Anti-human MxA antibodies or functional fragments thereof and uses thereof

Abstract

The present application relates to the technical field of biotechnology, in particular to anti-human MxA antibody or functional fragment thereof and application thereof. The present application provides a plurality of monoclonal antibodies with high affinity to human MxA protein through computer simulation technology, and verifies the affinity of each mutation and its related application, thereby providing a reference for the research in the field of immune detection of human MxA.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to genetic engineering products. More specifically, this invention discloses a high-affinity anti-human MxA antibody or a functional fragment thereof and its applications. Background Technology

[0002] Myxovirus resistance protein A (MxA) is a protein distributed in the cytoplasm, induced by type I interferon. The full-length MxA protein consists of 662 amino acids with a molecular weight of 76 kDa. Human cells treated with type I IFN produce two Mx proteins, named MxA and MxB. MxA has broad-spectrum antiviral activity and can be used as a specific indicator to differentiate whether an organism is infected with a virus, while MxB has no antiviral activity. Extensive research indicates that MxA has advantages such as stable biological activity, direct antiviral action, long half-life, good specificity, and convenient detection. Furthermore, MxA protein is highly sensitive to viral responses; even a very small amount of virus can induce cell expression of MxA protein. Therefore, MxA protein can be used for the early diagnosis of viral infections and for the differential diagnosis of clinical viral infections from bacterial or other microbial infections.

[0003] In emergency situations, rapid detection of MxA levels and quick differentiation between viral and bacterial infections are crucial for disease diagnosis, rational drug use, and evaluation of treatment efficacy. Current clinical methods for MxA detection include enzyme-linked immunosorbent assay (ELISA), fluorescence chromatography, and latex-enhanced immunoturbidimetry. Antibody affinity is particularly important in immunodiagnostics; therefore, developing a high-affinity MxA protein has significant clinical medical application value. Summary of the Invention

[0004] To address the problems existing in the prior art, the purpose of this invention is to provide a high-affinity anti-human MxA antibody or its functional fragment using computer-aided design, which can specifically bind to human MxA protein and be used for in vitro quantitative detection of MxA content in human samples (serum, plasma or whole blood), mainly for the auxiliary diagnosis of viral infections in clinical practice.

[0005] To achieve the above objectives, the present invention is implemented through the following technical solution, as detailed below:

[0006] Anti-human MxA antibodies or their functional fragments, including:

[0007] CDR-VH1, CDR-VH2, CDR-VH3, CDR-VL1, CDR-VL2, and CDR-VL3, characterized in that CDR-VH1 to CDR-VH3 are amino acid sequences consistent with CDR-VH1 to CDR-VH3 in the heavy chain variable region shown in SEQ ID NO:1, or are derived from their amino acid sequences by mutation, wherein the mutation sites are positions 29, 36, 62, 113, and 116 in the heavy chain variable region;

[0008] The CDR-VL1 to CDR-VL3 are the same amino acid sequences of the CDR-VL1 to CDR-VL3 of the light chain variable region shown in SEQ ID NO:2 or are derived from its amino acid sequence by mutation. The mutation sites are the positions of amino acids 35, 36, 56, and 115 in the light chain variable region.

[0009] The 29th, 36th, 62nd, 113th, and 116th positions of the heavy chain variable region and the 35th, 36th, 56th, and 115th positions of the light chain variable region are selected from the following combinations:

[0010] 1) Variable regions of heavy chains: 29V, 36S, 62D, 113N, 116S; and variable regions of light chains: 35G, 36W, 56K, 115P.

[0011] 2) Variable regions of heavy chains: 29T, 36R, 62D, 113N, 116K; and variable regions of light chains: 35G, 36N, 56K, 115P.

[0012] 3) Variable regions of heavy chains: 29T, 36R, 62D, 113N, 116D; and variable regions of light chains: 35G, 36N, 56K, 115P.

[0013] 4) Variable regions of heavy chains: 29V, 36S, 62Q, 113W, 116W and variable regions of light chains: 35G, 36R, 56K, 115P.

[0014] Furthermore, the CDR-VH1, CDR-VH2, CDR-VH3, CDR-VL1, CDR-VL2, and CDR-VL3 are defined by the Kabat, Chothia, IMGT, AbM, or Contact system.

[0015] Furthermore, CDR-VH1, CDR-VH2, CDR-VH3, CDR-VL1, CDR-VL2, and CDR-VL3 are defined by the IMGT system, and the antibody or its functional fragment includes the following complementarity-determining regions:

[0016] CDR-VH1: GY-X1-FT-X2-YW

[0017] CDR-VH2: VYPG-X1-GDT

[0018] CDR-VH3: ARGYD-X1-SF-X2-Y

[0019] CDR-VL1: QTIVHSN-X1-X2-TY

[0020] CDR-VL2: X1-VS

[0021] And CDR-VL3: FQGSHV-X1-PT,

[0022] in

[0023]

[0024] The present invention has found that when the above-mentioned mutation sites in each complementarity-determining region are the above-mentioned amino acid residues, the antibody has a better affinity for the MxA protein.

[0025] In this article, "CDR" refers to the "complementarity-determining region" within the variable sequence of the antibody. Each of the heavy and light chains has three CDRs, starting from the N-terminus of either the heavy or light chain.

[0026] Antigen binding sites may include six CDRs (CDR-VH1, CDR-VH2, CDR-VH3, CDR-VL1, CDR-VL2, and CDR-VL3 in this invention). A polypeptide containing a single CDR (e.g., CDR-VH1, CDR-VH2, CDR-VH3, CDR-VL1, CDR-VL2, and CDR-VL3) can be referred to as a "molecular recognition unit." Crystallographic analysis of antigen-antibody complexes has demonstrated that the amino acid residues of the CDRs form extensive contacts with the bound antigen, with the most extensive antigen contact being with the heavy chain CDR3. Therefore, the molecular recognition unit may primarily be responsible for the specificity of the antigen binding site. Generally, CDR residues directly and substantially participate in influencing antigen binding.

[0027] Further, the antibody includes heavy chain backbone regions FR1-H, FR2-H, FR3-H and FR4-H and light chain backbone regions FR1-L, FR2-L, FR3-L and FR4-L, wherein FR1-H is an amino acid sequence as shown in SEQ ID NO:3 or an amino acid sequence having at least 80% homology with it;

[0028] The FR2-H is an amino acid sequence as shown in SEQ ID NO:4 or an amino acid sequence having at least 80% homology with it;

[0029] The FR3-H is an amino acid sequence as shown in SEQ ID NO:5 or an amino acid sequence having at least 80% homology with it;

[0030] The FR4-H is an amino acid sequence as shown in SEQ ID NO:6 or an amino acid sequence having at least 80% homology with it;

[0031] The FR1-L is an amino acid sequence as shown in SEQ ID NO:7 or an amino acid sequence having at least 80% homology with it;

[0032] The FR2-L is an amino acid sequence as shown in SEQ ID NO:8 or an amino acid sequence having at least 80% homology with it;

[0033] The FR3-L is an amino acid sequence as shown in SEQ ID NO:9 or an amino acid sequence having at least 80% homology with it;

[0034] The FR4-L is an amino acid sequence as shown in SEQ ID NO:10 or an amino acid sequence having at least 80% homology with it.

[0035] Furthermore, the anti-MxA antibody or its functional fragment further includes a constant region;

[0036] Preferably, the constant region includes a heavy chain constant region and / or a light chain constant region;

[0037] Optionally, the heavy chain constant region is selected from the heavy chain constant regions of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; the light chain constant region is selected from the κ-type or λ-type light chain constant region.

[0038] Optionally, the species source of the constant region is cattle, horses, dairy cows, pigs, sheep, goats, rats, mice, dogs, cats, rabbits, camels, donkeys, deer, mink, chickens, ducks, geese, turkeys, fighting cocks, or humans; optionally, the species source of the constant region is mice.

[0039] Furthermore, the functional fragment is selected from any one of the antibody's F(ab')2, Fab', Fab, Fv, and scFv.

[0040] The term "antibody" includes various forms of antibody structures, including but not limited to complete antibodies and antibody fragments. Antibodies according to the invention are preferably goat, sheep, mouse, rabbit, or rat antibodies, chimeric antibodies, or further genetically engineered antibodies, provided they retain the characteristic properties according to the invention. An "antibody fragment" comprises a portion of a full-length antibody, preferably its variable domains, or at least its antigen-binding site. Examples of antibody fragments include biantibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; single-chain antibody molecules; scFv, sc(Fv)2; biantibodies; and multispecific antibodies formed from antibody fragments.

[0041] On the other hand, the present invention discloses an antibody conjugate comprising the anti-MxA antibody or a functional fragment thereof and a conjugated portion thereof;

[0042] Optionally, the coupling portion is selected from purified tags or detectable labels, such as colloidal gold, radioactive labels, luminescent substances, colored substances, enzymes such as fluorescent labels, chromophore labels, electron-dense labels such as radioactive isotopes, fluorophores, rhodamine and its derivatives, luciferase, luciferin, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucosylamylase, lysozyme, carbohydrate oxidase, glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, biotin / antibiotin protein, and one or more of spin labels;

[0043] Optionally, the coupling portion is selected from solid-phase carriers, such as magnetic microspheres, plastic microspheres, plastic microparticles, microporous plates, glass, capillaries, nylon, and nitrocellulose membranes.

[0044] On the other hand, the present invention discloses a reagent or kit containing the aforementioned anti-MxA antibody or a functional fragment thereof.

[0045] On the other hand, this invention discloses any of the following applications,

[0046] The application of the anti-MxA antibody or its functional fragment described in E1) in the preparation of reagents or kits for detecting inflammation or infection;

[0047] The application of the anti-MxA antibody or its functional fragment described in E2) in virus detection products containing influenza virus, adenovirus (ADV), respiratory syncytial virus (RSV), parainfluenza virus (PIV), EB virus, herpes virus, or myxovirus resistance protein A.

[0048] The use of the antibody-drug conjugate described in E3 in the preparation of reagents or kits for detecting inflammation or infection.

[0049] On the other hand, the present invention discloses a method for detecting MxA protein, the method comprising the process of binding the anti-MxA antibody or its functional fragment as described in claims 1-6 to the MxA protein.

[0050] Beneficial effects:

[0051] The purpose of this invention is to provide a high-affinity antibody against human MxA and a method for detecting MxA. The antibody provided by this invention can specifically bind to MxA, exhibiting excellent binding activity and affinity, which is beneficial for improving the specificity and sensitivity of detection. It can be used for the detection of MxA and the diagnosis of diseases with abnormal MxA levels, providing more diverse protein options for the detection of MxA and the diagnosis of diseases with abnormal MxA levels. Attached Figure Description

[0052] Figure 1 The three-dimensional structure of the MxA antigen;

[0053] Figure 2 The three-dimensional structure of the variable region of antibody 11#MxA;

[0054] Figure 3 This is a structural diagram of the antigen-antibody complex;

[0055] Figure 4 This is a fitting graph showing the affinity of antigen MxA for different antibodies. Detailed Implementation

[0056] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0057] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Any methods and materials similar to or equivalent to those described herein may be used in the practice or testing of formulations or unit doses herein. Unless otherwise stated, the techniques employed or considered herein are standard methods. Materials, methods, and examples are illustrative and not limiting in nature.

[0058] As used herein, the terms “comprising,” “including,” “having,” “may,” and their variations are intended to be open-ended transitional phrases, terms, or words that do not exclude the possibility of additional actions or structures.

[0059] Unless otherwise specified, the practice of this invention will employ conventional techniques of cell biology, molecular biology (including recombinant technologies), microbiology, biochemistry, and immunology, which are within the capabilities of those skilled in the art. Such techniques are well explained in the literature, such as *Molecular Cloning: A Laboratory Manual*, 2nd edition (Sambrook et al., 1989); *Oligonucleotide Synthesis* (edited by M.J. Gait, 1984); *Animal Cell Culture* (edited by R.R. Freshney, 1987); *Methods in Enzymology* (Academic Press, Inc.); and *Handbook of Experimental Immunology* (D.M. Weir and CC. Blackwyn). The references cited are: J.M. Miller and M.C. Salos (eds., 1987); F.M. Usubel et al. (eds., 1987); PCR: The Polymerase Chain Reaction (eds., Mullis et al., 1994); and J.E. C. Olgan et al. (eds., 1991), each of which is explicitly incorporated herein by reference.

[0060] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0061] Example 1: Preparation of Monoclonal Antibodies

[0062] A. Mouse immunization and antibody detection

[0063] Five 6-8 week old SPF-grade female BALB / c mice were selected. Freund's complete adjuvant was mixed with human MxA protein at a concentration of 1 mg / ml in equal volumes and emulsified. The emulsified antigen was used to immunize the 6-8 week old SPF-grade female BALB / c mice via paw injection or subcutaneous injection in the back, with each mouse receiving 40 μg of antigen protein. Two weeks after the initial immunization, the antigen protein was mixed with Freund's incomplete adjuvant and emulsified, and again injected via paw injection or subcutaneous injection in the back, with each mouse receiving 40 μg of antigen protein. Two weeks after the second immunization, the antigen protein was mixed with Freund's incomplete adjuvant and emulsified, and again injected via paw injection or subcutaneous injection in the back, with each mouse receiving 40 μg of antigen protein. Two weeks later, blood was collected via tail vein, the supernatant was collected by centrifugation, and serum titer was detected by ELISA. Immunization was repeated every two weeks, and serum titer was measured. After three immunizations, the serum titer, after a million-fold dilution, reached over 2.0. A serum titer of 10 was selected for screening. 6 Lymphocytes were isolated from the mice mentioned above for cell fusion.

[0064] B. Cell fusion and screening of positive hybridoma cells and subcloning

[0065] Lymphocytes from immunized mice were isolated and fused with cultured SP2 / 0 cells via PEG1500-mediated fusion or electrofusion. The fused cells were cultured in HAT-1640 medium containing 20% ​​FBS serum for selection. After one week, the medium was changed, and after another 7 days of culture, the culture supernatant was used for positive clone selection. Human MxA protein was used for screening positive wells. Wells with a high ELISA positive value to cell number ratio were selected for multiple subcloning. ELISA plates were coated with human MxA protein. The culture supernatant of the subclones was used to screen for monoclonal clones that showed affinity under antigen-coated conditions. The monoclonal hybridoma cell line with the highest affinity was selected, ultimately yielding a hybridoma cell line with a high antibody titer that secretes MxA protein monoclonal antibody, named 11#MxA, which exhibited good stability.

[0066] C. Production and purification of monoclonal antibodies

[0067] Two groups of 6-8 week old BALB / c mice were selected and injected intraperitoneally with 500 μL of paraffin oil to suppress the immune response. One week after injection, 0.5 ml of 11#MxA cells (approximately 1 × 10⁻⁶ cells) was injected intraperitoneally into the mice. 6 Ascites fluid collection began two weeks later. The collected ascites fluid was purified by ammonium sulfate precipitation and affinity purification with protein G to obtain the target antibody 11#MxA.

[0068] Example 2: Validation of antibody performance

[0069] 1. ELISA Affinity Test

[0070] Dilute MxA antigen to 1 μg / mL with carbonate coating buffer (pH 9.6), add 100 μL per well to a 96-well ELISA plate, and incubate overnight at 4°C. Remove the plate, wash three times with PBS buffer containing 0.05% Tween-20, and blot dry. Add 100 μL / well of diluent (1% BSA, 0.1% PBST) (excluding A-well). Dilute antibody to 200 ng / mL, add to A-well, and serially dilute 3-fold to G-well; H-well serves as a blank control. Incubate at 37°C for 30 min. Remove the plate, wash three times, and blot dry. Dilute goat anti-mouse-HRP 1:3000 with diluent, add 100 μL / well, and incubate at 37°C for 30 min. Remove the plate, wash three times, and blot dry. Mix colorimetric solutions A and B at a 1:1 ratio, and immediately add 100 μL of the mixture to each well. Incubate at room temperature for 3 minutes. Stop the reaction by adding 50 μL of 0.5 M sulfuric acid to each well. Place the plate in a microplate reader and read the OD450 value. The results are shown in Table 1 below.

[0071] Table 1 Valence Measurement

[0072]

[0073] As shown in Table 1, the 11#MxA antibody can recognize the MxA antigen, but the signal value is weak at lower antibody concentrations. The following will use computer-aided design to improve the affinity of the 11#MxA antibody.

[0074] Example 3: Enhanced Antibody Affinity

[0075] The heavy chain variable region of antibody 11#MxA in Example 1 is shown in SEQ ID NO:1, wherein the amino acid sequences of each complementarity-determining region on the heavy chain variable region are as follows:

[0076] CDR-VH1: GYV(X1)-FTS(X2)-YW

[0077] CDR-VH2: VYPGD(X1)-GDT

[0078] CDR-VH3: ARGYDN(X1)-SFS(X2)-Y

[0079] Its light chain variable region is shown in SEQ ID NO:2, wherein the amino acid sequences of each complementarity-determining region on the light chain variable region are as follows:

[0080] CDR-VL1:QTIVHSNG(X1)-R(X2)-TY

[0081] CDR-VL2: K(X1)-VS

[0082] CDR-VL3: FQGSHVP(X1)-PT

[0083] Based on antibody 11#MxA, mutations were made at sites related to antibody activity in the complementarity-determining region.

[0084] 1. Homologous modeling

[0085] The MxA antigen structure was modeled using AlphaFold2 for homology, and the resulting three-dimensional structure is shown below. Figure 1 As shown. Homology modeling of the variable region of antibody 11#MxA was performed using the ModelAntibody module in Discovery Studio, and the resulting 3D structure is shown below. Figure 2 As shown.

[0086] 2. Molecular docking

[0087] The structures of the antigen and antibody were pretreated: hydrogen was added, side chain residues were repaired, and the CHARMm force field was increased. Then, 5000 steps of Powell energy optimization were performed until energy convergence. The antigen and antibody were molecularly docked using the ZDOCK (http: / / zdock.umassmed.edu / ) online protein-protein docking software to obtain the antigen-antibody interaction complex. The complex was then optimized using molecular dynamics. Before the kinetic simulation, energy optimization was performed twice, sequentially on the solvent molecules and the entire system. During the first optimization, hydrogen was added to the solute molecules. To address the constraint limitations, the system underwent 2000 steps of optimization using the steepest descent method, followed by another 2000 steps using the conjugated gradient method. A second optimization, removing the constraints, involved 10,000 steps of steepest descent optimization and 5,000 steps of conjugated gradient method optimization on the entire complex. Subsequently, the entire system was further optimized. The system was constrained by a coupling coefficient of 1.0 / ps and heated from 0K to 300K over 100ps. The constraints were then removed and the system was equilibrated for 500ps. Finally, a 20ns MD simulation was performed in the NPT ensemble at 300K and 1.0 atm. During the kinetic simulation, the step size was set to 2 fs, and the conformation was acquired every 1000 steps. The kinetically equilibrated structure was selected as the final antigen-antibody complex structure, as shown in the results. Figure 3 As shown.

[0088] 3. Complex Interaction Analysis

[0089] The interaction between antigen and antibody complexes was analyzed using the Dock and Analyze Protein Complexes module in Discovery Studio to identify epitopes of antigens and antibodies and key interactions.

[0090] 4. Virtual amino acid mutation

[0091] Virtual amino acid mutations can determine the optimal combination of amino acid mutations through alanine scanning and saturation mutagenesis, thus providing guidance for site-directed amino acid mutagenesis in experiments. This example uses the CalculateMutation Energy (Binding) module in Discovery Studio to perform interaction-based virtual amino acid mutations on antigen-antibody complexes, selecting the ligands (i.e., antigens) surrounding the antigens. All antibody amino acids within the complex were selected as subsequent mutation sites. The first step involved ALA mutation of the complex. Following ALA mutation, sites with decreased affinity were selected, suggesting these amino acids are key to receptor-ligand interaction.

[0092] The sites of reduced affinity are amino acids at positions 29, 36, 62, 113, and 116 in the heavy chain variable region and positions 35, 36, 56, and 115 in the light chain variable region.

[0093] The second step is to perform saturation mutations, i.e. single-point mutations, on these sites with reduced affinity, as shown in Table 2. The mutation results are shown in Table 3. It can be seen that mutation 2 has the greatest increase in mutation energy (i.e. affinity). Therefore, mutation 2 is used as the backbone sequence for subsequent combined mutations.

[0094] The third step involves performing combined mutations based on the single-point mutation 2, and screening for other mutation sites with better affinity. The antibodies with increased affinity after combined mutations are shown in Table 4, and the effects are shown in Table 5. It can be seen that after combined mutations of 2-4, 2-6, 2-9, 2-7 and 2-16, the affinity was significantly improved compared to the single-point mutation.

[0095] Table 2 Saturation mutation sites related to antibody activity

[0096]

[0097] Table 3 Mutation energy at saturation mutation sites

[0098]

[0099]

[0100] Table 4. Combination mutation sites related to antibody activity

[0101]

[0102] Table 5 Mutation energy at saturation mutation sites

[0103]

[0104] 5. Valence determination

[0105] In this embodiment, mutant antibodies with a mutation energy less than -4 from Table 6 were selected for wet assay verification. MxA protein concentration was 1000 ng / ml, and the absorbance of the mutant antibody's OD450 was measured. The results are as follows:

[0106] Table 6. Antibody Mutation Titer Test

[0107]

[0108] It can be seen that mutations 2-4, 2-9, and 2-16 all exhibit high affinity. To further verify the affinity of mutations 2-4, 2-9, and 2-16, the applicant of this invention performed affinity tests on the aforementioned mutant antibodies. In this embodiment, a Biacore T200 Cytiva instrument was used, employing SPR technology to determine the affinity between the antigen and antibody (single-concentration affinity determination and ranking). The results are shown in Table 7.

[0109] Table 7 Affinity Test

[0110] Antigen MxA Mutation 2-16 1.37E+05 2.33E-06 1.70E-11 19.8 Antigen MxA Mutations 2-4 1.24E+05 9.96E-05 8.00E-10 10.7 Antigen MxA Mutations 2-9 1.94E+05 1.15E-07 5.93E-13 21.2 Antigen MxA 11#MxA 1.47E+05 1.33E-04 9.04E-10 27.3

[0111] Figure 4 The mutations in the samples are as follows: Anti-M1 corresponds to mutations 2-9 in Table 7; Anti-M2 corresponds to mutation 11#MxA in Table 7; Anti-M3 corresponds to mutations 2-4 in Table 7; and Anti-M6 corresponds to mutations 2-16 in Table 7.

[0112] As shown in the table above, the affinity between antigen MxA and different antibodies was detected and ranked using the Biacore T200. The affinity from high to low was antibody mutation 2-9, mutation 2-16, mutation 2-4, and 11#MxA. Mutations 2-9 and 2-16 had high affinity for the antigen, and their dissociation constants exceeded the detection range of the instrument.

[0113] 6. Stability testing

[0114] Antibodies 11#MxA, mutation 2-4, mutation 2-9, and mutation 2-16 were placed in a predetermined buffer (PBS, 0.05% ProClin). TMThe antibodies were accelerated at 37℃ for 7 and 14 days (300), and the accelerated antibodies were evaluated by indirect ELISA. A control was used at 4℃ to assess the long-term stability of the antibodies. Additionally, the antibodies underwent five freeze-thaw cycles at -20℃, and the results were shown as the deviation between the values ​​at 4℃ and the accelerated values. The measurement results are shown in Table 8. It can be seen that the deviations of antibodies 11#MxA, mutant 2-4, mutant 2-9, and mutant 2-16 were all within 10%, indicating that all four antibodies exhibited good stability.

[0115] Table 8 Stability Study

[0116] 11#MxA -5.3％ -6.6％ -4.9％ Mutations 2-4 -4.4％ -8.5％ -7.8％ Mutations 2-9 -5.2％ -6.9％ -4.9％ Mutation 2-16 -7.8％ -8.5％ -9.6％

[0117] Example 3: Antibody Application Research

[0118] This embodiment applies antibodies 11#MxA, mutation 2-4, mutation 2-9, and mutation 2-16 to magnetic particle chemiluminescence detection. The specific operation is as follows:

[0119] A) Magnetic bead coating

[0120] Tosyl magnetic microparticles containing toluenesulfonyl groups were selected, and anti-myxovirus antibody protein A (MxA) was coupled to the surface of the microparticles. The specific preparation method is as follows: First, the magnetic beads were washed with magnetic bead coating solution to remove impurities from the surface of the magnetic beads. Then, according to the coating ratio, the above four antibody strains and the coating enhancer were added respectively. After vortexing and mixing, the mixture was placed on a 37°C inverted mixer and coated for 24 hours under suitable rotation speed conditions. After the magnetic beads and antibodies formed stable covalent bonds, the mixture was washed and blocked for 24 hours with Tris buffer containing blocking protein to remove non-specifically adsorbed protein on the surface of the magnetic beads and block the remaining surface active groups. Finally, the mixture was washed three times with magnetic bead preservation solution, and the coated magnetic beads were stored at 4°C.

[0121] B) Enzyme-labeled antibody

[0122] To verify the affinity and reactivity of the four antibody strains, a single-factor experiment was conducted, maintaining the same preparation process and concentration of the enzyme-labeled antibody in the kit. The enzyme-labeled antibody solution used an alkaline phosphatase-labeled anti-MxA monoclonal monomer. The specific preparation method was as follows: After desalting the antibody and enzyme, the antibody was activated with Traut's reagent (2-iminothione). Traut's reagent reacts with primary amines to introduce thiol groups onto the protein. Sulfo-SMCC was added to the alkaline phosphatase-coupled portion. Sulfo-SMCC, a heterobifunctional protein cross-linking agent, contains a maleimide group and an N-hydroxysuccinimide active ester at both ends of its molecule. At pH ≥ 7, the active ester group forms a stable amide bond with the amino group, coupling with the enzyme. After activation of both the enzyme and antibody, a ligation reaction was performed. Under pH 6.5-7.5 conditions, the maleimide in the enzyme-coupled portion specifically reacts with the thiol group to form a stable thiol-ether bond. Finally, enzyme-labeled antibody polymers with different degrees of polymerization were separated using a purification instrument and stored in appropriate buffers for later use.

[0123] 3) Determination of chemiluminescence reactivity of magnetic particles

[0124] Following the magnetic microparticle chemiluminescence method, an MxA detection kit was fabricated and assembled. Except for the magnetic bead-conjugated antibody, which used the four antibody strains mentioned above, all other components of the kit were kept the same. Quality control samples of different concentrations were measured, and each quality control sample was measured three times (n=3). The affinity and reactivity of the four antibody strains were evaluated by comparing the differences in light intensity. The experimental results are shown in Table 9 below.

[0125] Table 9 Reactivity Measurement

[0126]

[0127]

[0128]

[0129]

[0130] In summary, when using the sandwich method for detection, mutation 2-9 exhibits the strongest binding affinity to the analyte, followed by mutation 2-16; that is, the experimental results show that the binding affinity to the analyte is: mutation 2-9 > mutation 2-16 > mutation 2-4 > 11#MxA. Therefore, the computer-aided design method used in this embodiment to improve antibody affinity helps us screen for high-affinity antibodies and saves significant time and costs.

[0131] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details and illustrations shown and described herein.

Claims

1. An anti-human MxA antibody, characterized in that, The antibody includes the following complementarity-determining regions: CDR-VH1: GYTFTRYW; CDR-VH2: VYPGDGDT; CDR-VH3: ARGYDNSFDY; CDR-VL1: QTIVHSNGNTY; CDR-VL2: KVS; And CDR-VL3: FQGSHVPPT; The CDR is defined by the IMGT system.

2. The anti-human MxA antibody as described in claim 1, characterized in that, The antibody comprises heavy chain backbone regions FR1-H, FR2-H, FR3-H and FR4-H and light chain backbone regions FR1-L, FR2-L, FR3-L and FR4-L, wherein FR1-H is an amino acid sequence as shown in SEQ ID NO:3 or an amino acid sequence having at least 80% homology with it. The FR2-H is an amino acid sequence as shown in SEQ ID NO:4 or an amino acid sequence having at least 80% homology with it; The FR3-H is an amino acid sequence as shown in SEQ ID NO:5 or an amino acid sequence having at least 80% homology with it; The FR4-H is an amino acid sequence as shown in SEQ ID NO:6 or an amino acid sequence having at least 80% homology with it; The FR1-L is an amino acid sequence as shown in SEQ ID NO:7 or an amino acid sequence having at least 80% homology with it; The FR2-L is an amino acid sequence as shown in SEQ ID NO:8 or an amino acid sequence having at least 80% homology with it; The FR3-L is an amino acid sequence as shown in SEQ ID NO:9 or an amino acid sequence having at least 80% homology with it; The FR4-L is an amino acid sequence as shown in SEQ ID NO:10 or an amino acid sequence having at least 80% homology with it.

3. The anti-human MxA antibody as described in claim 2, characterized in that, The anti-human MxA antibody also contains a constant region.

4. The anti-human MxA antibody as described in claim 3, characterized in that, The constant region includes the heavy chain constant region and / or the light chain constant region.

5. The anti-human MxA antibody as described in claim 4, characterized in that, The heavy chain constant region is selected from the heavy chain constant regions of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; the light chain constant region is selected from the κ-type or λ-type light chain constant region.

6. The anti-human MxA antibody as described in claim 5, characterized in that, The species source of the constant region is cattle, horses, pigs, sheep, goats, rats, mice, dogs, cats, rabbits, camels, donkeys, deer, minks, chickens, ducks, geese, or humans.

7. The anti-human MxA antibody as described in claim 6, characterized in that, The species source of the constant region is mice.

8. An antibody conjugate, characterized in that, The antibody conjugate comprises the anti-human MxA antibody as described in any one of claims 1-7 and a conjugated portion thereof, wherein the conjugated portion is selected from one or more of colloidal gold, luminescent substances, colored substances, enzymes or solid-phase carriers.

9. A reagent or kit, characterized in that, The reagent or kit contains an anti-human MxA antibody as described in any one of claims 1-7.

10. The following application, characterized in that, E1) The use of the anti-human MxA antibody as described in any one of claims 1-7 in the preparation of auxiliary diagnostic reagents or kits for viral infection; E2) The use of the antibody conjugate as described in claim 8 in the preparation of auxiliary diagnostic reagents or kits for viral infection.

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