Monoclonal antibodies for detecting A35R protein and their applications

Abstract

This invention relates to monoclonal antibodies for detecting A35R protein and their applications. This invention screened six antibodies, including antibody 4E7, antibody 11G12, antibody 6F10, antibody 6C11, antibody 18C3, and antibody 23A7. Specifically, this invention discloses the amino acid sequences of the heavy chain variable region and the light chain variable region of the above antibodies, as well as the amino acid sequences of their complementarity-determining regions and framework regions. Antibodies 4E7 and 11G12 can bind not only to the A35R-MPXV protein but also to its homologs A35R-CPXV, A35R-VTT8, and A35R-Variola. Antibodies 6F10, 6C11, 23A7, and 18C3 can specifically recognize the A35R-MPXV protein. The monoclonal antibodies described above all exhibit high binding capacity to the A35R protein, making them suitable for the sensitive and specific detection and prevention of monkeypox virus A35R protein. They can also be used clinically for the diagnosis and treatment of monkeypox virus infection-related diseases.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to monoclonal antibodies for detecting A35R protein and their applications. Background Technology

[0002] Monkeypox virus (MPXV) is an orthopoxvirus belonging to the large double-stranded DNA virus family of the Poxviridae family. It is closely related to smallpox virus, which has been eradicated worldwide, and has replaced smallpox virus as the most serious disease caused by orthopoxvirus (Thornhill JP, Barkati S, Walmsley S, et al. Monkeypox Virus Infectionin 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) (Rothenburg S, Yang Z, Beard P, et al. Monkeypox emergency: Urgent questions and perspectives [J]. Cell, 2022, 185(18): 3279-3281.). A35R is an envelope component of intracellular enveloped viral particles (IEVs) and extracellular enveloped viral particles (EEVs), and is homologous to the vaccinia virus protein A33R. It plays a crucial role in the intercellular transfer 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. In July 2022, the Director-General of the World Health Organization declared the monkeypox outbreak a Public Health Emergency of International Concern. Since June 2023, cases of infection have also appeared in various parts of my country. Currently, the most effective method for preventing monkeypox virus infection is vaccination. Diagnostic methods include nucleic acid amplification detection, enzyme-linked immunosorbent assay (ELISA), Western blotting, immunohistochemistry, and virus isolation and culture, but each has its own advantages and disadvantages. Therefore, it is necessary to develop neutralizing antibodies against one or more epitopes of the monkeypox virus to achieve good neutralization and provide a reserve of clinical diagnostic and preventive measures. Summary of the Invention

[0004] To address the problems existing in the prior art, this invention provides monoclonal antibodies for detecting the A35R protein and their applications. This invention prepares and screens six monoclonal antibodies that are highly specific, possess good A35R protein binding ability, and are easy to produce. Through this method, the antibodies 4E7, 6F10, 6C11, 11G12, 23A7, and 18C3 obtained by this invention can specifically react with the membrane protein A35R on monkeypox virus IMV particles, and can be applied clinically for the diagnosis and prevention of monkeypox virus infection-related diseases.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0031] The beneficial effects of this invention are: this invention discloses the above-mentioned antibody sequence, and when the sequence is known, the above-mentioned antibody can be repeatedly obtained based on gene recombination technology.

[0032] A second objective of this invention is to provide an application of a monoclonal antibody for detecting the A35R protein, wherein the monoclonal antibody is used in the preparation of detection or preventative products.

[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 with monkeypox virus A35R protein.

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

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

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

[0038] The beneficial effects of this invention are: the six antibodies screened by this invention are suitable for sensitive and specific detection of monkeypox virus A35R-MPXV protein, and can be used in clinical diagnosis of monkeypox virus infection-related diseases. Attached Figure Description

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

[0040] Figure 2 This is a concentration-effect curve of the A35R binding antibody screening in Example 1 of the present invention; where (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 to the A35R-MPXV homologous protein in Example 2 of the present invention is shown; where (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 This is a concentration-effect curve of A35R binding to biotinylated antibody screening in Example 3 of the present invention; where (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 bar chart of the competitive ELISA in 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 monoclonal antibody in Example 3 of this invention has the following effect curves on the detection of A35R-MPXV concentration: (a) 4E7-6F10, (b) 4E7-6C11, (c) 11G12-18C3, and (d) 11G12-23A7. Detailed Implementation

[0045] The principles and features of this invention are described below. The examples given are for illustrative purposes only and are not intended to limit the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they should be performed according to the techniques or conditions described in the literature in this field, or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0046] Experimental materials

[0047] 1. Key drugs and reagents are shown in Table 1 below. All other reagents are domestically produced analytical grade.

[0048]

[0049] 2. Main Instruments

[0050]

[0051] Example 1: Preparation and purification of monkeypox virus monoclonal antibody

[0052] 1. Expression and purification of monkeypox virus A35R protein

[0053] (1) The MPXV A35R protein gene sequence was modified by adding a His tag sequence to the C end and then constructing it into the eukaryotic expression vector PTT5 to prepare a recombinant expression plasmid.

[0054] (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.

[0055] (3) The recombinant cells were cultured in a 5% CO2 incubator at 37°C for 5 days at 120 rpm. Then, the supernatant was obtained by centrifugation for 30 min at 4°C and 3000 rpm. Subsequently, the target protein A35R-MPXV was obtained by affinity chromatography using a Ni-NTA column.

[0056] (4) Repeat steps (1), (2), and (3) above to prepare vaccinia virus protein A35R-CPXV, smallpox virus protein A35R-Variola, and vaccinia virus Tian Tan strain protein A35R-VTT8. The results are as follows: Figure 1 As shown.

[0057] 2. Obtaining Antibodies

[0058] 2.1 Mouse Immunization and Antiserum Detection

[0059] Mice were repeatedly immunized with A35R-MPXV antigen protein, and serum titer was tested after immunization. The specific experimental steps are as follows:

[0060] (1) Five 6-week-old SPF-grade female BalB / C mice (purchased from Zhejiang Vital River Laboratory Animal Technology Co., Ltd.) weighing 25±2 g were selected as immunization animals. The mice were marked and numbered sequentially as left anterior, right anterior, left posterior, right posterior, and none. 30-50 μl of blood was collected from the tail vein of each of the five mice. After coagulation, the pre-immunization serum was collected by centrifugation.

[0061] (2) First immunization: Calculate the volume of A35R protein required based on an immunization dose of 100 μg / mouse. After emulsifying the A35R protein with an equal volume of Freund's complete adjuvant, inject it subcutaneously into multiple points in mice.

[0062] (3) Second immunization: Two weeks after the first immunization, the volume of A35R protein required was calculated based on an immunization dose of 50 μg / mouse. The A35R protein was emulsified with an equal volume of Freund's incomplete adjuvant and then injected subcutaneously into mice at multiple points.

[0063] (4) Third immunization: One week after the second immunization, calculate the required volume of A35R protein based on an immunization dose of 50 μg / animal, emulsify the antigen with an equal volume of Freund's incomplete adjuvant, and inject it subcutaneously at multiple points;

[0064] (5) ELISA detection: One week after the third immunization, 30-50 μl of blood was collected from the tail vein of 5 mice. After coagulation, the serum was collected by centrifugation after three immunizations. The antigen (i.e., A35R protein) was diluted to a final concentration of 1 μg / ml with antigen coating buffer (i.e., carbonate buffer with pH 7.4). 100 μl was coated onto the ELISA plate and incubated overnight (12-16 h) at 4°C. After blocking with 0.1% casein blocking buffer at room temperature for 1 h, the plate was washed. The mouse serum was diluted at dilutions of 1:2000, 1:4000, 1:8000, 1:16000, 1:32000, 1:64000, and 1:128000. Antibody dilution buffer (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 ELISA reader. 450 Readings. The results are shown in Table 1:

[0065] Table 1

[0066]

[0067] Table 1 shows that the serum titers of mice were qualified after three immunizations, and unlabeled mice were fused.

[0068] 2.2 Preparation and Screening of Hybridoma Cells

[0069] (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, mice were boosted with A35R-MPXV recombinant protein, 50 μg / mouse, by intraperitoneal injection.

[0070] (2) Fusion: The mice were sacrificed and the spleens were removed under sterile conditions. The spleen cells were mechanically crushed and collected. Larger impurities were removed by 200 mesh sieve. The cells were washed three times with PBS buffer and counted to obtain the mouse spleen cells to be fused. SP2 / 0 myeloma cells in the logarithmic growth phase were collected and washed three times with PBS and counted. After cell counting, SP2 / 0 myeloma cells and mouse spleen cells to be fused were gently mixed at a ratio of 1:2.5. After centrifugation, the PBS was discarded. Cell fusion was performed using an electrofusion instrument to obtain fused cells. After centrifugation, complete culture medium containing HAT was added, the fused cells were resuspended and mixed, and then plated in 96-well plates.

[0071] (3) Change the medium: Perform a complete change of the medium 5 days after fusion;

[0072] (4) ELISA detection: Coated with the detection agent (A35R-MPXV), the cell culture supernatant was aspirated 7 days after fusion for ELISA detection, and the positive cell wells were recorded (judgment criteria: three times the number of negative results), marked and the medium was changed halfway;

[0073] (5) ELISA retesting and screening: The next day, the positive cell wells after changing the medium were retested and the coating screening agents (A35R-MPXV, A35R-CPXV, A35R-Variola, A35R-VTT8) were screened. The required clone number was recorded and subcloning was performed.

[0074] 2.3 Establishment of stable cell lines

[0075] (1) First subcloning: Subcloning was performed in the positive cell wells of the above ELISA retest. The cells were limitedly diluted with complete culture medium and plated in 96-well plates at a rate of 1 cell / well. After 7 days, the cells were observed under a microscope. Wells with clonal growth were marked and registered. The registered clones were tested by ELISA. The clones that turned negative were discarded. The positive clones with vigorous growth were selected and the medium was changed once. The cells were retested and screened the next day.

[0076] (2) Secondary subcloning: The positive single clone wells selected in the previous step are screened for secondary subcloning. The cells are limitedly diluted using complete culture medium and plated in 96-well plates at a rate of 1 cell / well. After 7 days, the cells are observed under a microscope. Wells with clonal growth are detected, single clone wells are marked and registered, and ELISA is performed on the registered clones.

[0077] (3) After multiple subclonings, when the ELISA test shows 100% positivity, select vigorous single-cloning wells for expanded culture and fixation, and freeze-store the seed, thus establishing a stable hybridoma cell line.

[0078] 3. Antibody gene sequencing

[0079] 3.1 Extraction of total RNA from hybridoma cell lines

[0080] (1) Add sufficient TRIPure Reagent to the established hybridoma cell line and repeatedly pipette to obtain a homogenate.

[0081] (2) Let the above homogenate stand at room temperature for 5 min to allow the nucleic acids and proteins in the homogenate to fully dissociate. Add 0.2 mL of chloroform to each 1 mL of TRIPure Reagent reagent volume of homogenate, tighten the cap, shake vigorously by hand for 15 s, and then let stand at room temperature for 2-3 min.

[0082] (3) After centrifuging at 12000 g for 10 min at 4℃, add the upper colorless aqueous phase to a new test tube and calculate the volume of the upper colorless aqueous phase in the new test tube.

[0083] (4) Add an equal volume of pre-cooled isopropanol to the colorless water in the upper layer of the new test tube, tighten the cap, shake gently, and let stand at room temperature for 10 min until the RNA is fully precipitated.

[0084] (5) After centrifuging at 12000 g for 10 min at 4℃, discard the supernatant, add 1 mL of 80% ethanol to each tube for rinsing, tighten the cap, and gently shake the centrifuge tube to remove residual isopropanol and salt.

[0085] (6) After centrifuging at 7500 g for 5 min at 4℃, discard the supernatant. Open the tube cap and dry the RNA precipitate (evaporate at room temperature or vacuum dry) to obtain the dried RNA precipitate. Note that the RNA precipitate should not be completely dried, otherwise it will be difficult to dissolve.

[0086] (7) Dissolve the dried RNA precipitate in 50 μl RNase-free ddH2O to obtain a solution.

[0087] 3.2 RNA reverse transcription into cDNA

[0088] The RNA extracted in Example 3.2 was reverse transcribed into cDNA using the cDNA Synthesis Kit and following the kit's instructions.

[0089] (1) Genomic DNA removal

[0090] ① Prepare the following mixture in an RNase-free centrifuge tube:

[0091]

[0092] Incubate at 65℃ for 5 minutes, then in an ice bath for 2 minutes.

[0093]

[0094] ③ Gently mix the reaction solution from the previous step using a pipette, incubate at 42°C for 2 minutes.

[0095] (2) Reverse transcription reaction:

[0096] Prepare the following reaction mixture in a PCR tube:

[0097]

[0098] The reverse transcription reaction conditions were set as follows: 25℃ for 5 min, 37℃ for 45 min, and 85℃ for 5 s. After cooling to 4℃, the product (i.e., cDNA) was stored at -80℃.

[0099] 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:

[0100] 3.3.1 PCR reaction system

[0101]

[0102] PCR reaction procedure

[0103]

[0104] 3.3.2 TA Cloning and Sequencing

[0105] The antibody fragment was TA cloned and sequenced.

[0106] ① Prepare the following reaction mixture in a PCR tube:

[0107]

[0108] ② Add 5 µL of Solution I and react at 16°C overnight.

[0109] ③ The ligation product was added to 100 µL of DH5α competent cells and placed on ice for 30 min.

[0110] ④ Heat at 42℃ for 45 seconds, then ice bath for 1 minute.

[0111] ⑤ Add 890 µL of SOC medium and incubate at 37℃ and 200 rpm for 60 min to revive the culture.

[0112] ⑥ Spread the culture onto LB-Amp medium and incubate overnight at 37°C to form single colonies.

[0113] ⑦ Select a single colony and perform sequencing verification.

[0114] After the above steps, 17 antibody sequences were obtained.

[0115] 4. Preparation of antibodies

[0116] (1) Hybridoma cell thawing: The hybridoma cells frozen in liquid nitrogen were taken out and quickly placed in a 37°C water bath to thaw rapidly. After the cell suspension was fully thawed, it was added to Gibico DMEM medium mixed with 10% FBS. After centrifugation at 300×g for 5 min, the supernatant was discarded, the cell pellet was resuspended in fresh medium, and transferred to a 10cm dish. The cells were then cultured in a 5% CO2 incubator at 37°C.

[0117] (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 × 10⁶ cells / mL are inoculated into a 15 cm dish. 6 Collect the cells and continue culturing. Continuously monitor the cell status during the culturing process.

[0118] (3) Antibody collection and purification: Collect cell supernatant after 5 days of culture. Purify the culture supernatant with Protein L agarose.

[0119] (4) Solution replacement: The antibody obtained in step 3 was ultrafiltered using a 50 kDa ultrafiltration tube, and the solution was replaced with PBS buffer to obtain the antibody solution. The antibody concentration was measured using an ultra-micro UV spectrophotometer after replacement.

[0120] (5) Antibody purity identification: SDS-PAGE electrophoresis is used to detect whether the obtained protein is an antibody and to identify its purity.

[0121] Example 2: ELISA detection of antigen-antibody binding ability

[0122] (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. 25 μL of the diluted antigen solution was coated into each well of a 384-well ELISA plate and incubated overnight at 4°C.

[0123] (2) Blocking: The next day, discard the supernatant and wash each well three times with 100 μl of PBST (1×PBS, 0.3% TWEEN-20). Then, add 100 μl of 5% BSA blocking solution to each well and incubate at 37°C for 2 h. Discard the blocking solution and wash three times with PBST solution to obtain the blocked ELISA plate.

[0124] (3) Primary antibody incubation: Discard the supernatant and perform three-fold serial dilutions of the antibody with PBS solution containing 0.5% BSA, with an initial antibody concentration of 80 μg / mL. Dilute to a total of 11 gradients and add to the blocked ELISA plate. Incubate the ELISA plate in a 37°C incubator for 1 h;

[0125] (4) Secondary antibody incubation: Discard the supernatant, wash 5 times with PBST solution, and pat dry on absorbent paper. Add 50 μL of HRP-coupled anti-mouse IgG-Fc antibody (diluted 1:50000 with PBS solution containing 0.5% BSA) to each well of the ELISA plate. Incubate the ELISA plate in a 37°C incubator for 1 h;

[0126] (5) Color development: After the secondary antibody incubation, discard the supernatant, wash 5 times with PBST solution, and pat dry on absorbent paper. Add 50 μL of TMB color development solution to each well, incubate at room temperature for 5 min, and then add 25 μL of stop solution to each well. Read the absorbance value of each well at 450 nm using a microplate reader; and plot the concentration-effect curve, as shown below. Figure 2 , 3 As shown in Table 2, the antibody concentration at which binding activity is 50%, i.e., the BD50 value of antibody affinity to antigen, was calculated using GraphPad Prism 9.5 software.

[0127] Table 2

[0128]

[0129] From Table 2, Figure 2-3 We can obtain:

[0130] The experimental results show that the two selected antibodies, 4E7 and 11G12, can bind not only to the A35R-MPXV protein, but also to its homologous proteins A35R-CPXV, A35R-VTT8, and A35R-Variola; the results are as follows. Figure 3 As shown, the four selected antibodies, 6F10, 6C11, 23A7 and 18C3, can specifically recognize the A35R-MPXV protein.

[0131] Example 3: Antigen-antibody affinity test and competition experiment

[0132] 1. Antibody biotin labeling

[0133] (1) Prepare biotin labeling reagent (Thermo EZ-Link™ NHS-PEG4-Biotin labeling reagent); calculate the required volume of biotin labeling reagent (0.67 μl) based on 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 h;

[0134] (2) After the reaction is complete, add 100 μl of 50 mM glycine solution of equal volume to the reaction system to remove unreacted labeling reagents and let stand for 10 min. Then use a 50 kDa ultrafiltration tube to ultrafilter the labeled antibody solution to remove excess labeling reagents and glycine in the solution to obtain labeled biotinylated antibody.

[0135] 2. Biotinylated antibody affinity assay

[0136] (1) Antigen coating: Dilute A35R-MPXV protein with coating buffer to a final concentration of 1 ng / μl. Coat each well of a 384-well ELISA plate with 25 μl of the diluted solution and incubate overnight at 4 ℃.

[0137] (2) Blocking: The next day, discard the supernatant and wash each well three times with 100 μl of PBST. Then, add 100 μl of 5% BSA blocking solution to each well and incubate at 37 ℃ for 2 h. Discard the blocking solution and wash three times with PBST solution.

[0138] (3) Primary antibody incubation: Discard the supernatant and dilute the biotinylated antibody three-fold with PBS solution containing 0.5% BSA, with an initial antibody concentration of 10 μg / mL. Dilute to a total of 11 gradients and add to the blocked ELISA plate. Incubate the ELISA plate in a 37 ℃ incubator for 1 h.

[0139] (4) Secondary antibody incubation: Discard the supernatant, wash 5 times with PBST solution, and pat dry on absorbent paper. Add 50 μL of HRP-Conjugated-Streptavidin (1:5000 dilution in PBS solution containing 0.5% BSA) to each well. Incubate the ELISA plate in a 37 ℃ incubator for 1 h.

[0140] (5) Color development: After the secondary antibody incubation is complete, discard the supernatant, wash 5 times with PBST solution, and pat dry on absorbent paper. Add 50 μL of TMB color development solution to each well, incubate at room temperature for 5 min, and then add 25 μL of stop solution to each well. Read the absorbance value of each well at 450 nm using a microplate reader and plot the concentration-effect curve, as shown below. Figure 4 As shown.

[0141] 3. Competitive ELISA assay

[0142] (1) Antigen coating: Dilute A35R-MPXV protein with coating buffer to a final concentration of 1 ng / μl. Coat each well of a 384-well ELISA plate with 25 μl of the diluted solution and incubate overnight at 4 ℃.

[0143] (2) Blocking: The next day, discard the supernatant and wash each well three times with 100 μl of PBST. Then, add 100 μl of 5% BSA blocking solution to each well and incubate at 37 ℃ for 2 h. Discard the blocking solution and wash three times with PBST solution.

[0144] (3) Primary antibody incubation: Based on the results of the biotinylated antibody affinity experiment above, the labeled antibody was diluted to the corresponding IC50 concentration. 90 Biotinylated 4E7, 11G12, 6F10, 6C11, 23A7, and 18C3 antibodies were paired one-to-one with unbiotinylated 4E7, 11G12, 6F10, 6C11, 23A7, and 18C3 antibodies. The concentrations of the unbiotinylated antibodies were adjusted to 10 μg / mL, 1 μg / mL, and 0.1 μg / mL. An equal volume of unlabeled and labeled antibodies was mixed and added to the blocked ELISA plate, which was then incubated at 37 °C for 0.5 h.

[0145] (4) Secondary antibody incubation: Discard the supernatant, wash 5 times with PBST solution, and pat dry on absorbent paper. Add 50 μL of HRP-Conjugated-Streptavidin (1:5000 dilution in PBS solution containing 0.5% BSA) to each well. Incubate the ELISA plate in a 37 ℃ incubator for 1 h;

[0146] (5) Color development: After the secondary antibody incubation, discard the supernatant, wash 5 times with PBST solution, and pat dry on absorbent paper. Add 50 μL of TMB color development solution to each well, incubate at room temperature for 5 min, and then add 25 μL of stop solution to each well. Read the absorbance value of each well at 450 nm using a microplate reader, and plot the competitive ELISA column chart as shown below. Figure 5 As shown.

[0147] 4. Monoclonal antibody paired ELISA assay

[0148] (1) Coating antibodies: Based on the results of competitive ELISA, paired antibodies 4E7-6F10 and 4E7-6C11, and 11G12-23A7 and 11G12-18C3 were selected. The coating antibodies were diluted with coating buffer to a final concentration of 1 ng / μl. 100 μl of the diluted solution was coated into each well of a 96-well ELISA plate and incubated overnight at 4 ℃.

[0149] (2) Blocking: The next day, discard the supernatant and wash each well three times with 200 μl of PBST. Then, add 200 μl of 5% BSA blocking solution to each well and incubate at 37 ℃ for 2 h. Discard the blocking solution, wash three times with PBST solution, and pat dry on absorbent paper.

[0150] (3) Antigen incubation: A35R-MPXV protein was serially diluted with coating buffer, starting at a concentration of 1.6 μg / mL, followed by 2-fold serial dilutions, for a total of 12 concentration gradients. Each well was coated with 100 μl of antigen diluted at different gradient concentrations and incubated at 37 ℃ for 1 h. The solution in the plate was discarded, and the plate was washed 5 times with PBST solution and patted dry on absorbent paper.

[0151] (4) Primary antibody incubation: Dilute the biotinylated antibodies 6F10, 6C11, 4E7, 11G12, 18C3 and 23A7 to a final concentration of 1 μg / mL, and add 100 μl of the corresponding antibody to each well. Incubate at 37 ℃ for 1 h.

[0152] (5) Secondary antibody incubation: Discard the supernatant, wash 5 times with PBST solution, and pat dry on absorbent paper. Add 100 μL of HRP-Conjugated-Streptavidin (1:5000 dilution in PBS solution containing 0.5% BSA) to each well, and incubate the ELISA plate in a 37 ℃ incubator for 1 h.

[0153] (6) Color development: After the secondary antibody incubation is complete, discard the supernatant, wash 5 times with PBST solution, and pat dry on absorbent paper. Add 100 μL of TMB color development solution to each well, let it stand at room temperature for 5 min, and then add 50 μL of stop solution to each well.

[0154] (7) Data Reading and Analysis: Use a microplate reader to read the absorbance value of each well at a wavelength of 450 nm, and plot the concentration-effect curve as shown in the figure. Figure 6 .

[0155] Depend on Figure 5-6 We can obtain:

[0156] The experimental results show that the two selected antibodies, 4E7 and 11G12, can bind not only to the A35R-MPXV protein, but also to its homologous proteins A35R-CPXV, A35R-VTT8, and A35R-Variola; while the paired antibodies 4E7-6F10 and 4E7-6C11, and 11G12-23A7 and 11G12-18C3 can specifically recognize the A35R-MPXV protein.

[0157] In summary, this invention provides monkeypox virus detection antibodies and their applications. This invention uses high-purity A35R-MPXV protein, A35R-CPXV, A35R-VTT8, and A35R-Variola as antigens to immunize mice. After immunization, the serum titers of the mice are detected using ELISA, and mice with the highest serum titers are selected. Myeloma cells Sp2 / 0 are then fused with spleen cells from the selected mice with the highest serum titers. Positive hybridoma cells are screened using ELISA for subcloning, preparing hybridoma cell lines capable of producing monoclonal antibodies. The antibodies are then sequenced. Subsequently, monoclonal antibodies with high specificity, good A35R protein binding ability, and ease of production are prepared and screened. Through the above methods, the antibodies 4E7, 6F10, 6C11, 11G12, 23A7, and 18C3 obtained by this invention can specifically react with the membrane protein A35R on monkeypox virus IMV particles and can be applied clinically for the diagnosis and prevention of monkeypox virus infection-related diseases.

[0158] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to 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 is antibody 4E7; the monoclonal antibody contains a heavy chain variable region and a light chain variable region; The complement determination regions CDR1, CDR2, and CDR3 of the heavy chain variable region of antibody 4E7 are SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3, respectively; the complement determination regions CDR1, CDR2, and CDR3 of the light chain variable region of antibody 4E7 are SEQ ID NO.4, SEQ ID NO.5, and SEQ ID NO.6, respectively.

2. The monoclonal antibody for detecting A35R protein according to claim 1, characterized in that, The frame regions FR1, FR2, FR3, and FR4 of the heavy chain variable region of antibody 4E7 are, in order, SEQ ID NO.37, SEQ ID NO.38, SEQ ID NO.39, and SEQ ID NO.

40.

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 antibody 4E7 are, in order, SEQ ID NO.61, SEQ ID NO.62, SEQ ID NO.63, and SEQ ID NO.

64.

4. The monoclonal antibody for detecting A35R protein according to claim 1, characterized in that, The sequence of the heavy chain variable region of antibody 4E7 is SEQ ID NO.85; the sequence of the light chain variable region of antibody 4E7 is SEQ ID NO.

86.

5. The application of monoclonal antibodies for detecting A35R protein, characterized in that, The monoclonal antibody as described in any one of claims 1-4 is used in the preparation of a detection product; the detection product is a product for detecting monkeypox virus.

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