Monkeypox virus A29L monoclonal antibody, kit and application

Abstract

The present invention relates to the field of monoclonal antibodies, and specifically to monkeypox virus A29L monoclonal antibodies, kits and applications. The monkeypox virus A29L monoclonal antibody comprises a heavy chain and a light chain, the amino acid sequence of the heavy chain is shown in SEQ ID NO: 13, and the amino acid sequence of the light chain is shown in SEQ ID NO: 14; or the amino acid sequence of the heavy chain is shown in SEQ ID NO: 15, and the amino acid sequence of the light chain is shown in SEQ ID NO: 16. The present invention also provides a kit comprising a monkeypox virus A29L monoclonal antibody or a conjugate thereof, and provides the use of the monkeypox virus A29L monoclonal antibody or the kit in detecting monkeypox virus or preparing a product for detecting monkeypox virus. The monoclonal antibody in the present invention has high purity, high binding activity with monkeypox virus A29L, and good specificity; it has high sensitivity for detecting monkeypox virus A29L, a wide linear range, and a minimum detection concentration of 37.8 pg / mL; it lays a foundation for establishing an immunological detection method for monkeypox virus.

Description

Technical Field

[0001] The present invention relates to the field of monoclonal antibodies, and in particular to monkeypox virus A29L monoclonal antibodies, kits and applications. Background Art

[0002] Monkeypox is a zoonosis caused by the monkeypox virus (MPXV). Common symptoms include fever, rash, headache, muscle aches and swollen lymph nodes. The incubation period of infection is 5-21 days. The main route of infection is contact with infected people, animals or materials. In 1958, the monkeypox virus was first isolated from crab-eating macaques in Copenhagen and was named monkeypox because of its similar symptoms to other poxviruses. According to different epidemiological characteristics and clinical symptoms, the monkeypox virus is divided into two major branches, namely the Central African branch (Congo Basin branch) and the West African branch. It is generally believed that the Central African branch is more toxic, with an average mortality rate of 10.6%, while the average mortality rate of the West African branch is 3.6%. From July 2022 to May 2023, an outbreak of monkeypox caused by a variant of the West African branch of the monkeypox virus involved 116 countries around the world and was declared by the World Health Organization to constitute an "international public health emergency". Only on August 14, 2024, not long after, the monkeypox epidemic was once again declared an "emergency public health event of international concern", and the General Administration of Customs of my country immediately issued the "Announcement on Preventing the Introduction of Monkeypox Epidemics into my country". This monkeypox epidemic was caused by the Central African branch with a higher mortality rate, and the development of monkeypox virus detection technology is crucial for the effective prevention and control of the epidemic. Monkeypox virus is a large, enveloped, double-stranded DNA virus belonging to the Poxviridae, the Chordata Poxvirinae, and the Orthopoxvirus genus. Common laboratory detection methods for monkeypox mainly include virus culture and isolation identification, electron microscopy observation, PCR detection, serological detection, and monkeypox antigen detection. There are two different forms of infection of monkeypox virus, namely intracellular mature virus (IMV) and extracellular enveloped virus (EEV). IMV mediates transmission between hosts, and EEV mediates transmission within the same host. Different surface proteins are distributed on the surface of IMV and EMV. Among them, the A29L protein on the surface of IMV is a homologous protein of vaccinia virus A27, which plays multiple roles in the viral life cycle, including binding to cell surface heparin sulfate, regulating cell membrane fusion, mediating IMV transport and the formation of EEV. It is currently a hot antigen in the fields of monkeypox neutralizing antibody research and immunological detection method development. Summary of the invention

[0003] In order to solve the above technical problems, the present invention uses A29L as an antigen to immunize mice, screens two specific antibodies, and verifies their application in the detection of monkeypox virus, laying the foundation for establishing an immunological detection method for monkeypox virus.

[0004] Herein, the term "complementarity determining region (CDR)" refers to the amino acid sequence within the variable region of an antibody that confers antigen specificity and binding affinity. There are three CDRs (HCDR1, HCDR2, HCDR3) in each heavy chain variable region, and three CDRs (LCDR1, LCDR2, LCDR3) in each light chain variable region.

[0005] The present invention provides a monkeypox virus A29L monoclonal antibody, comprising:

[0006] (1) a heavy chain and a light chain, the heavy chain variable region comprising HCDR1 as set forth in SEQ ID NO:1 (positions 26-33 of the amino acid sequence set forth in SEQ ID NO:13), HCDR2 as set forth in SEQ ID NO:2 (positions 51-58 of the amino acid sequence set forth in SEQ ID NO:13), and HCDR3 as set forth in SEQ ID NO:3 (positions 97-107 of the amino acid sequence set forth in SEQ ID NO:13), and the light chain variable region comprising LCDR1 as set forth in SEQ ID NO:4 (positions 27-32 of the amino acid sequence set forth in SEQ ID NO:14), LCDR2 as set forth in SEQ ID NO:5 (positions 50-52 of the amino acid sequence set forth in SEQ ID NO:14), and LCDR3 as set forth in SEQ ID NO:6 (positions 89-97 of the amino acid sequence set forth in SEQ ID NO:14); or (2) a heavy chain and a light chain, the heavy chain variable region comprising HCDR1 as set forth in SEQ ID NO:7 (positions 26-33 of the amino acid sequence set forth in SEQ ID NO:15), HCDR2 as set forth in SEQ ID NO:8 (positions 26-33 of the amino acid sequence set forth in SEQ ID NO:15), NO: 15 (positions 51-58 of the amino acid sequence shown in SEQ ID NO: 15) and HCDR3 shown in SEQ ID NO: 9 (positions 97-103 of the amino acid sequence shown in SEQ ID NO: 15), and the light chain variable region comprises LCDR1 shown in SEQ ID NO: 10 (positions 27-31 of the amino acid sequence shown in SEQ ID NO: 16), LCDR2 shown in SEQ ID NO: 11 (positions 49-51 of the amino acid sequence shown in SEQ ID NO: 16) and LCDR3 shown in SEQ ID NO: 12 (positions 88-96 of the amino acid sequence shown in SEQ ID NO: 16).

[0007] HCDR1, HCDR2 and HCDR3 are the complementarity determining regions (CDRs) of the heavy chain, and LCDR1, LCDR2 and LCDR3 are the complementarity determining regions of the light chain; the complementarity determining regions can be defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering systems. Preferably, the complementarity determining regions of the present invention are defined according to the IMGT numbering system.

[0008] Furthermore, the amino acid sequence of the heavy chain is shown in SEQ ID NO: 13, and the amino acid sequence of the light chain is shown in SEQ ID NO: 14; or the amino acid sequence of the heavy chain is shown in SEQ ID NO: 15, and the amino acid sequence of the light chain is shown in SEQ ID NO: 16.

[0009] Furthermore, the monkeypox virus A29L monoclonal antibody also comprises a constant region, and the constant region comprises a heavy chain constant region and / or a light chain constant region.

[0010] Furthermore, the heavy chain constant region is selected from the heavy chain constant region of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE and IgD; and the light chain constant region is selected from the κ type or λ type light chain constant region.

[0011] Furthermore, the species origin of the constant region is any one of cattle, horses, pigs, sheep, goats, rats, mice, dogs, cats, rabbits, donkeys, deer, minks, chickens, ducks, geese and humans.

[0012] The present invention also provides a kit comprising the monkeypox virus A29L monoclonal antibody or a conjugate thereof.

[0013] Furthermore, the conjugate is a monkeypox virus A29L monoclonal antibody conjugated with biotin or a marker.

[0014] Furthermore, the marker is selected from at least one of fluorescent dyes, enzymes, radioactive isotopes, chemiluminescent reagents and nanoparticle markers.

[0015] Furthermore, the kit also includes a second antibody, which binds to the monkeypox virus A29L monoclonal antibody or to monkeypox virus A29L.

[0016] Furthermore, the kit also includes any one or more of a monkeypox virus A29L standard, a sample diluent, a washing solution, and a stop solution.

[0017] The present invention also provides the use of the monkeypox virus A29L monoclonal antibody or the kit in detecting monkeypox virus or preparing a product for detecting monkeypox virus.

[0018] Compared with the prior art, the beneficial effects of the present invention include at least:

[0019] 1. The monoclonal antibody of the present invention has high purity, high binding activity with monkeypox virus A29L and good specificity.

[0020] 2. The monoclonal antibody of the present invention has high sensitivity in detecting monkeypox virus A29L, a wide linear range, and a minimum detection concentration of 37.8 pg / mL. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 To detect the serum titer of immunized mice;

[0022] Figure 2 is the number corresponding to the cell;

[0023] Figure 3 is the ELISA detection value of cells;

[0024] Figure 4 The results are verified by duplicate ELISA of 1M1G3 and 1M2F7 cell supernatants;

[0025] Figure 5 The SDS-PAGE gel electrophoresis results of antibodies 1M1G3 and 1M2F7;

[0026] Figure 6 The results of indirect ELISA antibody specificity tests for antibodies 1M1G3 and 1M2F7;

[0027] Figure 7 The results of western blot detection of antigens;

[0028] Figure 8 Standard curves for 1M2F7 and 1M1G3 antigen testing. DETAILED DESCRIPTION

[0029] In order to make the purpose, technical scheme and beneficial effects of the present invention clearer, the present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments. Examples of the embodiments are shown in the accompanying drawings. It should be understood that the specific embodiments described in the following embodiments of the present invention are only exemplary descriptions of the specific embodiments of the present invention, and are intended to be used to explain the present invention, but do not constitute a limitation of the present invention.

[0030] The endpoints of ranges and any values ​​disclosed herein are not limited to the exact range or value, and these ranges or values ​​should be understood to include approximations to these ranges.

[0031] Example 1, Monoclonal Antibody Screening

[0032] 1. Mouse Immunization

[0033] Balb / c mice were selected as immunized subjects. The antigen was diluted with physiological saline, mixed thoroughly with the immune adjuvant at a volume ratio of 1:1, and the mice were immunized by intraperitoneal injection.

[0034] 2. Serum titer detection

[0035] After the final immunization, a small amount of tail blood was collected from the immunized mice, and the serum titer was determined by indirect ELISA:

[0036] (1) Antigen coating: Dilute the A29L antigen to 2 μg / mL with PBS buffer, add 100 μL per well to a 96-well ELISA plate, and incubate overnight at 4°C. Wash the coated ELISA plate three times with a plate washer, add 100 μL of blocking solution per well, incubate in a 37°C incubator for 2 h, then wash the blocked ELISA plate three times with a plate washer and dry.

[0037] (2) Incubation with primary antibody: Dilute the immune mouse serum with PBS buffer at the dilution ratio of 1:1000, 1:2000, 1:4000, 1:8000, 1:16000, 1:32000, 1:64000, 1:128000, 1:256000, 1:512000 (named as d-1 group, d-2 group...d-256 group, d-512 group, respectively). Add 100 μL of the diluted serum into the corresponding wells. PBS buffer was used as the control (ctrl group). Incubate at 37°C for 1 h. After incubation, wash the ELISA plate in a plate washer three times and then dry it.

[0038] (3) Incubation with secondary antibody: Add 100 μL of HRP-labeled goat anti-mouse secondary antibody to the corresponding wells, incubate at 37°C for 30 min, and wash the ELISA plate three times in a plate washer.

[0039] (4) Color development: Add 100 μL of the mixed color development solution to each well of a 96-well plate and incubate at 37°C for 15 min.

[0040] (5) Stop and read: Add stop solution to the 96-well plate, 100 μL per well, and then place it in an ELISA reader for reading. Set the detection wavelength to 450 nm and read the test results. An OD value greater than 2.1 times that of the negative control is considered positive. Serum titer test results Figure 1 shown.

[0041] 3. Shock immunity

[0042] A certain amount of antigen was taken and injected intraperitoneally into the above immunized mice as the last impact immunization before sorting.

[0043] 4. B cell sorting

[0044] Take the spleen of the immunized mouse, remove the surrounding connective tissue, place the spleen on the cell filter, gently press the spleen with the needle of the syringe to obtain the cell suspension, add Ficoll-Paque PLUS cell separation solution and cell suspension into the test tube at a volume ratio of 1:2, and centrifuge at 1500 rpm for 15 min. The middle layer of the test tube after centrifugation is generally the mononuclear cell layer. Aspirate the mononuclear cell layer, add SOL024 solution, centrifuge at 1500 rpm for 10 min, remove the supernatant after centrifugation, and add 500 µL SOL024 solution to resuspend the cells.

[0045] 5. Flow cytometry sorting of memory B cells (MBC)

[0046] Taking advantage of the fact that BCR on the surface of MBC can specifically bind to antigen proteins, the antigen proteins are fluorescently labeled, and the MBCs that specifically bind to the antigen proteins can be sorted out one by one from the cell suspension by flow cytometry.

[0047] 6. Construction of monoclonal antibody high-throughput expression plasmid

[0048] The single cells obtained by sorting were lysed to amplify the full length of the light chain and the Fab region fragment of the heavy chain. The amplified antibody heavy chain Fab base sequence and light chain sequence were connected to the pcDNA3.4 vector containing the mouse IgG1 Fc fragment to construct the light and heavy chain recombinant expression plasmids. The plasmids were extracted using a large extraction kit.

[0049] 7. High-throughput expression of antibodies

[0050] Cells were seeded one day before transfection to ensure that the cells grew to 3.0-5.0×10 6 Cells / mL, viability 98%, dilute the cell suspension with culture medium, and plate cells in 96 deep-well plates, 400 μL per well. Plate transfection buffer in 96-well plates, 100 μL per well, then add the plasmid to the transfection buffer, pipette and mix, then add PEI max to the buffer, pipette and mix, incubate at 37°C for 10 min, and add to the 96 deep-well plate cells for continued culture. 25 μL of feed was added to each well 20 h after transfection, and after 4-5 days of expression, centrifuge at 4000 rpm for 10 min, and collect the supernatant.

[0051] 8. ELISA detection of cell supernatant and screening of candidate clones

[0052] The cell supernatant was tested for A29L-specific antibodies by indirect ELISA.

[0053] (1) Antigen coating: dilute the antigen to 2 μg / mL with PBS buffer, add 100 μL per well to a 96-well ELISA plate, and incubate overnight at 4°C. Wash the coated ELISA plate three times with a plate washer and tap dry, add 100 μL of blocking solution per well, incubate in a 37°C incubator for 2 h, and then wash the blocked ELISA plate three times with a plate washer and tap dry.

[0054] (2) Incubation with primary antibody: Place 100 μL of cell supernatant in each well, take an equal amount of immune serum as a positive control, and PBS buffer as a negative control, and incubate at 37°C for 1 h. After incubation, wash the ELISA plate three times in a plate washer and dry.

[0055] (3) Incubation with secondary antibody: Add 100 μL of HRP-labeled goat anti-mouse secondary antibody to the corresponding wells, incubate at 37°C for 30 min, wash the plate three times in a plate washer and dry.

[0056] (4) Color development: Add 100 μL of the mixed color development solution to each well of a 96-well plate and incubate at 37°C for 15 min.

[0057] (5) Stop and read: Add stop solution to the 96-well plate, 100 μL per well, and then place it in an ELISA reader for reading. Set the detection wavelength to 450 nm and read the test results. An OD value greater than 2.1 times that of the negative control well is considered positive. The corresponding cell number is as follows: Figure 2 As shown, the ELISA test values ​​are as follows Figure 3 The supernatants of 1M1G3 and 1M2F7 cells were selected for repeated ELISA validation, and the results are shown in Figure 4 shown.

[0058] 9. ELISA detection of cell supernatant and screening of candidate clones

[0059] The light and heavy chain "signal peptide + variable region" base sequencing of the two antibodies 1M1G3 and 1M2F7 was performed. The amino acid sequences of the heavy chain variable region and light chain variable region of 1M1G3 and the amino acid sequences of the heavy chain variable region and light chain variable region of 1M2F7 are shown in Table 1.

[0060] Table 1: Amino acid sequences of antibodies 1M1G3 and 1M2F7

[0061]

[0062] Example 2, Monoclonal Antibody Expression and Specificity Detection

[0063] The antibody sequence was cloned into a eukaryotic expression vector and expressed using Expi-293F cells. The obtained antibody was subjected to SDS-PAGE gel electrophoresis. Figure 5 As shown in the figure, the antibody bands are around 25 kDa and 50 kDa, respectively, which is in line with expectations.

[0064] The A29L protein was coated and BSA was used as a control. The antibody specificity was detected by indirect ELISA. The results were as follows Figure 6 As shown, both antibodies 1M1G3 and 1M2F7 showed good specificity.

[0065] Example 3, Western Blot Detection

[0066] A 10% protein gel was prepared, and 0.5 μg and 1 μg (labeled as 0.5 and 1.0, respectively) of A29L protein were loaded for SDS-PAGE gel electrophoresis. After the electrophoresis, the membrane was transferred to the PVDF membrane at 200 mA for 35 min. After blocking with 5% skim milk at room temperature for 1 h, primary antibodies 1M1G3 and 1M2F7 were added at 1:1000, respectively, and incubated at 4°C overnight. After washing 5 times with TBST, HRP-labeled goat anti-mouse secondary antibody was added at 1:6000 and incubated at room temperature for 1 h. After washing 6 times with TBST, developer was added and developed using a chemiluminescence imaging system. The results are shown in Figure 7 As shown, both 1M1G3 antibody and 1M2F7 antibody can recognize A29L antigen well.

[0067] Example 4, ELISA antigen detection standard curve

[0068] The A29L protein standard was diluted 2-fold from 2000 pg / mL to 7 gradients of 2000, 1000, 500, 250, 125, 62.5, and 31.25. 100 μL per well was coated on the ELISA plate and incubated at 4°C overnight. After blocking, 1M2F7 and 1M1G3 antibodies were added at 2 μg / mL respectively for 1 h at room temperature. After washing 6 times with PBST, HRP-labeled goat anti-mouse secondary antibody was added at 1:4000 and incubated at room temperature for 1 h. After washing the plate, the color developing solution was added, the color development was terminated for 3 min, and the OD450 was read using an ELISA reader. A linear regression standard curve was drawn with the A29L protein concentration as the horizontal axis and the OD450 value as the vertical axis, as shown in the figure. Figure 8As shown. 1M2F7 presents a good standard curve, the linear range of detection is 0-2000 pg / mL, and the minimum detection concentration is 37.8 pg / mL. The calculation method of the minimum detection concentration is the zero-hole OD450 absorbance value plus the A29L concentration corresponding to 3 times the standard deviation. Finally, it should be noted that the above embodiments are only used to illustrate the technical scheme of the present invention and do not constitute a limitation on the content of the present invention. Within the scope of the technical concept of the present invention, the technical scheme of the present invention can be subjected to a variety of simple variations, including the combination of various technical features in any other suitable manner. These simple variations and combinations should also be regarded as the contents disclosed by the present invention and belong to the protection scope of the present invention.

Claims

1. A monkeypox virus A29L monoclonal antibody, characterized in that Include: (1) a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising HCDR1 as shown in SEQ ID NO: 1, HCDR2 as shown in SEQ ID NO: 2, and HCDR3 as shown in SEQ ID NO: 3, and the light chain variable region comprising LCDR1 as shown in SEQ ID NO: 4, LCDR2 as shown in SEQ ID NO: 5, and LCDR3 as shown in SEQ ID NO: 6; or (2) a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising HCDR1 as shown in SEQ ID NO: 7, HCDR2 as shown in SEQ ID NO: 8, and HCDR3 as shown in SEQ ID NO: 9, and the light chain variable region comprising LCDR1 as shown in SEQ ID NO: 10, LCDR2 as shown in SEQ ID NO: 11, and LCDR3 as shown in SEQ ID NO:

12.

2. The monkeypox virus A29L monoclonal antibody according to claim 1, characterized in that: The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 13, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 14; or the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 15, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

16.

3. A monkeypox virus A29L monoclonal antibody according to claim 1 or 2, characterized in that: The monkeypox virus A29L monoclonal antibody further comprises a constant region, and the constant region comprises a heavy chain constant region and / or a light chain constant region.

4. A monkeypox virus A29L monoclonal antibody according to claim 3, characterized in that: The heavy chain constant region is selected from the heavy chain constant region of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE and IgD; the light chain constant region is selected from the κ type or λ type light chain constant region.

5. A kit, characterized in that: The invention comprises: the monkeypox virus A29L monoclonal antibody or a conjugate thereof according to any one of claims 1 to 4, wherein the conjugate is a monkeypox virus A29L monoclonal antibody conjugated with biotin or a marker, and the marker is selected from at least one of a fluorescent dye, an enzyme, a radioactive isotope, a chemiluminescent agent and a nanoparticle marker.

6. A kit according to claim 5, characterized in that The kit further comprises a second antibody, which binds to the monkeypox virus A29L monoclonal antibody or to monkeypox virus A29L.

7. A kit according to claim 5, characterized in that The kit further comprises any one or more of a monkeypox virus A29L standard, a sample diluent, a washing solution and a stop solution.

8. Use of the monkeypox virus A29L monoclonal antibody according to any one of claims 1 to 4 or the kit according to any one of claims 5 to 7 in the preparation of a product for detecting monkeypox virus.

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