A monoclonal antibody targeting monkeypox virus protein a29l and uses thereof

Abstract

The present application relates to a kind of artificially synthesized recombinant monoclonal antibody with human antibody amino acid sequence as framework.The present application uses phage display technology, and obtains one monoclonal antibody H11 from Tomlinson I+J phage library, and these new amino acid sequences constitute CDR1, CDR2 and CDR3 region, can be combined with A29L protein specific binding.It shows good stability, high specificity and high affinity and other characteristics, provide tool support for subsequent monkeypox virus diagnosis and detection with A29L protein as target.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of antibody engineering, and particularly relates to a monoclonal antibody targeting monkeypox virus protein A29L and application thereof. BACKGROUND

[0002] Monkeypox virus (MPXV) belongs to the Orthopoxvirus genus of the Chordopoxvirinae subfamily of the Poxviridae family of the Choripoviridae order in the classification of virology. A29L is an intracellular mature virus surface envelope protein, which plays an important role in promoting the fusion of monkeypox virus and cells, and can induce B cells to produce immunogens as one of the important antibody targets. Therefore, the research and development of A29L detection technology is particularly important for the diagnosis, prognosis evaluation and treatment detection of monkeypox virus infection in the clinic.

[0003] Based on the characteristics of viral epidemiology and monitoring needs, the diagnosis of monkeypox virus infection is more suitable for specifically detecting monkeypox virus DNA by PCR method. However, the nucleic acid amplification detection based on antigen-antibody recognition has higher sensitivity and accuracy. However, the traditional antibody has defects in specificity and stability, and the interference with the function of the target protein also limits its practical application. Therefore, it is necessary to develop A29L targeting antibody with high specificity and high sensitivity to improve the detection accuracy. SUMMARY

[0004] The strong specificity, good stability and strong affinity of the monoclonal antibody make it more suitable for immunodetection and treatment. In view of the above shortcomings of the prior art, the present application uses phage display technology to screen antibodies, and obtains a monoclonal antibody targeting A29L protein, and identifies the affinity and specificity of the antibody. The antibody can be used as an important technical tool for detecting the level of monkeypox virus protein A29L in the human body.

[0005] Therefore, the present application provides a monoclonal antibody targeting monkeypox virus protein A29L and application thereof. The antibody can specifically bind to A29L protein and exhibit good stability, high specificity and high affinity, thereby providing a powerful tool support for the diagnosis and detection of monkeypox virus targeting A29L protein.

[0006] Heavy chain and light chain are two polypeptide chains of antibody (immunoglobulin) molecules, which together constitute the basic structure of the antibody. The heavy chain is the main part of the antibody and has a large molecular weight. The structure of the heavy chain includes a variable region (VH) and a constant region (CH); the light chain also has a variable region (VL) and a constant region (CL), and each light chain is paired with a heavy chain to form a functional structure of the antibody. The light chain includes two types: kappa (Kappa) chain and lambda (Lambda) chain.

[0007] The term "complementarity-determining region (CDR)" refers to the amino acid sequences within antibody variable regions that confer antigen specificity and binding affinity, which directly interact with antigens and determine the specificity of the antibody. There are three CDRs in each heavy chain variable region (referred to herein as CDR-H1, CDR-H2, CDR-H3, respectively) and three CDRs in each light chain variable region (referred to herein as CDR-L1, CDR-L2, CDR-L3, respectively).

[0008] Thus, the first aspect of the present application provides a monoclonal antibody targeting monkeypox virus protein A29L, comprising: a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises CDR-H1 as shown in SEQ ID NO: 2, CDR-H2 as shown in SEQ ID NO: 3, and CDR-H3 as shown in SEQ ID NO: 4, and the light chain variable region comprises CDR-L1 as shown in SEQ ID NO: 6, CDR-L2 as shown in SEQ ID NO: 7, and CDR-L3 as shown in SEQ ID NO: 8.

[0009] The complementarity-determining regions can be defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering system. Preferably, the complementarity-determining regions of the present application are defined according to the IMGT numbering system.

[0010] Further, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 5.

[0011] Further, the monoclonal antibody targeting monkeypox virus protein A29L further comprises a constant region, which comprises a heavy chain constant region, and / or a light chain constant region.

[0012] Further, 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 kappa type or lambda type light chain constant region.

[0013] The second aspect of the present application provides a polynucleotide sequence encoding the above-mentioned monoclonal antibody targeting monkeypox virus protein A29L.

[0014] The third aspect of the present application provides an expression vector loaded with the above-mentioned polynucleotide sequence.

[0015] The fourth aspect of the present application provides a host cell obtained by transforming the expression vector.

[0016] The target monkeypox virus protein A29L monoclonal antibody is obtained by synthesizing a polynucleotide sequence encoding the target monkeypox virus protein A29L monoclonal antibody, inserting the polynucleotide sequence into a vector to construct an expression vector, transforming the expression vector into a competent cell to obtain a transformed host cell, and separating and purifying the target monkeypox virus protein A29L monoclonal antibody from the host cell by culturing the host cell. The host cell can be a prokaryotic cell such as Escherichia coli, Bacillus subtilis, Streptomyces and Pseudomonas, or a eukaryotic cell such as yeast, or a higher eukaryotic cell such as an insect cell.

[0017] The fifth aspect of the present application provides a detection kit or an antibody chip or an antibody probe, which comprises the target monkeypox virus protein A29L monoclonal antibody or a conjugate thereof as described in any one of the above aspects, wherein the conjugate is obtained by conjugating the target monkeypox virus protein A29L monoclonal antibody with biotin or a label selected from at least one of a fluorescent dye, an enzyme, a radioisotope, a chemiluminescent reagent and a nanoparticle label.

[0018] The sixth aspect of the present application provides use of the target monkeypox virus protein A29L monoclonal antibody or the detection kit or the antibody chip or the antibody probe in the preparation of a product for detecting monkeypox virus.

[0019] The beneficial effects of the present application include:

[0020] 1. The present application provides a target monkeypox virus protein A29L monoclonal antibody, which has high specificity and affinity in binding with the A29L protein of monkeypox virus, and can effectively reduce the detection limit of the A29L protein (LOD is 1.683 ng / mL), which provides a powerful tool for studying the function of the A29L protein and detecting the concentration of A29L.

[0021] 2. The monoclonal antibody H11 of the present application has high sensitivity and wide linear range in detecting monkeypox virus A29L. The half-inhibition concentration (IC50) of the antibody in detecting A29L based on the competition method is 500.3 ng / mL, and the minimum detection limit (LOD) is 1.683 ng / mL. The half-effect concentration (EC50) of the antibody in detecting A29L based on the sandwich method is 495.8 ng / mL, and the minimum detection limit (LOD) of the antibody in detecting A29L based on the standard curve is 0.382 ng / mL. The antibody has high affinity with the A29L protein, and can be used as an important technical tool for detecting the level of monkeypox virus protein A29L in human body. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 ELISA results of phage library R0 and phage libraries R1-R3 obtained by panning.

[0023] Figure 2The results of screening for monoclonal antibodies against A29L using the phage library R3 are presented.

[0024] Figure 3 The results are from a competitive enzyme-linked immunosorbent assay (ELISA) of monoclonal antibody H11.

[0025] Figure 4 The OD curve is used to detect the concentration of A29L protein by competitive ELISA using the monoclonal antibody H11.

[0026] Figure 5 The results of the detection of A29L protein using a sandwich enzyme-linked immunosorbent assay (ELISA) with monoclonal antibody H11 and mouse antibody 1M1G3 were obtained.

[0027] Figure 6 A standard curve was prepared for detecting A29L protein concentration using a sandwich enzyme-linked immunosorbent assay (ELISA) with monoclonal antibody H11 and mouse antibody 1M1G3. Detailed Implementation

[0028] To better understand the above technical solutions, the present invention will be described in detail below with reference to the accompanying drawings and specific embodiments. Unless otherwise specified, the percentages (%) used in the various embodiments refer to mass concentration.

[0029] While specific embodiments of the invention are shown in the accompanying drawings and examples, it should be understood that the invention can be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided to enable a clearer and more thorough understanding of the invention and to fully convey the scope of the invention to those skilled in the art.

[0030] Example 1

[0031] This embodiment obtains a monoclonal antibody H11 by panning phage-display antibodies. Sequencing of this antibody determines the amino acid sequences of the antibody, its three complementarity-determining regions (CDRs) of the heavy chain variable region, and its three CDRs of the light chain variable region. The steps of this embodiment are as follows:

[0032] (1) Amplification of phage display antibody library

[0033] E. coli stock solution containing the Tomlinson I+J phage library was added to 25 mL of 2YT liquid medium (2YTAG) containing 100 μg / mL ampicillin (Amp) and 1% glucose, and incubated overnight at 37°C and 250 rpm. 5 mL of the overnight culture was added to 50 mL of 2YTAG and incubated in an Erlenmeyer flask at 37°C and 250 rpm until OD reached [value missing]. 600 =0.4, add 25 μL of helper phage M13K07 (titer: 10) 12cfu / mL), after 30 min of infection at 37°C, the infected E. coli solution was centrifuged at 5000 rpm for 30 min, the supernatant was discarded, and the centrifugation was repeated twice to completely remove the supernatant. The bacterial pellet was resuspended in 100 mL of 2YT liquid medium (2YT AGK) containing 100 μg / mL Amp, 50 μg / mL Kanamycin and 0.1% glucose, and the solution was placed in a conical flask and incubated at 30°C, 250 rpm for 20 h. The next day, the solution was centrifuged at 5000 rpm for 30 min, and the supernatant was transferred to a new 50 mL sterile centrifuge tube. 20 mL of PEG / NaCl (20% polyethylene glycol 6000, 2.5 M NaCl) solution was added to the supernatant, mixed well and placed on ice for 1 h. The solution was centrifuged at 5000 rpm for 1 h, the supernatant was discarded, and the centrifugation was repeated twice to completely remove the supernatant. 4 mL of sterilized PBS buffer was added to dissolve the pellet, which was used as the phage display antibody library solution. The concentration of the prepared antibody library was 10 13 cfu / mL.

[0034] (2) Screening of phage display antibody library

[0035] ① 5 μg / mL of A29L protein (antigen) diluted in PBS buffer (all PBS used in this experiment was sterilized) was coated in a 96-well microplate, 100 μL per well, a total of 16 wells, and incubated at 4°C overnight. The next day, the antigen solution was discarded, and 200 μL of PBS solution containing 2% skim milk (MPBS) was added, and the plate was incubated at room temperature for 2 h. The Tomlinson I phage library and the Tomlinson J phage library were mixed at a ratio of 1:1, and diluted with MPBS to a concentration of 10 10 cfu / mL, and used as the phage library R0.

[0036] ② The MPBS solution containing the phage library R0 was added to the microplate at a concentration of 100 μL per well, and shaken at room temperature for 1 h, and then incubated at room temperature for 1 h. The plate was washed 10 times with PBST solution, 100 μL of glycine hydrochloride buffer (Glycine-HCl, pH=2.2) was added to each well, and shaken at room temperature for 10 min, and then incubated at room temperature for 10 min. The phage bound to the A29L protein was eluted, and then 50 μL of tris-hydroxymethyl aminomethane hydrochloride buffer (Tris-HCl, pH=7.4) was added to each well to neutralize.

[0037] ③ 20 μL of E. coli TG-1 strain was added to 8 mL of 2YT liquid medium without antibiotics and glucose, and incubated at 37°C, 250 rpm until the OD 600=0.4, add 1.6 mL of eluted phage solution to 8 mL of bacterial culture, incubate at 37°C for 30 min, centrifuge at 5000 rpm for 30 min, discard the supernatant, resuspend the bacteria in 2YT liquid medium (2YTAG) containing 100 μg / mL ampicillin and 1% glucose, and incubate overnight at 37°C and 250 rpm. The next day, add 40 μL of the overnight bacteria to 4 mL of 2YTAG liquid medium, and incubate at 37°C and 250 rpm until OD reaches 0.4. 600 =0.4. Add 10 μL of helper phage M13K07 (titer: 5 × 10⁻⁴). 11 After inoculating the bacteria at 37°C for 30 min, centrifuge the infected E. coli solution at 4°C and 5000 rpm for 30 min using a high-speed centrifuge. Discard the supernatant and centrifuge again to completely remove the supernatant. Resuspend the bacteria in 20 mL of 2YT liquid medium (2YTAGK) containing 100 μg / mL Amp, 50 μg / mL kanamycin, and 0.1% glucose, and incubate in an Erlenmeyer flask at 30°C and 250 rpm for 20 h.

[0038] ④ Centrifuge the overnight bacteria at 5000 rpm for 30 min at 4℃, separate and recover the supernatant, add 5 mL of PEG / NaCl solution to the supernatant, mix well, place on ice for 1 h, centrifuge at 5000 rpm for 1 h at 4℃, discard the supernatant, centrifuge again to completely remove the supernatant. Add 200 μL of sterile PBS solution as the antibody library (R1) for the second round of panning; repeat the above steps three times to obtain phages R2 and R3 respectively. After the third panning, use enzyme-linked immunosorbent assay (ELISA) to verify the binding specificity of the original antibody library R0 and the panned R1, R2, and R3 to the A29L protein.

[0039] The titers of the four phage libraries were determined, and the titers of the four antibody libraries were calculated. Bovine serum albumin (BSA) and A29L protein diluted in PBS buffer (5 μg / mL) were coated into 96-well microplates, 100 μL per well for each of the four groups, and incubated overnight at 4°C. The next day, the solution in the microplates was discarded, and 200 μL of PBS solution containing 5% skim milk powder (MPBS) was added, and the plates were incubated at room temperature for 2 hours to block the infection. The microplates were washed three times with PBST solution, and 100 μL of PBS containing 10% skim milk powder was added to each of the four wells. 10The phage library (R0, R1, R2, R3) of 5% MPBS solution of cfu was incubated at room temperature for 1 h. The microplate was washed with PBST solution for 6 times, then mouse anti-M13-HRP antibody was added at a dilution of 1:5000 (volume ratio) and incubated at room temperature for 1 h. The microplate was washed with PBST for 12 times, 100 μL TMBZ was added to each well for color development, and after reaction at 37 °C for 10 min, 50 μL 10% sulfuric acid solution was added to each well to stop the reaction, and the iMark TM microplate reader (Bio-Rad) was used to measure the absorbance at 450 nm and 630 nm, and a column chart was drawn.

[0040] The results of the enzyme-linked immunosorbent assay are shown in Figure 1 As shown, when comparing the binding ability of the phage libraries R0, R1, R2, R3 obtained by panning with the A29L protein, it was found that with the increase of the number of panning, the binding ability of the phage solution R3 with the A29L protein was significantly increased, while the binding performance of the four groups of phage libraries to BSA was very weak and did not change, indicating that the antibody against A29L protein in the constructed phage display antibody library was successfully enriched.

[0041] (3) Screening of monoclonal antibody

[0042] Titer determination was performed on R3, and the next day, 96 single colonies were randomly picked into a 96-deep well plate. 1.5 mL of 2YTAG liquid medium was added to each of the 96 wells, and the 96-well plate was sealed with a sealing film to prevent cross contamination caused by shaking out of the bacterial solution. The plate was incubated at 37 °C, 200 rpm overnight. The next day, 10 μL of overnight bacteria was added to a new 96-well plate containing 90 μL of 2YTAG liquid medium, and the plate was sealed with a sealing film. The plate was shaken at 37 °C, 200 rpm until the OD 600 = 0.2, 75 μL of helper phage M13K07 (titer: 2.5 x 10 11 cfu / mL) was mixed with 5 mL of 2YTAG liquid medium, and 150 μL was added to each well of the 96-well plate, which was sealed with a sealing film and incubated at 37 °C for 30 min. After the incubation, 1.35 mL of 2YTAK (without glucose) was added to each well, which was sealed with a sealing film and incubated at 30 °C, 250 rpm for 20 h.

[0043] respectively, and incubated overnight at 4°C. The next day, the solution in the microplate was discarded, 200 μL of PBS solution containing 5% skimmed milk powder (MPBS) was added, and the microplate was sealed and incubated at room temperature for 2 h. The bacterial solution cultured for 20 h was taken out into a 2 mL centrifuge tube, centrifuged at 5000 rpm for 30 min at 4°C, and the MPBS-sealed 96-well plate was washed with PBST for 3 times, 50 μL of MPBS solution was added to each well, and then 50 μL of the centrifuged phage supernatant was added to each well in correspondence, and the mixture was gently mixed and incubated at room temperature for 1 h. The microplate was washed with PBST for 6 times, 100 μL of mouse anti-M13-HRP antibody diluted at a ratio of 1:5000 was added to each well, and the mixture was incubated at room temperature for 1 h. The microplate was washed with PBST for 12 times, 100 μL of TMBZ was added to each well for color development, and after reaction at 37°C for 10 min, 50 μL of 10% sulfuric acid solution was added to each well to stop the reaction, and the iMark TM The absorbance at 450 nm and 630 nm was measured by a microplate reader (Bio-Rad), and a bar graph was plotted as shown in FIG. 2. Figure 2 The antibody with high A29L binding activity was screened, and the positive clone was verified again to further determine the positive clone.

[0044] (4) Antibody sequence comparison analysis

[0045] According to the above experimental results, 8 positive clones were selected, the plasmid was extracted and gene sequencing was performed, 1 strain of antibody was obtained and named H11, and no same sequence as the antibody gene described in the application was found by comparing with the registered antibody sequence in the antibody gene library, so the antibody is a new type of antibody. The amino acid sequence of the H11 antibody is as follows.

[0046] The sequence of the heavy chain variable region of the H11 antibody is SEQ ID NO: 1, the sequence of CDR-H1 is SEQ ID NO: 2, the sequence of CDR-H2 is SEQ ID NO: 3, and the sequence of CDR-H3 is SEQ ID NO: 4;

[0047] The sequence of the light chain variable region of the H11 antibody is SEQ ID NO: 5, the sequence of CDR-L1 is SEQ ID NO: 6, the sequence of CDR-L2 is SEQ ID NO: 7, and the sequence of CDR-L3 is SEQ ID NO: 8.

[0048] The amino acid sequence of the antibody is shown in the following table:

[0049]

[0050]

[0051] Example 2

[0052] This example further verifies the antigen specificity of antibody H11. The experimental method is as follows.

[0053] BSA and A29L protein were coated in 96-well microplates at 5 μg / mL, 3 wells for BSA and 6 wells for A29L protein, 100 μL per well, and incubated at 4°C overnight. The next day, the solution in the microplate was discarded, and 200 μL of PBS solution containing 5% skimmed milk powder (MPBS) was added, and the microplate was incubated at room temperature for 2 h. The microplate was washed 3 times with PBST solution, 50 μL of MPBS and 50 μL of phage display antibody supernatant mixture were added to each well, 3 wells coated with A29L protein were added with the above mixture, and at the same time, 10 μg / mL of A29L protein was added for competition, and incubated at room temperature for 1 h. The microplate was washed 6 times with PBST solution, and then 1:5000 diluted mouse anti-M13-HRP antibody was added, and incubated at room temperature for 1 h. The microplate was washed 12 times with PBST, 100 μL of TMBZ was added to each well for color development, and after 10 min of reaction at 37°C, 50 μL of 10% sulfuric acid solution was added to each well to stop the reaction, and the iMark TM The absorbance at 450 nm and 630 nm was measured using a microplate reader (Bio-Rad), and a bar graph was drawn.

[0054] The results of the competitive enzyme-linked immunosorbent assay are shown in Figure 3 H11 antibody specifically binds to A29L protein, and does not bind to coated BSA, and when free A29L protein competes, the binding ability of H11 antibody to A29L protein is significantly reduced, indicating that H11 antibody is indeed a specific antibody against A29L protein, and the binding to A29L protein is specific.

[0055] Example 3

[0056] This example is to detect the concentration of A29L protein using monoclonal antibody H11 by competitive ELISA method, and to determine the detection limit of the antibody to A29L protein. The experimental method is as follows.

[0057] BSA and A29L protein were coated in 96-well microplates at 1 μg / mL, 100 μL per well, and incubated at 4°C overnight. The next day, the solution in the microplate was discarded, and 200 μL of PBS solution containing 5% skimmed milk powder (MPBS) was added, and the microplate was incubated at room temperature for 2 h. The microplate was washed 3 times with PBST solution, and 10 10The phage-displayed antibody H11 was used to detect the presence of free A29L in solution by competitive ELISA (results shown in Figure 6). When the concentration of free A29L in solution is low, the phage-displayed antibody binds to the A29L protein coated on the microplate, resulting in a higher absorbance. As the concentration of free A29L protein in solution increases, the phage-displayed antibody competes with the free A29L protein for binding, resulting in a decrease in binding to the coated A29L protein on the microplate and a decrease in absorbance.

[0058] The phage-displayed antibody H11 was used to detect the presence of free A29L in solution by competitive ELISA (results shown in Figure 6). When the concentration of free A29L in solution is low, the phage-displayed antibody binds to the A29L protein coated on the microplate, resulting in a higher absorbance. As the concentration of free A29L protein in solution increases, the phage-displayed antibody competes with the free A29L protein for binding, resulting in a decrease in binding to the coated A29L protein on the microplate and a decrease in absorbance. Figure 4

[0059] The half-inhibitory concentration (IC50) of the H11 antibody was determined by the dose-response curve to be 500.3 ng / mL, and the lowest detection limit (LOD) of the A29L protein was calculated to be 1.683 ng / mL. 50

[0060] Example 4

[0061] In this example, the phage-displayed antibody H11 and the specific antibody 1M1G3 (the relevant content of antibody 1M1G3 can be found in CN118772268B) were used to bind to the target antigen, forming a double antibody "sandwich" structure, to achieve specific detection of the antigen. The experimental method is as follows.

[0062] ​​The 1M1G3 anti-A29L Fab (anti-A29L mouse Fab antibody stored in our lab) was coated at 10 μg / mL in 96-well plates, 6 wells in total, 100 μL per well, and incubated at 4°C overnight. The next day, the solution in the microplate was discarded, and 200 μL of PBS containing 5% skim milk (MPBS) was added, and incubated at room temperature for 2 h. After washing the microplate 3 times with PBST solution, 10 μg / mL BSA was coated in 3 wells, and 10 μg / mL A29L was coated in the remaining 3 wells, and incubated at room temperature for 1 h. The microplate was washed 6 times with PBST solution, 50 μL of MPBS and 50 μL of phage display antibody H11 supernatant mixture was added to each well, and incubated at room temperature for 1 h. The microplate was washed 8 times with PBST, and then 1:5000 diluted mouse anti-M13-HRP antibody was added, and incubated at room temperature for 1 h. The microplate was washed 12 times with PBST, 100 μL of TMBZ was added to each well for color development, and after 10 min of reaction at 37°C, 50 μL of 10% sulfuric acid solution was added to each well to stop the reaction, and the iMark TM The absorbance at 450 nm and 630 nm was measured using a microplate reader (Bio-Rad), and a bar graph was drawn.

[0063] The results of the sandwich ELISA are shown in FIG. 6. The binding between the capture antibody 1M1G3 anti-A29L Fab and the target antigen A29L and the detection phage H11 was stable, and a stable sandwich structure was formed. Moreover, in the control experiment with the non-target antigen BSA, no obvious signal was detected. The above indicates that this method has good specificity. Figure 5

[0064] Example 5

[0065] In this example, the phage display antibody H11 and the specific antibody 1M1G3 were used to quantitatively detect the antigen by sandwich ELISA. The experimental method is as follows.

[0066] The 1M1G3 anti-A29L Fab was coated at 5 μg / mL in 96-well plates, 100 μL per well, and incubated at 4°C overnight. The next day, the solution in the microplate was discarded, and 200 μL of MPBS was added, and incubated at room temperature for 2 h. After washing the microplate 3 times with PBST solution, 100 μL of MPBS containing different concentrations of BSA and A29L (0, 1, 10, 100, 1000, 10000 and 100000 ng / mL) was added to each well, and each concentration was repeated three times, and incubated at room temperature for 1 h. After incubation at room temperature for 1 h, the microplate was washed 6 times with PBST solution, and 100 μL of MPBS diluted phage display antibody supernatant solution (2 x 1011cfu / mL) was added to each well, and incubated at room temperature for 1 h. The microplate was washed 8 times with PBST, and then 1:5000 diluted mouse anti-M13-HRP antibody was added, and incubated at room temperature for 1 h. The microplate was washed 12 times with PBST, 100 μL of TMBZ was added to each well for color development, and after 10 min of reaction at 37°C, 50 μL of 10% sulfuric acid solution was added to each well to stop the reaction, and the iMark 9 ​The microplate was incubated at room temperature for 1 hour with cfu / mL. The microplate was washed 8 times with PBST solution, then mouse anti-M13-HRP antibody diluted 1:5000 was added, and the microplate was incubated at room temperature for 1 hour. The microplate was washed 12 times with PBST, and 100 μL of TMBZ was added to each well for color development. After reacting at 37°C for 10 minutes, 50 μL of 10% sulfuric acid solution was added to each well to stop the reaction. The reaction was then analyzed using iMark. TM A microplate reader (Bio-Rad) was used to measure absorbance at 450 nm and 630 nm to plot a standard curve.

[0067] The results of the sandwich-method enzyme-linked immunosorbent assay (ELISA) for detecting A29L concentration are as follows: Figure 6 As shown, no obvious signal was detected in the absence of antigen or at antigen concentrations of 1 and 10 ng / mL. However, the signal gradually increased with increasing concentration. The limit of detection (LOD) of this detection method was determined to be 0.382 ng / mL and the half-maximal effective concentration (EC50) was 495.8 ng / mL using a standard curve. This indicates that antibody H11 can be used in combination with other antibodies to detect the concentration of A29L in solution.

[0068] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A monoclonal antibody targeting monkeypox virus protein A29L, characterized in that, include: Heavy chain variable region and light chain variable region, among which, The heavy chain variable region includes CDR-H1 as shown in SEQ ID NO:2, CDR-H2 as shown in SEQ ID NO:3, and CDR-H3 as shown in SEQ ID NO:4, and the light chain variable region includes CDR-L1 as shown in SEQ ID NO:6, CDR-L2 as shown in SEQ ID NO:7, and CDR-L3 as shown in SEQ ID NO:

8.

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

5.

3. The monoclonal antibody targeting monkeypox virus protein A29L according to claim 1 or 2, characterized in that, The monoclonal antibody targeting monkeypox virus protein A29L also includes a constant region, which comprises a heavy chain constant region and / or a light chain constant region.

4. The monoclonal antibody targeting monkeypox virus protein A29L according to claim 3, characterized in that, The heavy chain constant region is selected from the heavy chain constant regions 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 polynucleotide encoding a monoclonal antibody targeting monkeypox virus protein A29L as described in any one of claims 1-4.

6. An expression vector loaded with the polynucleotide of claim 5.

7. A host cell obtained by transformation of the expression vector according to claim 6.

8. A detection kit, antibody chip, or antibody probe comprising a monoclonal antibody or conjugate thereof targeting monkeypox virus protein A29L as described in any one of claims 1-4, wherein the conjugate is obtained by conjugating the monoclonal antibody targeting monkeypox virus protein A29L with a label, wherein the label is selected from at least one of fluorescent dyes, enzymes, radioisotopes, chemiluminescent reagents, biotin, and nanoparticle-based labels.

9. The use of the monoclonal antibody targeting monkeypox virus protein A29L as described in any one of claims 1-4, or the detection kit, antibody chip, or antibody probe as described in claim 8, in the preparation of products for detecting monkeypox virus.

Images