Monoclonal antibody targeting monkey pox virus protein A29L and application thereof

Abstract

The invention relates to an artificially synthesized recombinant monoclonal antibody taking a human antibody amino acid sequence as a framework. The monoclonal antibody H11 is obtained by screening from a Tomlinson I + J phage library by utilizing a phage display technology, and CDR1, CDR2 and CDR3 regions formed by the new amino acid sequences can be specifically combined with A29L protein. The kit shows the characteristics of good stability, high specificity, high affinity and the like, and provides a tool support for subsequent diagnosis and detection of the monkey pox virus taking A29L protein as a target spot.

Description

Technical Field

[0001] The present invention relates to the technical field of antibody engineering, and particularly to a monoclonal antibody targeting the monkeypox virus protein A29L and its application. Background Art

[0002] Monkeypox virus (MPXV) belongs to the genus Orthopoxvirus, subfamily Chordopoxvirinae, family Poxviridae, order Chuviridae in virological classification. A29L is a surface envelope protein of mature virus in cells, which plays an important role in promoting the fusion of monkeypox virus and cells. As one of the important antibody targets, it can induce B cells to produce immunogenicity. Therefore, the research and development of A29L detection technology is particularly important in the diagnosis, prognosis evaluation and treatment detection of monkeypox virus infection in clinical practice.

[0003] Based on the epidemiological characteristics of the virus and the monitoring requirements, the diagnosis of monkeypox virus infection is more suitable for specifically detecting monkeypox virus DNA by PCR method. However, the nucleic acid amplification detection generally requires large and expensive special equipment and professional laboratory equipment and technical personnel, while the immunological method based on antigen-antibody recognition has higher sensitivity and accuracy. However, traditional antibodies have defects in terms of specificity and stability, and the interference with the function of target proteins also limits their practical applications. Therefore, it is necessary to develop A29L-targeted antibodies with strong specificity and high sensitivity to improve the detection accuracy. Summary of the Invention

[0004] The strong specificity, good stability and strong affinity of monoclonal antibodies make them more suitable for immunoassay and treatment. In view of the above deficiencies of the prior art, the present invention uses phage display technology to screen antibodies and obtains a monoclonal antibody targeting the A29L protein, and identifies its affinity and specificity. This 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 invention provides a monoclonal antibody targeting the monkeypox virus protein A29L and its application. The antibody can specifically bind to the A29L protein and exhibits characteristics such as good stability, high specificity and high affinity, providing strong tool support for the diagnosis and detection of monkeypox virus targeting the A29L protein in the future.

[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 larger 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). Each light chain pairs with a heavy chain to form the functional structure of the antibody. The light chain includes two types: κ (Kappa) chain and λ (Lambda) chain.

[0007] The term "Complementarity-Determining Region (CDR)" refers to the amino acid sequences within the variable regions of an antibody that confer antigen specificity and binding affinity, directly interact with the antigen, 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, and CDR-H3, respectively), and three CDRs in each light chain variable region (referred to herein as CDR-L1, CDR-L2, and CDR-L3, respectively).

[0008] Accordingly, a first aspect of the present invention provides a monoclonal antibody targeting the 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 Region can be defined according to the Kabat, IMGT, Chothia, AbM, or Contact numbering system. Preferably, the Complementarity-Determining Region of the present invention is defined according to the IMGT numbering system.

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

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

[0012] Furthermore, 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 regions.

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

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

[0015] A fourth aspect of the present invention provides a host cell transformed by the above-mentioned expression vector.

[0016] By synthesizing the polynucleotide sequence encoding the monoclonal antibody targeting the monkeypox virus protein A29L, inserting it into a vector to construct an expression vector, transferring the expression vector into competent cells to obtain successfully transformed host cells, and culturing the above host cells, the monoclonal antibody targeting the monkeypox virus protein A29L is isolated and purified from the host cells. The host cells can be prokaryotic cells such as Escherichia coli, Bacillus subtilis, Streptomyces, and Pseudomonas, eukaryotic cells such as yeast, or higher eukaryotic cells such as insect cells.

[0017] The fifth aspect of the present invention provides a detection kit, antibody chip, or antibody probe, which comprises the monoclonal antibody targeting the monkeypox virus protein A29L or its conjugate described in any one of the above, and the conjugate is obtained by conjugating the monoclonal antibody targeting the monkeypox virus protein A29L with biotin or a labeling agent, and the labeling agent is selected from at least one of fluorescent dyes, enzymes, radioisotopes, chemiluminescent reagents, and nanoparticle-based labeling agents.

[0018] The sixth aspect of the present invention provides the application of the monoclonal antibody targeting the monkeypox virus protein A29L, the detection kit, antibody chip, or antibody probe in the preparation of products for detecting monkeypox virus.

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

[0020] 1. The present invention provides a monoclonal antibody targeting the monkeypox virus protein A29L, which has specific binding to the monkeypox virus A29L protein and high affinity, 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 invention has high sensitivity and a wide linear range for detecting the monkeypox virus A29L. The half-inhibitory concentration (IC50) for detecting A29L using this antibody based on the competitive method is 500.3 ng / mL, and the lowest detection limit (LOD) is 1.683 ng / mL; the half-maximal effective concentration (EC50) for detecting A29L based on the sandwich method is 495.8 ng / mL, and the lowest detection limit (LOD) for detecting A29L by the sandwich method obtained from the standard curve is 0.382 ng / mL. It has high affinity with the A29L protein, and this antibody can be used as an important technical tool for detecting the level of the monkeypox virus protein A29L in the human body. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 It is the results of enzyme-linked immunosorbent assay for phage library R0 and phage libraries R1-R3 obtained by panning.

[0023] Figure 2Results of screening for monoclonal antibody against A29L using phage library R3.

[0024] Figure 3 Results of competitive ELISA for monoclonal antibody H11.

[0025] Figure 4 OD curve for detecting A29L protein concentration by competitive ELISA using monoclonal antibody H11.

[0026] Figure 5 Experimental results of detecting A29L protein by sandwich ELISA using monoclonal antibody H11 and murine antibody 1M1G3.

[0027] Figure 6 Standard curve for detecting A29L protein concentration by sandwich ELISA using monoclonal antibody H11 and murine antibody 1M1G3. Detailed implementation mode

[0028] To better understand the above technical solution, the present invention will be described in detail below in conjunction with the accompanying drawings through specific implementation modes. When there is no special description, the % of concentration involved in each example represents mass concentration.

[0029] Although the specific implementation modes of the present invention are shown in the accompanying drawings and examples, it should be understood that the present invention can be implemented in various forms and should not be limited by the examples described herein. On the contrary, these examples are provided to enable a clearer and more thorough understanding of the present invention and to fully convey the scope of the present invention to those skilled in the art.

[0030] Example 1

[0031] In this example, a monoclonal antibody H11 was obtained by panning the phage-displayed antibody. The antibody was sequenced to determine the amino acid sequences of the three complementarity-determining regions of the antibody and its heavy-chain variable region and the three complementarity-determining regions of the light-chain variable region. The steps of this example are as follows:

[0032] (1) Amplification of phage-displayed antibody library

[0033] Add the Escherichia coli stock solution containing Tomlinson I+J phage library to 25 mL of 2YT liquid medium (2YTAG) containing 100 μg / mL ampicillin (Amp) and 1% glucose (Glucose), and culture overnight at 37 °C and 250 rpm. Take 5 mL of the overnight culture and add it to 50 mL of 2YTAG, and culture in a conical flask at 37 °C and 250 rpm until OD 600 = 0.4, and add 25 μL of helper phage M13K07 (titer: 10 12(cfu / mL). After infecting at 37°C for 30 min, the infected Escherichia coli solution was centrifuged at 5000 rpm for 30 min using a high-speed centrifuge. The supernatant was discarded, and the centrifugation was repeated twice to completely remove the supernatant. The cells were resuspended in 100 mL of 2YT liquid medium (2YTAGK) containing 100 μg / mL Amp, 50 μg / mL kanamycin, and 0.1% glucose, and the solution was placed in an Erlenmeyer flask and cultured at 30°C and 250 rpm for 20 h. The next day, it was centrifuged at 5000 rpm for 30 min, and the supernatant was taken and placed in 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. After mixing evenly, it was left standing on ice for 1 h, 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 precipitate, which was used as the phage display antibody library solution. The phage display antibody library was titrated using Escherichia coli, and the concentration of the prepared antibody library was 10 13 cfu / mL.

[0034] (2) Panning of the phage display antibody library

[0035] ① Coat 5 μg / mL of A29L protein (antigen) diluted with PBS buffer (all PBS used in this experiment has been sterilized) in a 96-well microplate, 100 μL per well, for a total of 16 wells, and incubate overnight at 4°C. The next day, discard the antigen solution, add 200 μL of PBS solution containing 2% skim milk powder (MPBS), and let it stand at room temperature for 2 h for blocking. Wash the microplate 3 times with PBST solution (PBS containing 0.1% Tween 20). Mix the Tomlinson I phage library and the Tomlinson J phage library at a ratio of 1:1, and dilute it with MPBS to 10 10 cfu / mL as phage library R0.

[0036] ② Add 100 μL of the MPBS solution containing phage library R0 to each well of the microplate, shake it on a horizontal shaker at room temperature for 1 h, and then let it stand at room temperature for 1 h. Wash it 10 times with PBST solution. Add 100 μL of glycine hydrochloride buffer (Glycine-HCl, pH = 2.2) to each well, shake it on a horizontal shaker at room temperature for 10 min, and then let it stand at room temperature for 10 min to elute the phages bound to the A29L protein. Then add 50 μL of tris(hydroxymethyl)aminomethane hydrochloride buffer (Tris-HCl, pH = 7.4) to each well for neutralization.

[0037] ③ Take 20 μL of Escherichia coli TG-1 strain and add it to 8 mL of 2YT liquid medium without antibiotics and glucose, and shake it at 37°C and 250 rpm until OD 600= 0.4, 1.6 mL of the eluted phage solution was added to 8 mL of the bacterial solution, and the mixture was infected at 37 °C for 30 min. Then, it was centrifuged at 5000 rpm for 30 min at 4 °C. The supernatant was discarded, and the bacterial pellet was resuspended in 2YT liquid medium (2YTAG) containing 100 μg / mL ampicillin and 1% glucose, and shaken overnight at 37 °C and 250 rpm. The next day, 40 μL of the overnight culture was added to 4 mL of 2YTAG liquid medium, and shaken at 37 °C and 250 rpm until OD 600 = 0.4. 10 μL of helper phage M13K07 (titer: 5 × 10 11 cfu / mL) was added, and after infecting at 37 °C for 30 min, the infected Escherichia coli solution was centrifuged at 5000 rpm for 30 min at 4 °C using a high-speed centrifuge. The supernatant was discarded, and centrifuged again to completely remove the supernatant. The bacterial pellet was resuspended in 20 mL of 2YT liquid medium (2YTAGK) containing 100 μg / mL Amp, 50 μg / mL kanamycin, and 0.1% glucose, and cultured in a conical flask at 30 °C and 250 rpm for 20 h.

[0038] ④ The overnight culture was centrifuged at 5000 rpm for 30 min at 4 °C to separate and recover the supernatant. 5 mL of PEG / NaCl solution was added to the supernatant solution, and after mixing evenly, it was placed on ice for 1 h. Then, it was centrifuged at 5000 rpm for 1 h at 4 °C. The supernatant was discarded, and centrifuged again to completely remove the supernatant. 200 μL of sterile PBS solution was added to obtain the antibody library (R1) for the second round of panning; the above steps were repeated three times to obtain phages R2 and R3 respectively. After the third round of panning, enzyme-linked immunosorbent assay was used 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 measured, and the titers of the four antibody libraries were calculated. 5 μg / mL of bovine serum albumin (BSA) and A29L protein diluted with PBS buffer were coated onto a 96-well microplate, with 3 wells coated for each, 100 μL per well, a total of 4 groups, and incubated overnight at 4 °C. The next day, the solution in the microplate was discarded, and 200 μL of PBS solution containing 5% skim milk powder (MPBS) was added, and left at room temperature for 2 h for blocking. The microplate was washed 3 times with PBST solution, and 100 μL containing 10 10A 5% MPBS solution of the phage library (R0, R1, R2, R3) of cfu was incubated at room temperature for 1 h. The microplate was washed 6 times with PBST solution, and then a mouse anti-M13-HRP antibody diluted 1:5000 (volume ratio) 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. After reacting at 37 °C for 10 min, 50 μL of 10% sulfuric acid solution was added to each well to terminate the reaction, and iMark TM A microplate reader (Bio-Rad) was used to measure the absorbance at 450 nm and 630 nm, and a bar chart was drawn.

[0040] The results of the enzyme-linked immunosorbent assay are as Figure 1 shown. When comparing the binding ability of the phage libraries R0, R1, R2, and R3 obtained by panning with the A29L protein, it was found that with the increase in the number of panning cycles, the binding ability of the phage solution R3 to the A29L protein increased significantly, while the binding performance of the four phage libraries to BSA was very weak and unchanged, indicating that the antibodies against the A29L protein in the constructed phage display antibody library were successfully enriched.

[0041] (3) Screening of monoclonal antibodies

[0042] The titer of R3 was determined. The next day, 96 single colonies were randomly picked into a 96-well 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 the bacterial liquid from shaking out and causing cross-contamination. It was placed in a shaker at 37 °C and 200 rpm overnight. The next day, 10 μL of the overnight culture was taken from each well and added one by one to a new 96-well plate containing 90 μL of 2YTAG liquid medium. The plate was sealed with a sealing film and shaken at 37 °C and 200 rpm until OD 600 = 0.2. 75 μL of the helper phage M13K07 (titer: 2.5×10 11 cfu / mL) was mixed with 5 mL of 2YTAG liquid medium and added to the 96-well plate at 150 μL per well. The plate was sealed with a sealing film and infected at 37 °C for 30 min. After the infection, 1.35 mL of 2YTAK (without glucose) was added to each well, the plate was sealed with a sealing film, and it was placed in a shaker at 30 °C and 250 rpm for 20 h.

[0043] Add 100 μL of PBS solution containing BSA (5 μg / mL) and A29L protein (5 μg / mL) into 96-well microplates respectively in a one-to-one correspondence, and incubate overnight at 4°C. The next day, pour out the solution in the microplate, add 200 μL of PBS solution containing 5% skim milk powder (MPBS), and let it stand at room temperature for 2 h for blocking. Take out the bacterial solution cultured for 20 h into a 2 mL centrifuge tube, centrifuge at 5000 rpm for 30 min at 4°C. During this period, wash the 96-well microplate blocked with MPBS 3 times with PBST. Add 50 μL of MPBS solution to each well, then add 50 μL of the phage supernatant after centrifugation in a one-to-one correspondence, and gently pipette and mix well. Let it stand at room temperature for 1 h for incubation. Wash the microplate 6 times with PBST solution, add 100 μL of mouse anti-M13-HRP antibody diluted at a ratio of 1:5000 to each well, and let it stand at room temperature for 1 h for incubation. Wash the microplate 12 times with PBST, add 100 μL of TMBZ to each well for color development. After reacting at 37°C for 10 min, add 50 μL of 10% sulfuric acid solution to each well to terminate the reaction, and use iMark TM microplate reader (Bio-Rad) to measure the absorbance at 450 nm and 630 nm, and draw a histogram as shown in Figure 2 shown. Screen out the antibodies with high binding activity to A29L, re-verify them, and further determine the positive clones.

[0044] (4) Comparative analysis of antibody sequences

[0045] According to the above experimental results, select 8 positive clones, extract plasmids and perform gene sequencing to obtain 1 antibody named H11. By comparing with the antibody sequences registered in the antibody gene bank, no sequence identical to the antibody gene described in the present invention is found. Therefore, this antibody is a novel antibody. The detailed amino acid sequence of H11 antibody is described as follows.

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

[0047] The variable region sequence of the light chain of H11 antibody is SEQ ID NO:5, the CDR-L1 sequence is SEQ ID NO:6; the CDR-L2 sequence is SEQ ID NO:7; the CDR-L3 sequence 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] Coat 5 μg / mL of BSA and A29L protein in a 96-well microplate. Coat 3 wells with BSA and 6 wells with A29L, 100 μL per well, and incubate overnight at 4°C. The next day, pour out the solution in the microplate, add 200 μL of PBS solution containing 5% skim milk powder (MPBS), and let it stand at room temperature for 2 h for blocking. Wash the microplate 3 times with PBST solution. Add 50 μL of MPBS and 50 μL of the phage display antibody supernatant mixture to each well. For the 3 wells coated with A29L protein, add the above mixture and at the same time add A29L protein with a final concentration of 10 μg / mL for competition, and let it stand at room temperature for 1 h for incubation. Wash the microplate 6 times with PBST solution, then add mouse anti-M13-HRP antibody diluted 1:5000, and let it stand at room temperature for 1 h for incubation. Wash the microplate 12 times with PBST, add 100 μL of TMBZ to each well for color development. After reacting at 37°C for 10 min, add 50 μL of 10% sulfuric acid solution to each well to terminate the reaction, and use iMark TM Microplate reader (Bio-Rad) to measure the absorbance at 450 nm and 630 nm, and draw a bar graph.

[0054] The results of the competitive enzyme-linked immunosorbent assay are as Figure 3 shown. Antibody H11 specifically binds to A29L protein and does not bind to the coated BSA. At the same time, when there is free A29L protein for competition, the binding ability of antibody H11 to A29L protein decreases significantly, indicating that antibody H11 is indeed a specific antibody against A29L protein and has specificity in binding to A29L protein.

[0055] Example 3

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

[0057] Coat 1 μg / mL of BSA and A29L protein in a 96-well microplate, 100 μL per well, and incubate overnight at 4°C. The next day, pour out the solution in the microplate, add 200 μL of PBS solution containing 5% skim milk powder (MPBS), and let it stand at room temperature for 2 h for blocking. Wash the microplate 3 times with PBST solution. Add the phage display antibody solution diluted with MPBS (10 10cfu / mL), a series of diluted solutions of phage-displayed antibodies with final concentrations of 0, 8, 40, 200, 1000, 5000, and 25000 ng / mL of A29L were added to the microwells coated with A29L protein, with each concentration repeated three times, and incubated at room temperature for 1 h. Washed 8 times with PBST, and HRP-labeled mouse anti-M13 antibody was added. The microwell plate was washed 6 times with PBST solution, then a 1:5000 diluted mouse anti-M13-HRP antibody was added and incubated at room temperature for 1 h. The microwell plate was washed 12 times with PBST, 100 μL of TMBZ was added to each well for color development, after reacting at 37 °C for 10 min, 50 μL of 10% sulfuric acid solution was added to each well to terminate the reaction, and the absorbance at 450 nm and 630 nm was measured, and a dose-response curve was plotted.

[0058] The presence of free A29L in the solution was detected by competitive ELISA using phage-displayed antibodies (the results are as Figure 4 shown). When the concentration of free A29L in the solution is low, the phage-displayed antibody binds to the A29L protein coated on the microwell plate, resulting in a higher absorbance intensity. As the concentration of free A29L protein in the solution increases, the phage-displayed antibody competes with the free A29L protein for binding, resulting in a decrease in the binding to the A29L protein coated in the microwell plate and a decrease in absorbance.

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

[0060] Example 4

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

[0062] Coat 10 μg / mL of 1M1G3 anti-A29L Fab (a mouse-derived Fab antibody against A29L stored in our laboratory) in a 96-well microplate, with a total of 6 wells, 100 μL per well, and incubate overnight at 4°C. The next day, pour out the solution in the microplate, add 200 μL of PBS solution containing 5% skim milk powder (MPBS), and let it stand at room temperature for 2 h for blocking. After washing the microplate 3 times with PBST solution, coat 10 μg / mL BSA in 3 of the wells and 10 μg / mL A29L in the remaining 3 wells, and let it stand at room temperature for 1 h for incubation. Wash the microplate 6 times with PBST solution, add 50 μL of the mixture of MPBS and 50 μL of the supernatant of phage-displayed antibody H11 to each well, and let it stand at room temperature for 1 h for incubation. Wash the microplate 8 times with PBST solution, then add a 1:5000 diluted mouse anti-M13-HRP antibody, and let it stand at room temperature for 1 h for incubation. Wash the microplate 12 times with PBST, add 100 μL of TMBZ to each well for color development, after reacting at 37°C for 10 min, add 50 μL of 10% sulfuric acid solution to each well to terminate the reaction, and use iMark TM Measure the absorbance at 450 nm and 630 nm using a microplate reader (Bio-Rad) and plot a bar graph.

[0063] The results of the sandwich enzyme-linked immunosorbent assay are as Figure 5 shown. The binding between the capture antibody 1M1G3 anti-A29L Fab and the target antigen A29L and the detection phage H11 is stable, forming a stable sandwich structure. 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.

[0064] Example 5

[0065] In this example, the phage-displayed antibody H11 and the specific antibody 1M1G3 are used to quantitatively detect the antigen by sandwich enzyme-linked immunosorbent assay. The experimental method is as follows.

[0066] Coat 5 μg / mL of 1M1G3 anti-A29L Fab in a 96-well microplate, 100 μL per well, and incubate overnight at 4°C. The next day, pour out the solution in the microplate, add 200 μL of MPBS, and let it stand at room temperature for 2 h for blocking. After washing the microplate 3 times with PBST solution, add 100 μL of MPBS containing different concentrations of BSA and A29L (0, 1, 10, 100, 1000, 10000, and 100000 ng / mL) to each well, repeat each concentration three times, and let it stand at room temperature for 1 h for incubation. Let it stand at room temperature for 1 h for incubation. Wash the microplate 6 times with PBST solution, add 100 μL of the supernatant solution of the phage-displayed antibody diluted with MPBS (2×10 9(cfu / mL), incubated at room temperature for 1 h. Wash the microplate 8 times with PBST solution, then add mouse anti-M13-HRP antibody diluted 1:5000 and incubate at room temperature for 1 h. Wash the microplate 12 times with PBST, add 100 μL of TMBZ to each well for color development, after reacting at 37 °C for 10 min, add 50 μL of 10% sulfuric acid solution to each well to terminate the reaction, and use iMark TM Microplate reader (Bio-Rad) to measure the absorbance at 450 nm and 630 nm and plot the standard curve.

[0067] The results of detecting the concentration of A29L by sandwich enzyme-linked immunosorbent assay are as Figure 6 shown. No obvious signal was detected when there was no antigen and when the antigen concentration was 1 and 10 ng / mL, while the signal gradually increased with the continuous increase of the concentration. The lowest detection limit (LOD) of this detection method was obtained as 0.382 ng / mL and the half-maximal effective concentration (EC50) was 495.8 ng / mL through the standard curve. The above 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, rather than to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions 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: 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.

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 further comprises a constant region, and the constant region 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 region of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE and IgD; the light chain constant region is selected from the κ type or λ type light chain constant region.

5. A polynucleotide sequence encoding the monoclonal antibody targeting monkeypox virus protein A29L according to any one of claims 1 to 4.

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

7. A host cell transformed by the expression vector according to claim 6.

8. A detection kit or antibody chip or antibody probe, comprising the monoclonal antibody targeting monkeypox virus protein A29L or its conjugate according to any one of claims 1 to 4, wherein the conjugate is obtained by coupling the monoclonal antibody targeting monkeypox virus protein A29L with biotin or a marker, and the marker is selected from at least one of a fluorescent dye, an enzyme, a radioactive isotope, a chemiluminescent reagent and a nanoparticle marker.

9. Use of the monoclonal antibody targeting monkeypox virus protein A29L according to any one of claims 1 to 4 or the detection kit or antibody chip or antibody probe according to claim 8 in the preparation of a product for detecting monkeypox virus.

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