PBST per well. Then, add 200 μl 5% BSA blocking solution to each well and place in a 37°C incubator for 2 h. Discard the blocking solution, wash three times with PBST solution, and pat dry on absorbent paper.

[0153] (3) Antigen incubation: Use coating buffer to dilute A35R-MPXV protein in a gradient manner, with a starting concentration of 1.6 μg / mL, 2-fold gradient dilution, and a total of 12 concentration gradients. Each well is coated with 100 μl of antigen diluted in different gradient concentrations and incubated in a 37°C incubator for 1 hour. Discard the solution in the plate, wash 5 times with PBST solution, and pat dry on absorbent paper.

[0154] (4) Primary antibody incubation: Dilute the 6F10, 6C11, 4E7, 11G12, 18C3 and 23A7 biotinylated antibodies to a final concentration of 1 μg / mL, add 100 μl of the corresponding antibody to each well and incubate in a 37°C incubator for 1 h.

[0155] (5) Secondary antibody incubation: discard the supernatant, wash five times with PBST solution, and pat dry on absorbent paper. Add 100 μL HRP-Conjugated-Streptavidin (1:5000 dilution in PBS solution containing 0.5% BSA) to each well, and incubate the ELISA plate in a 37°C incubator for 1 h.

[0156] (6) Color development: After the secondary antibody incubation is completed, discard the supernatant, wash with PBST solution 5 times, and pat dry on absorbent paper. Add 100 μL TMB color development solution to each well, let it react at room temperature for 5 minutes, and then add 50 μL stop solution to each well.

[0157] (7) Data reading and analysis: Use an ELISA reader to read the absorbance value of each well at a wavelength of 450 nm and draw a concentration effect curve. Figure 6 .

[0158] Depend on Figure 5-6 We can get:

[0159] From the experimental results, it can be seen that the two screened antibodies 4E7 and 11G12 can not only bind to A35R-MPXV protein, but also bind to its homologous proteins A35R-CPXV, A35R-VTT8, and A35R-Variola; and the paired antibodies 4E7-6F10 and 4E7-6C11, 11G12-23A7 and 11G12-18C3 can specifically recognize A35R-MPXV protein results.

[0160] In summary, the present invention provides monkeypox virus detection antibodies and their applications. The present invention prepares high-purity A35R-MPXV protein, A35R-CPXV, A35R-VTT8, and A35R-Variola as antigens for mouse immunization, and uses ELISA to detect the mouse serum titer after immunization, and screens out mice with the highest serum titer; then myeloma cells Sp2 / 0 are fused with the spleen cells of the screened mice with the highest serum titer, and the positive hybridoma cells are screened out by ELISA for subcloning, and a hybridoma cell line capable of producing monoclonal antibodies is prepared, and the antibodies are sequenced. Subsequently, monoclonal antibodies with strong specificity, good A35R protein binding ability and easy production are prepared and screened. In the above manner, the antibodies 4E7, 6F10, 6C11, 11G12, 23A7, and 18C3 obtained by the present invention can react specifically with the membrane protein A35R on the monkeypox virus IMV particles, and can be used in the clinical diagnosis and prevention of diseases related to monkeypox virus infection.

[0161] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A monoclonal antibody for detecting A35R protein, characterized in that: The monoclonal antibody comprises at least one of antibody 4E7, antibody 11G12, antibody 6F10, antibody 6C11, antibody 18C3, and antibody 23A7; the monoclonal antibody comprises a heavy chain variable region and a light chain variable region; The complementary determining regions CDR1, CDR2, and CDR3 of the heavy chain variable region of the antibody 4E7 are SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3, respectively; the complementary determining regions CDR1, CDR2, and CDR3 of the light chain variable region of the antibody 4E7 are SEQ ID NO.4, SEQ ID NO.5, and SEQ ID NO.6, respectively; The complementary determining regions CDR1, CDR2, and CDR3 of the heavy chain variable region of the antibody 11G12 are SEQ ID NO.7, SEQ ID NO.8, and SEQ ID NO.9, respectively; the complementary determining regions CDR1, CDR2, and CDR3 of the light chain variable region of the antibody 11G12 are SEQ ID NO.10, SEQ ID NO.11, and SEQ ID NO.12, respectively; The complementary determining regions CDR1, CDR2, and CDR3 of the heavy chain variable region of the antibody 6F10 are SEQ ID NO.13, SEQ ID NO.14, and SEQ ID NO.15, respectively; the complementary determining regions CDR1, CDR2, and CDR3 of the light chain variable region of the antibody 6F10 are SEQ ID NO.16, SEQ ID NO.17, and SEQ ID NO.18, respectively; The complementary determining regions CDR1, CDR2, and CDR3 of the heavy chain variable region of the antibody 6C11 are SEQ ID NO.19, SEQ ID NO.20, and SEQ ID NO.21, respectively; the complementary determining regions CDR1, CDR2, and CDR3 of the light chain variable region of the antibody 6C11 are SEQ ID NO.22, SEQ ID NO.23, and SEQ ID NO.24, respectively; The complementary determining regions CDR1, CDR2, and CDR3 of the heavy chain variable region of the antibody 18C3 are SEQ ID NO.25, SEQ ID NO.26, and SEQ ID NO.27, respectively; the complementary determining regions CDR1, CDR2, and CDR3 of the light chain variable region of the antibody 18C3 are SEQ ID NO.28, SEQ ID NO.29, and SEQ ID NO.30, respectively; The complementary determining regions CDR1, CDR2, and CDR3 of the heavy chain variable region of the antibody 23A7 are SEQ ID NO.31, SEQ ID NO.32, and SEQ ID NO.33, respectively; the complementary determining regions CDR1, CDR2, and CDR3 of the light chain variable region of the antibody 23A7 are SEQ ID NO.34, SEQ ID NO.35, and SEQ ID NO.36, respectively.

2. The monoclonal antibody for detecting A35R protein according to claim 1, characterized in that: The framework regions FR1, FR2, FR3, and FR4 of the heavy chain variable region of the antibody 4E7 are SEQ ID NO.37, SEQ ID NO.38, SEQ ID NO.39, and SEQ ID NO.40, respectively; The framework regions FR1, FR2, FR3, and FR4 of the heavy chain variable region of the antibody 11G12 are SEQ ID NO.41, SEQ ID NO.42, SEQ ID NO.43, and SEQ ID NO.44, respectively; The framework regions FR1, FR2, FR3, and FR4 of the heavy chain variable region of the antibody 6F10 are SEQ ID NO.45, SEQ ID NO.46, SEQ ID NO.47, and SEQ ID NO.48, respectively; The framework regions FR1, FR2, FR3, and FR4 of the heavy chain variable region of the antibody 6C11 are SEQ ID NO.49, SEQ ID NO.50, SEQ ID NO.51, and SEQ ID NO.52, respectively; The framework regions FR1, FR2, FR3, and FR4 of the heavy chain variable region of the antibody 18C3 are SEQ ID NO.53, SEQ ID NO.54, SEQ ID NO.55, and SEQ ID NO.56, respectively; The framework regions FR1, FR2, FR3, and FR4 of the heavy chain variable region of the antibody 23A7 are SEQ ID NO.57, SEQ ID NO.58, SEQ ID NO.59, and SEQ ID NO.60, respectively.

3. The monoclonal antibody for detecting A35R protein according to claim 1, characterized in that: The framework regions FR1, FR2, FR3, and FR4 of the light chain variable region of the antibody 4E7 are SEQ ID NO.61, SEQ ID NO.62, SEQ ID NO.63, and SEQ ID NO.64, respectively; The framework regions FR1, FR2, FR3, and FR4 of the light chain variable region of the antibody 11G12 are SEQ ID NO.65, SEQ ID NO.66, SEQ ID NO.67, and SEQ ID NO.68, respectively; The framework regions FR1, FR2, FR3, and FR4 of the light chain variable region of the antibody 6F10 are SEQ ID NO.69, SEQ ID NO.70, SEQ ID NO.71, and SEQ ID NO.72, respectively; The framework regions FR1, FR2, FR3, and FR4 of the light chain variable region of the antibody 6C11 are SEQ ID NO.73, SEQ ID NO.74, SEQ ID NO.75, and SEQ ID NO.76, respectively; The framework regions FR1, FR2, FR3, and FR4 of the light chain variable region of the antibody 18C3 are SEQ ID NO.77, SEQ ID NO.78, SEQ ID NO.79, and SEQ ID NO.80, respectively; The framework regions FR1, FR2, FR3, and FR4 of the light chain variable region of the antibody 23A7 are SEQ ID NO.81, SEQ ID NO.82, SEQ ID NO.83, and SEQ ID NO.84, respectively.

4. The monoclonal antibody for detecting A35R protein according to claim 1, characterized in that: The sequence of the heavy chain variable region of the antibody 4E7 is SEQ ID NO.85; the sequence of the light chain variable region of the antibody 4E7 is SEQ ID NO.86; The sequence of the heavy chain variable region of the antibody 11G12 is SEQ ID NO.87; the sequence of the light chain variable region of the antibody 11G12 is SEQ ID NO.88; The sequence of the heavy chain variable region of the antibody 6F10 is SEQ ID NO.89; the sequence of the light chain variable region of the antibody 6F10 is SEQ ID NO.90; The sequence of the heavy chain variable region of the antibody 6C11 is SEQ ID NO.91; the sequence of the light chain variable region of the antibody 6C11 is SEQ ID NO.92; The sequence of the heavy chain variable region of the antibody 18C3 is SEQ ID NO.93; the sequence of the light chain variable region of the antibody 18C3 is SEQ ID NO.94; The sequence of the heavy chain variable region of the antibody 23A7 is SEQ ID NO.95; the sequence of the light chain variable region of the antibody 23A7 is SEQ ID NO.

96.

5. The use of a monoclonal antibody for detecting A35R protein, characterized in that: The monoclonal antibody according to any one of claims 1 to 4 is used in the preparation of a detection product or a prevention product.

6. The use of the monoclonal antibody for detecting A35R protein according to claim 5, characterized in that: The detection product is a product for detecting monkeypox virus.

7. The use of the monoclonal antibody for detecting A35R protein according to claim 5, characterized in that: The preventive product is a product for preventing monkeypox virus infection.

8. The use of the monoclonal antibody for detecting A35R protein according to claim 5, characterized in that: The antibody 6F10, the antibody 6C11, the antibody 18C3, and the antibody 23A7 are used in the preparation of products for detecting monkeypox virus or preventing monkeypox virus infection by specifically recognizing the monkeypox virus protein A35R-MPXV.

9. The use of the monoclonal antibody for detecting A35R protein according to claim 5, characterized in that: The antibody 4E7 and the antibody 6F10, or the antibody 4E7 and the antibody 6C11 are paired in combination and can be used in the preparation of products for detecting monkeypox virus or preventing monkeypox virus infection by specifically recognizing the monkeypox virus protein A35R-MPXV.

10. The use of the monoclonal antibody for detecting A35R protein according to claim 9, characterized in that: The antibody 11G12 and the antibody 18C3, and the antibody 11G12 and the antibody 23A7 are paired in combination and can be used in the preparation of products for detecting monkeypox virus or preventing monkeypox virus infection by specifically recognizing the monkeypox virus protein A35R-MPXV.

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