Monkeypox virus detection antibodies and uses thereof

Abstract

This invention relates to antibodies for detecting monkeypox virus and their applications, belonging to the field of biotechnology. This invention screened three antibodies, including antibody 18F7, antibody 27C3, and antibody 27F1; all antibodies contain a heavy chain variable region and a light chain variable region. The heavy chain variable region of antibody 18F7 contains amino acid sequences as shown in SEQ ID NO. 1-3, and the light chain variable region contains amino acid sequences as shown in SEQ ID NO. 4-6; the heavy chain variable region of antibody 27C3 contains amino acid sequences as shown in SEQ ID NO. 7-8 and VH-CDR3:WDY, and the light chain variable region contains amino acid sequences as shown in SEQ ID NO. 9-11; the heavy chain variable region of antibody 27F1 contains amino acid sequences as shown in SEQ ID NO. 12-14, and the light chain variable region contains amino acid sequences as shown in SEQ ID NO. 15-17. Antibody 18F7 can bind not only to the A29L protein but also to its homologs A28L, 192, and A30L. Antibodies 27C3 and 27F1 specifically recognize the A29L protein. All three antibodies exhibit high binding affinity to the A29L protein, making them suitable for sensitive and specific detection of monkeypox virus A29L protein and applicable for the clinical diagnosis of monkeypox virus infection-related diseases.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to antibodies for detecting monkeypox virus and their applications. Background Technology

[0002] Monkeypox virus (MPV) is a type of enveloped double-stranded DNA virus belonging to the genus Orthopoxvirus in the family Poxviridae. It exists in two distinct forms: intracellular mature virus (IMV) and extracellular enveloped virus (EEV). Since the first reported case of MPV infection in 1970, over 30,000 cases have been reported in West and Central Africa. In recent years, the number of confirmed MPV cases has been gradually increasing, and MPV has become a significant threat to public health.

[0003] Currently, commonly used detection methods for monkeypox virus include virus isolation and identification, molecular diagnostics, and immunological methods. Immunological methods include detecting antibody levels in serum and direct detection of antigens. Due to the homology within the orthopoxvirus genus, most antigen-antibody tests can only identify down to the genus level and cannot further differentiate at a more precise level, thus failing to achieve specific detection of monkeypox virus.

[0004] A29L is a membrane protein on the monkeypox virus IMV particle, playing a crucial role in the invasion of host cells and exhibiting good immunogenicity. Although A29L is highly homologous to A28L of vaccinia virus, A30L of smallpox virus, and 192 protein of vaccinia virus, there are still differential sites in its amino acid sequence, which can induce the production of specific antibodies, thereby enabling the specific detection of monkeypox virus. Summary of the Invention

[0005] To address the aforementioned technical problems, the purpose of this invention is to provide a monkeypox virus detection antibody and its application. This invention provides an antibody against monkeypox virus protein to achieve specific diagnosis of monkeypox virus, helping patients and medical personnel to confirm monkeypox infection more quickly.

[0006] The technical solution of the present invention to solve the above-mentioned technical problems is as follows:

[0007] The first objective of this invention is to provide a monkeypox virus detection antibody selected from antibody 18F7, antibody 27C3, or antibody 27F1;

[0008] The antibody 18F7, the antibody 27C3, or the antibody 27F1 all include a heavy chain variable region;

[0009] The antibody 18F7, the antibody 27C3, or the antibody 27F1 all include a light chain variable region;

[0010] The three complementarity-determining regions in the heavy chain variable region of antibody 18F7 are as follows: the amino acid sequence of VH-CDR1 is shown in SEQ ID NO.1; the amino acid sequence of VH-CDR2 is shown in SEQ ID NO.2; and the amino acid sequence of VH-CDR3 is shown in SEQ ID NO.3.

[0011] The three complementarity-determining regions in the light chain variable region of antibody 18F7 are as follows: the amino acid sequence of VL-CDR1 is shown in SEQ ID NO.4; the amino acid sequence of VL-CDR2 is shown in SEQ ID NO.5; and the amino acid sequence of VL-CDR3 is shown in SEQ ID NO.6.

[0012] The three complementarity-determining regions in the heavy chain variable region of antibody 27C3 are as follows: the amino acid sequence of VH-CDR1 is shown in SEQ ID NO.7; the amino acid sequence of VH-CDR2 is shown in SEQ ID NO.8; the amino acid sequence of VH-CDR3 is as follows: VH-CDR3: WDY;

[0013] The three complementarity-determining regions in the light chain variable region of antibody 27C3 are as follows: the amino acid sequence of VL-CDR1 is shown in SEQ ID NO.9; the amino acid sequence of VL-CDR2 is shown in SEQ ID NO.10; and the amino acid sequence of VL-CDR3 is shown in SEQ ID NO.11.

[0014] The three complementarity-determining regions in the heavy chain variable region of antibody 27F1 are as follows: the amino acid sequence of VH-CDR1 is shown in SEQ ID NO.12; the amino acid sequence of VH-CDR2 is shown in SEQ ID NO.13; and the amino acid sequence of VH-CDR3 is shown in SEQ ID NO.14.

[0015] The three complementarity-determining regions in the light chain variable region of antibody 27F1 are as follows: the amino acid sequence of VL-CDR1 is shown in SEQ ID NO.15; the amino acid sequence of VL-CDR2 is shown in SEQ ID NO.16; and the amino acid sequence of VL-CDR3 is shown in SEQ ID NO.17.

[0016] Based on the above technical solution, the present invention can be further improved as follows.

[0017] Furthermore, the heavy chain variable region of antibody 18F7, antibody 27C3, or antibody 27F1 includes the frame region FR region;

[0018] The four frame regions in the heavy chain variable region of the antibody 18F7 are as follows: the amino acid sequence of VH-FR1 is shown in SEQ ID NO. 18; the amino acid sequence of VH-FR2 is shown in SEQ ID NO. 19; the amino acid sequence of VH-FR3 is shown in SEQ ID NO. 20; and the amino acid sequence of VH-FR4 is shown in SEQ ID NO. 21.

[0019] The four frame regions in the heavy chain variable region of antibody 27C3 are as follows: the amino acid sequence of VH-FR1 is shown in SEQ ID NO. 22; the amino acid sequence of VH-FR2 is shown in SEQ ID NO. 23; the amino acid sequence of VH-FR3 is shown in SEQ ID NO. 24; and the amino acid sequence of VH-FR4 is shown in SEQ ID NO. 25.

[0020] The four frame regions in the heavy chain variable region of antibody 27F1 are as follows: the amino acid sequence of VH-FR1 is shown in SEQ ID NO. 26; the amino acid sequence of VH-FR2 is shown in SEQ ID NO. 27; the amino acid sequence of VH-FR3 is shown in SEQ ID NO. 28; and the amino acid sequence of VH-FR4 is shown in SEQ ID NO. 29.

[0021] Furthermore, the light chain variable region of antibody 18F7, antibody 27C3, or antibody 27F1 includes the frame region FR region;

[0022] The four framework regions in the light chain variable region of antibody 18F7 are as follows: the amino acid sequence of VL-FR1 is shown in SEQ ID NO. 30; the amino acid sequence of VL-FR2 is shown in SEQ ID NO. 31; the amino acid sequence of VL-FR3 is shown in SEQ ID NO. 32; and the amino acid sequence of VL-FR4 is shown in SEQ ID NO. 33.

[0023] The four framework regions in the light chain variable region of antibody 27C3 are as follows: the amino acid sequence of VL-FR1 is shown in SEQ ID NO. 34; the amino acid sequence of VL-FR2 is shown in SEQ ID NO. 35; the amino acid sequence of VL-FR3 is shown in SEQ ID NO. 36; and the amino acid sequence of VL-FR4 is shown in SEQ ID NO. 37.

[0024] The four frame regions in the light chain variable region of antibody 27F1 are as follows: the amino acid sequence of VL-FR1 is shown in SEQ ID NO. 38; the amino acid sequence of VL-FR2 is shown in SEQ ID NO. 39; the amino acid sequence of VL-FR3 is shown in SEQ ID NO. 40; and the amino acid sequence of VL-FR4 is shown in SEQ ID NO. 41.

[0025] Furthermore, the amino acid sequence of the heavy chain variable region of the antibody 18F7 is shown in SEQ ID NO.42, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.43;

[0026] The amino acid sequence of the heavy chain variable region of antibody 27C3 is shown in SEQ ID NO.44, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.45.

[0027] The amino acid sequence of the heavy chain variable region of antibody 27F1 is shown in SEQ ID NO.46, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.47.

[0028] The second objective of this invention is to provide an application of monkeypox virus detection antibodies, specifically applying monkeypox virus detection antibodies 18F7, 27C3, or 27F1 to the preparation of products for detecting orpoxvirus in samples.

[0029] Furthermore, the product includes reagents, test plates, or kits.

[0030] Furthermore, the poxvirus is a monkeypox virus.

[0031] Furthermore, the combination of antibody 18F7 and antibody 27C3 can be used to specifically recognize monkeypox virus and thus be applied in the preparation of products for detecting monkeypox virus.

[0032] Furthermore, the combination of antibody 18F7 and antibody 27F1, after pairing, specifically recognizes the A29L protein and can be used in the preparation of products for detecting monkeypox virus.

[0033] Furthermore, antibodies 27C3 and 27F1 specifically recognize the A29L protein and can be used in the preparation of products for detecting monkeypox virus.

[0034] The beneficial effects of this invention are:

[0035] The three antibodies screened in this invention include the FR region and CDR region. Antibody 18F7 can bind not only to the A29L protein but also to its homologs A29L, A28L, 192, and A30L. Antibodies 27C3 and 27F1 can specifically recognize the A29L protein. These three antibodies are suitable for the sensitive and specific detection of monkeypox virus A29L protein and can be used clinically to diagnose monkeypox virus infection-related diseases. Attached Figure Description

[0036] 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;

[0037] Figure 2 This is a graph showing the antibody expression and purification results of Example 1 of the present invention; in the graph, Maker is a molecular weight indicator band, R is denatured protein-like protein, and N is non-denatured protein-like protein;

[0038] Figure 3 This is a concentration-effect curve of the A29L binding antibody screening in Example 2 of the present invention; where (a) is antibody 18F7; (b) is antibody 27C3; and (c) is antibody 27F1.

[0039] Figure 4 The graphs show the binding curves of antibodies to A29L homologs 192, A28L, and A30L in Example 2 of this invention; where (a) is antibody 18F7; (b) is antibody 27C3; and (c) is antibody 27F1. Detailed Implementation

[0040] 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.

[0041] Example 1: Preparation of Antibody

[0042] Experimental reagents: PEI transfection reagent (purchased from MCE); Expi293 cells (purchased from Thermo Scientific); Freund's complete adjuvant (purchased from Sigma); Freund's incomplete adjuvant (purchased from Sigma); DMEM high-glucose medium (purchased from Thermo Scientific); Protein L agarose (purchased from MCE); mouse myeloma cells SP2 / 0 (purchased from ATCC); Trizol reagent (purchased from Merck).

[0043] Experimental instruments: CO2 incubator (3111; Thermo); ELISA reader (Multiskan FC; Thermo Fisher Scientific (Shanghai) Instruments Co., Ltd.); electrofusion apparatus (ECFG21; NEPA GENE); Nanodrop spectrophotometer (Nanodrop 2000; Thermo); ultrafiltration tubes (10 kDa; Millipore); inverted biological microscope (MI11; Mshot).

[0044] (1) Acquisition of antigens

[0045] ① Construction of recombinant plasmid: The MPXV A29L protein and the C-terminal 6×His tag were constructed into the vector pCAGGS to form a recombinant expression plasmid.

[0046] ② Gently mix the recombinant expression plasmid with the transfection reagent PEI, incubate at room temperature for 15 min, and transfect Expi293 cells to obtain recombinant cells for protein expression.

[0047] The recombinant cells were cultured in an 8% CO2, 37°C incubator at 120 rpm for 5 days, and then centrifuged at 4°C at 3000 rpm for 30 min. The supernatant was collected and purified by affinity chromatography using Ni-NTA agarose gel to obtain the target protein A29L.

[0048] ③ Using the above steps, the A28L protein of vaccinia virus, the A30L protein of smallpox virus, and the 192 protein of vaccinia virus Tiantan strain were prepared, such as... Figure 1 As shown.

[0049] (2) Obtaining antibodies

[0050] a. Mouse immunization and antiserum detection

[0051] ① Five 6-week-old, 25±2 g, female SPF-grade BalB / C mice were selected. The mice were labeled with picric acid and numbered (none, left anterior, left posterior, right posterior, right anterior). 30-50 μL of blood was collected from each mouse via the tail vein. After coagulation, the pre-immune serum was collected by centrifugation.

[0052] ② First immunization: Calculate the volume of A29L antigen based on an immunization dose of 100μg per mouse, emulsify the antigen with an equal volume of Freund's complete adjuvant, and immunize by multiple subcutaneous injections;

[0053] ③ Second immunization: Two weeks later, a second immunization was performed. The volume of A29L antigen was calculated based on an immunization dose of 50 μg per mouse. An equal volume of Freund's incomplete adjuvant was used to emulsify the antigen, and multiple subcutaneous injections were administered for immunization.

[0054] ④ Third immunization: One week later, a third immunization was performed. The volume of A29L antigen was calculated based on an immunization dose of 50 μg per mouse. An equal volume of Freund's incomplete adjuvant was used to emulsify the antigen, and multiple subcutaneous injections were administered for immunization.

[0055] ⑤ Fourth immunization: One week later, a fourth immunization was performed. The volume of A29L antigen was calculated based on an immunization dose of 50 μg per mouse. An equal volume of Freund's incomplete adjuvant was used to emulsify the antigen, and multiple subcutaneous injections were administered for immunization.

[0056] ⑥ Serum collection: One week after the fourth immunization, 30-50 μL of blood was collected from the tail vein, and the serum was collected by centrifugation after coagulation.

[0057] ⑦ ELISA detection of serum titer: Immunogen and selection antigen (A29L recombinant protein) were coated separately with CBS buffer to a final concentration of 1 μg / mL, 100 μL / well. After incubation at 4°C overnight, the plates were blocked with 0.1% casein blocking buffer. After blocking at room temperature for 1 h, the plates were washed, and mouse serum was serially diluted to an initial dilution of 1:2000. Antibody dilution buffer (1×PBS + 0.5% casein) served as a negative control. OD450 readings were measured using a microplate reader. The results are shown in Table 1.

[0058] Table 1: OD450 readings of mouse serum titers after four immunizations

[0059]

[0060] b. Hybridoma cell preparation and screening

[0061] ①One week before fusion, prepare mouse myeloma cells SP2 / 0, adjust the cell state to the logarithmic growth phase, select mice with the highest ELISA titer in a, and immunize the mice with A29L recombinant protein 3 days before fusion, 50μg / mouse, intraperitoneal injection.

[0062] ② Fusion: Mouse spleens were harvested, mechanically disrupted, and spleen cells were collected. The cells were filtered through a 200-mesh sieve and washed three times with PBS. SP2 / 0 cells were collected, washed three times with PBS, and cell counts were performed. After cell counts, the cells were mixed at a ratio of SP2 / 0:spleen cells = 1:2.5. After centrifugation, the PBS was discarded, and cell fusion was performed using an electrofusion apparatus. After fusion, the cells were centrifuged and added to complete culture medium containing HAT. The cells were resuspended, mixed, and plated into 96-well plates.

[0063] ③ Change the medium: Perform a complete change of the medium 5 days after fusion.

[0064] ④ ELISA detection: Coat the detection source (A29L protein), collect the cell culture supernatant 7 days after fusion for ELISA detection, record the positive wells, mark them and perform half medium replacement;

[0065] ⑤ ELISA retesting and screening: The next day, the positive wells after the medium change are retested and the coated screening materials are screened. The clone number is recorded and subcloning is performed.

[0066] c. Establishment of stable cell lines

[0067] ① First subcloning: Select positive cells from the previous step and use the limiting dilution method with complete culture medium for subcloning screening. Plate the cells into the aforementioned 96-well plate at a density of 1 cell / well and an inoculation volume of 150 μL / well. Then, incubate at 37°C in a 5% CO2 incubator.

[0068] ② After 7 days, observe under an inverted biological microscope, mark the single clone wells, perform ELISA detection, discard the clones that turn negative, and for positive clones, select the wells with vigorous growth and perform a half-change of medium, and re-examine and screen the next day;

[0069] ③ Secondary subcloning: Select positive clones from the previous step, perform secondary subcloning screening using the limiting dilution method with complete culture medium, plate them into 96-well plates, with an inoculation density of 1 cell / well and an inoculation volume of 150 μL / well, and then incubate them in a 37°C, 5% CO2 incubator.

[0070] ④ After 7 days, observe under a biological inverted microscope, mark and register the single clone wells, perform ELISA detection on the registered clones, and select the wells with vigorous growth for one half-change of medium, and re-examine and screen the next day.

[0071] After multiple subclonings until the ELISA test showed a 100% positive rate, wells with vigorous growth were selected for expanded culture to obtain hybridoma cell lines.

[0072] (3) Antibody gene sequencing

[0073] Experimental reagents: cDNA Synthesis Kit (R312-02; Nanjing Novizan Biotechnology); TRIPURE Reagent (15596026; Life); Green Taq Mix (P131-01; Nanjing Novizan Biotechnology); pMD™19-T VectorCloning Kit (6013; Takara); GelStain (GS101-01; Beijing TransGen Biotechnology); Agarose (91622; Biowest (Company); all other reagents are domestically produced analytical grade.

[0074] Experimental apparatus: pipettes (Eppendorf); centrifuge tubes (311-01-051; Axygen); pipette tips (Axygen);

[0075] (1) Extraction of total RNA from hybridoma cell lines

[0076] ① Tissue homogenization: Add sufficient TRIPure Reagent to the cell sample and repeatedly pipette to lyse the cells to obtain a homogenate.

[0077] ② Let the above homogenate stand at room temperature for 5 minutes to allow the nucleic acids and proteins to fully dissociate. Add 0.2 mL of chloroform to each 1 mL of TRIPure Reagent reagent volume of homogenate, shake vigorously manually for 15 seconds, and then let stand at room temperature for 2–3 minutes.

[0078] ③ After centrifuging at 4℃ and 12000 g for 10 min, carefully aspirate the upper aqueous phase (colorless) into a new test tube and calculate the volume of the aspirated aqueous phase. Add an equal volume of pre-cooled isopropanol to the aspirated aqueous phase, tighten the cap, and gently shake to mix.

[0079] ④ Let stand at room temperature for 10 min to allow the RNA to fully precipitate. Centrifuge at 12000 g for 10 min at 4℃, discard the supernatant, add 1 mL of 80% ethanol to each tube to rinse, tighten the cap, and gently shake the centrifuge tube to remove residual isopropanol and salt.

[0080] ⑤ Centrifuge at 7500 g for 5 min at 4℃, discard the supernatant, open the tube cap, dry the RNA precipitate, and dissolve it in an appropriate amount of RNase-free ddH2O. Take 1 μL and perform electrophoresis on a 1% agarose gel, stain with GelStain, and then perform the detection.

[0081] (2) RNA was reverse transcribed into cDNA using the cDNA Synthesis Kit.

[0082] ① Residual genomic DNA removal: Prepare the following mixture in an RNase-free centrifuge tube:

[0083]

[0084] Incubate at 65℃ for 5 minutes, then in an ice bath for 2 minutes.

[0085]

[0086] Gently mix the reaction solution from the previous step using a pipette, incubate at 42°C for 2 minutes.

[0087] ② Reverse transcription reaction: Prepare the following reaction mixture in a PCR tube as shown below:

[0088]

[0089] The reverse transcription reaction conditions were set as follows: 25℃ for 5 min, 37℃ for 45 min, and 85℃ for 5 s. After reverse transcription, the product was cooled to 4℃ and stored at -80℃.

[0090] (3) Antibody fragments were obtained by PCR amplification using cDNA as a template. The PCR reaction system and reaction program were set as follows:

[0091] ①PCR reaction system:

[0092]

[0093] ②PCR reaction procedure:

[0094]

[0095] (4) Perform TA cloning and sequencing on the antibody fragment.

[0096] ① Prepare the following reaction mixture in a PCR tube:

[0097]

[0098] ② Add 5µL of Solution I and react at 16℃ overnight.

[0099] ③ The ligation product was added to 100 µL of DH5α competent cells and placed on ice for 30 min.

[0100] ④ Heat at 42℃ for 45 seconds, then ice bath for 1 minute.

[0101] ⑤ Add 890µL of SOC medium and incubate at 37℃ and 200 rpm for 60 min to revive the culture.

[0102] ⑥ Spread the culture onto LB-Amp medium and incubate overnight at 37°C to form single colonies.

[0103] ⑦ Select a single colony and perform sequencing verification.

[0104] After the above steps, the final antibody sequence is obtained.

[0105] All antibodies contain both heavy chain variable regions and light chain variable regions; the heavy chain variable region of antibody 18F7 contains amino acid sequences as shown in SEQ ID NO. 1-3, and the light chain variable region contains amino acid sequences as shown in SEQ ID NO. 4-6; the heavy chain variable region of antibody 27C3 contains amino acid sequences as shown in SEQ ID NO. 7-9, and the light chain variable region contains amino acid sequences as shown in SEQ ID NO. 10-12; the heavy chain variable region of antibody 27F1 contains amino acid sequences as shown in SEQ ID NO. 13-15, and the light chain variable region contains amino acid sequences as shown in SEQ ID NO. 16-18.

[0106] (4) Antibody preparation

[0107] ① Hybridoma cell thawing: Hybridoma cells frozen in liquid nitrogen were removed and rapidly thawed in a 37°C water bath. Once fully thawed, the cells were added to DMEM medium mixed with 10% FBS. After centrifugation at 300g for 5 minutes, the supernatant was discarded, and the cell pellet was resuspended in fresh medium. The pellet was then transferred to a 10cm culture dish and cultured in a 5% CO2 incubator at 37°C.

[0108] ② Cell expansion culture: After the cells in the 10cm culture dish have grown sufficiently, collect the cell suspension and count them. After counting, inoculate 1.2×10⁶ cells into a 15cm culture dish. 6 Each cell was cultured and its condition was continuously monitored during the culture process.

[0109] ③ Antibody collection and purification: Collect cell supernatant after 5 days of culture. Purify the antibody from the culture supernatant using Protein L agarose.

[0110] ④ Solution replacement: The antibody purified 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 then measured using a Nanodrop spectrophotometer.

[0111] ⑤ Antibody purity identification: The purity of the obtained protein is determined by detecting whether it is an antibody and by SDS-PAGE electrophoresis.

[0112] The results are as follows Figure 2 As shown:

[0113] Monkeypox virus monoclonal antibodies can be obtained through the above method.

[0114] To test the performance of the antibody prepared in this embodiment, the following experiment was conducted.

[0115] Example 2: Antigen-antibody affinity test and competition experiment

[0116] Experimental reagents: HRP-coupled anti-mouse IgG-Fc antibody (purchased from Sinopharm); TMB chromogenic solution (purchased from Beyotime); HRP-coupled streptavidin (purchased from Genscript).

[0117] Experimental equipment: CO2 incubator (150i; Thermo); microplate reader (EnSight; Perkin Elmer); ELISA plate (Immuno 384-well plate; Thermo)

[0118] (1) Antibody screening

[0119] ① Antigen coating: The A29L protein and its homologs 192, A28L and A30L prepared in Example 1 were diluted with 1×PBS to a final concentration of 1 ng / μL. 25 μL of the diluted protein solution was coated into each well of a 384-well ELISA plate and incubated overnight at 4°C.

[0120] ② Blocking: The next day, discard the supernatant and wash each well once with 100 μL of PBST (PBS, 0.1% Tween-20) solution. Then, add 100 μL of 5% BSA blocking solution to each well, incubate at 37°C for 2 hours, discard the blocking solution, and wash once with PBST solution to obtain the blocked ELISA plate.

[0121] ③ Add primary antibody: Discard the supernatant and dilute the antibody prepared in Example 1 with PBS solution containing 0.5% BSA in a gradient. The initial concentration is 60 μg / mL, and there are 11 gradients of three-fold dilution. Add the antibody to the blocked ELISA plate and incubate the ELISA plate in a 37°C incubator for 1 hour.

[0122] ④ Add secondary antibody: 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, and incubate the ELISA plate in a 37°C incubator for 1 h.

[0123] 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 10 min, and then add 25 μL of stop solution to each well.

[0124] ⑥ Read the absorbance value of each well at 450 nm wavelength using a microplate reader. The results are as follows: Figure 3-4 As shown in Table 3, the antibody concentration at which the A29L binding activity is 50% was calculated using GraphPadPrism 8 software, which is the BD50 value of the antibody affinity for the antigen.

[0125] Table 3: BD50 and maximum absorbance values ​​of A29L antibody binding to A29L.

[0126]

[0127] The results are as follows Figure 3-4 As shown in Table 3:

[0128] From 17 test antibodies, three antibodies with strong binding ability to A29L protein were selected based on a BD50 of less than 0.1 μg / mL. Of these three antibodies, 18F7 not only binds to A29L protein but also to its homologs A29L, A28L, 192, and A30L. The other two antibodies, 27C3 and 27F1, specifically recognize A29L protein.

[0129] (2) Antibody biotin labeling

[0130] ① Take 200 μL of each antibody strain and add it to a 1.5 ml EP tube.

[0131] ② Add 2 μL of biotin-labeled reagent (purchased from Thermo) to each tube, mix well, and react on ice in the dark for 1 h.

[0132] ③ After the reaction is complete, add 200 μl of 50 mM glycine solution to each tube and react for 10 min.

[0133] ④ After the reaction is complete, add the solution to a 50 kDa ultrafiltration tube, centrifuge at 12,000 g for 10 min, add 1×PBS after centrifugation, mix well and continue centrifugation, repeat 3 times.

[0134] ⑤ After centrifugation, the labeled antibody is precipitated and stored at 4°C.

[0135] (3) Sandwich ELISA to verify antibody pairing for A29L protein detection:

[0136] ① Antibody coating: Dilute 18F7, 27C3, and 27F1 antibodies to 1 ng / μL with coating buffer, coat 100 μL in each well of a 96-well ELISA plate, and incubate overnight at 4°C.

[0137] ② Blocking: The next day, discard the supernatant and wash each well once with 200 μL of PBST solution. Then, add 200 μL of 5% BSA blocking solution to each well, incubate at 37°C for 2 hours, discard the blocking solution, and wash once with PBST solution to obtain the blocked ELISA plate.

[0138] ③ Add antigen: Discard the supernatant and serially dilute the A29L antigen with PBS solution containing 0.5% BSA. The initial concentration is 0.2 ng / μL. Dilute it twice for a total of 7 gradients. Add 100 μL to each well of the blocked ELISA plate and incubate the ELISA plate in a 37°C incubator for 1 h.

[0139] ④ Add paired antibodies: Discard the supernatant, wash 5 times with PBST solution, and pat dry on absorbent paper. Add 100 μL of biotin-labeled 18F7, 27C3, and 27F1 antibodies to each well of the ELISA plate, diluted with PBS solution containing 0.5% BSA to a final concentration of 1 ng / μL. Then incubate the ELISA plate in a 37°C incubator for 1 h.

[0140] ⑤ Add secondary antibody: Discard the supernatant, wash 5 times with PBST solution, and pat dry on absorbent paper. Add 100 μL of HRP-coupled streptavidin diluted 1:5000 with PBS solution containing 0.5% BSA to each well of the ELISA plate, and incubate the ELISA plate in a 37°C incubator for 1 h.

[0141] 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, incubate at room temperature for 10 min, and then add 50 μL of stop solution to each well.

[0142] The absorbance values ​​at 450 nm wavelength for each well were read using a microplate reader, and the results are as follows: Figure 5 As shown.

[0143] Depend on Figure 5 We can obtain:

[0144] Antibody 18F7 can be combined with antibodies 27C3 and 27F1 to sensitively and specifically detect A29L protein.

[0145] In summary, this invention provides antibodies for detecting monkeypox virus and their applications. This invention involves preparing high-purity A29L, A28L, A30L, and 192 proteins as antigens for mouse immunization. After immunization, the serum titer of the mice is detected using ELISA, and mice with the highest serum titer are selected. Myeloma cells Sp2 / 0 are then fused with spleen cells from the selected mice with the highest serum titer. Positive hybridoma cells are screened using ELISA for subcloning, resulting in a hybridoma cell line capable of producing monoclonal antibodies. The antibodies are then sequenced. Subsequently, monoclonal antibodies with high specificity, good A29L protein binding ability, and ease of production are prepared and screened. Through the above methods, the antibodies 18F7, 27C3, and 27F1 obtained by this invention can specifically react with the membrane protein A29L on monkeypox virus IMV particles and can be applied to immunoblotting, immunohistochemical detection, and immunofluorescence detection. 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. Antibodies for monkeypox virus detection, characterized in that, The monkeypox virus detection antibodies were selected from antibody 18F7, antibody 27C3 and antibody 27F1; The antibody 18F7, the antibody 27C3, or the antibody 27F1 all include a heavy chain variable region; The antibody 18F7, the antibody 27C3, or the antibody 27F1 all include a light chain variable region; The three complementarity-determining regions in the heavy chain variable region of antibody 18F7 are as follows: the amino acid sequence of VH-CDR1 is shown in SEQ ID NO.1; the amino acid sequence of VH-CDR2 is shown in SEQ ID NO.2; and the amino acid sequence of VH-CDR3 is shown in SEQ ID NO.

3. The three complementarity-determining regions in the light chain variable region of antibody 18F7 are as follows: the amino acid sequence of VL-CDR1 is shown in SEQ ID NO.4; the amino acid sequence of VL-CDR2 is shown in SEQ ID NO.5; and the amino acid sequence of VL-CDR3 is shown in SEQ ID NO.

6. The three complementarity-determining regions in the heavy chain variable region of antibody 27C3 are as follows: the amino acid sequence of VH-CDR1 is shown in SEQ ID NO.7; the amino acid sequence of VH-CDR2 is shown in SEQ ID NO.8; the amino acid sequence of VH-CDR3 is as follows: VH-CDR3: WDY; The three complementarity-determining regions in the light chain variable region of antibody 27C3 are as follows: the amino acid sequence of VL-CDR1 is shown in SEQ ID NO.9; the amino acid sequence of VL-CDR2 is shown in SEQ ID NO.10; and the amino acid sequence of VL-CDR3 is shown in SEQ ID NO.

11. The three complementarity-determining regions in the heavy chain variable region of antibody 27F1 are as follows: the amino acid sequence of VH-CDR1 is shown in SEQ ID NO.12; the amino acid sequence of VH-CDR2 is shown in SEQ ID NO.13; and the amino acid sequence of VH-CDR3 is shown in SEQ ID NO.

14. The three complementarity-determining regions in the light chain variable region of antibody 27F1 are as follows: the amino acid sequence of VL-CDR1 is shown in SEQ ID NO.15; the amino acid sequence of VL-CDR2 is shown in SEQ ID NO.16; and the amino acid sequence of VL-CDR3 is shown in SEQ ID NO.

17.

2. The monkeypox virus detection antibody according to claim 1, characterized in that, The heavy chain variable region of antibody 18F7, antibody 27C3, or antibody 27F1 includes the frame region FR region; The four frame regions in the heavy chain variable region of the antibody 18F7 are as follows: the amino acid sequence of VH-FR1 is shown in SEQ ID NO. 18; the amino acid sequence of VH-FR2 is shown in SEQ ID NO. 19; the amino acid sequence of VH-FR3 is shown in SEQ ID NO. 20; and the amino acid sequence of VH-FR4 is shown in SEQ ID NO.

21. The four frame regions in the heavy chain variable region of antibody 27C3 are as follows: the amino acid sequence of VH-FR1 is shown in SEQ ID NO. 22; the amino acid sequence of VH-FR2 is shown in SEQ ID NO. 23; the amino acid sequence of VH-FR3 is shown in SEQ ID NO. 24; and the amino acid sequence of VH-FR4 is shown in SEQ ID NO.

25. The four frame regions in the heavy chain variable region of antibody 27F1 are as follows: the amino acid sequence of VH-FR1 is shown in SEQ ID NO. 26; the amino acid sequence of VH-FR2 is shown in SEQ ID NO. 27; the amino acid sequence of VH-FR3 is shown in SEQ ID NO. 28; and the amino acid sequence of VH-FR4 is shown in SEQ ID NO.

29.

3. The monkeypox virus detection antibody according to claim 2, characterized in that, The light chain variable region of antibody 18F7, antibody 27C3, or antibody 27F1 includes the frame region FR region; The four framework regions in the light chain variable region of antibody 18F7 are as follows: the amino acid sequence of VL-FR1 is shown in SEQ ID NO. 30; the amino acid sequence of VL-FR2 is shown in SEQ ID NO. 31; the amino acid sequence of VL-FR3 is shown in SEQ ID NO. 32; and the amino acid sequence of VL-FR4 is shown in SEQ ID NO.

33. The four framework regions in the light chain variable region of antibody 27C3 are as follows: the amino acid sequence of VL-FR1 is shown in SEQ ID NO. 34; the amino acid sequence of VL-FR2 is shown in SEQ ID NO. 35; the amino acid sequence of VL-FR3 is shown in SEQ ID NO. 36; and the amino acid sequence of VL-FR4 is shown in SEQ ID NO.

37. The four frame regions in the light chain variable region of antibody 27F1 are as follows: the amino acid sequence of VL-FR1 is shown in SEQ ID NO. 38; the amino acid sequence of VL-FR2 is shown in SEQ ID NO. 39; the amino acid sequence of VL-FR3 is shown in SEQ ID NO. 40; and the amino acid sequence of VL-FR4 is shown in SEQ ID NO.

41.

4. The monkeypox virus detection antibody according to claim 3, characterized in that, The amino acid sequence of the heavy chain variable region of antibody 18F7 is shown in SEQ ID NO.42, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.43; The amino acid sequence of the heavy chain variable region of antibody 27C3 is shown in SEQ ID NO.44, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.

45. The amino acid sequence of the heavy chain variable region of antibody 27F1 is shown in SEQ ID NO.46, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.

47.

5. The application of the monkeypox virus detection antibody according to any one of claims 1-4, characterized in that, At least one of the monkeypox virus detection antibodies 18F7, 27C3 and 27F1 as described in any one of claims 1-4 is used in the product for preparing the test sample of orthopoxvirus; wherein the orthopoxvirus is monkeypoxvirus.

6. The application of the monkeypox virus detection antibody according to claim 5, characterized in that, The products include reagents, test plates, or kits.

7. The application of the monkeypox virus detection antibody according to claim 5, characterized in that, The combination of antibody 18F7 and antibody 27C3, when paired, specifically recognizes monkeypox virus and can be used in the preparation of products for detecting monkeypox virus.

8. The application of the monkeypox virus detection antibody according to claim 5, characterized in that, The combination of antibody 18F7 and antibody 27F1, after pairing, specifically recognizes the A29L protein and can be used in the preparation of products for detecting monkeypox virus.

9. The application of the monkeypox virus detection antibody according to claim 5, characterized in that, The antibodies 27C3 and 27F1 specifically recognize the A29L protein and can be used in the preparation of products for detecting monkeypox virus.

Images