An anti-mxa antibody or functional fragment thereof and uses thereof

Abstract

The application discloses an anti-MxA antibody or a functional fragment thereof and application thereof. The anti-MxA antibody or the functional fragment thereof disclosed by the application comprises a heavy chain complementarity determining region and a light chain complementarity determining region, has good specificity and sensitivity to MxA, and can be used for detecting MxA and diagnosing or assisting in diagnosing related diseases taking MxA as a marker, and has important clinical value.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to an anti-MxA antibody or a functional fragment thereof and its applications. Background Technology

[0002] MxA protein belongs to the dynamin superfamily of large GTPases and is one of the antiviral proteins induced by type I interferon (IFNα / β) to be produced by host cells, exhibiting broad antiviral activity. The full-length MxA protein contains 662 amino acids and has a molecular weight of 76 kDa. Its protein structure includes monomeric and oligomeric forms such as dimers, trimers, and tetramers. MxA protein typically accumulates in the cytoplasm and endoplasmic reticulum. The membrane compartments of the endoplasmic reticulum provide an interaction platform that facilitates viral target recognition. MxA detects viral infection by sensing and capturing nucleocapsid-like structures, playing a crucial role in antiviral activity. Therefore, many viruses (including influenza, parainfluenza virus, adenovirus, herpes simplex virus, Epstein-Barr virus, measles virus, Coxsackie virus, hepatitis B virus, and Thogoto virus) induce the production of large amounts of MxA protein in cells after infecting the human body, while bacterial infection does not induce high expression of MxA protein. In clinical practice, rapid differentiation between bacterial and viral infections is crucial to guide treatment. Detecting MxA protein expression can help distinguish between viral and bacterial infections. Furthermore, MxA protein is highly sensitive to viral responses; even very small viral loads can induce its expression in cells. Therefore, detecting MxA protein expression can also be used for the early diagnosis of viral infections.

[0003] In emergency situations, rapid differentiation between viral and bacterial infections is crucial for timely and accurate medication and ultimately, saving lives. In immunodiagnostics, the sensitivity and linearity of detection are often determined by the antibody or antibody pair used; therefore, the specificity, sensitivity, and linearity of the antibody are particularly important. Currently, there are few reported MxA antibodies both domestically and internationally, and the clinical diagnostic efficacy of these antibodies has not been fully confirmed. Therefore, developing antibodies with high recognition sensitivity to the MxA protein induced by viral infection in humans has significant clinical medical application value. Summary of the Invention

[0004] The purpose of this invention is to provide an MxA antibody or a functional fragment thereof with high affinity and high specificity for MxA protein and its application. The antibody or the functional fragment thereof has good affinity for MxA, and the detection of MxA using the antibody or the functional fragment thereof has good sensitivity and specificity.

[0005] Specifically as follows: On one hand, the present invention provides an anti-MxA antibody or a functional fragment thereof, wherein the anti-MxA antibody or the functional fragment thereof has the following complementarity-determining region: CDR-VL1: RSSQSI-X1-HSNGN-X2-YLE, where X1 is V or T, and X2 is R or T; CDR-VL2: K-X1-SN-X2-FS, where X1 is V or H, and X2 is R or A; CDR-VL3: F-X1-VSHVP-X2-T, where X1 is Q and X2 is L or W; CDR-VH1: T-X1-TL-X2, where X1 is Y or D, and X2 is H; CDR-VH2: FI-X1-PTSSY-X2-NYN-X3-KFKD, where X1 is N or I, X2 is T or S, and X3 is Q or S; CDR-VH3: GS-X1-LR-X2-SMDY, where X1 is Y or W, and X2 is P or Y.

[0006] Furthermore, in the complementary determination region, X1 of CDR-VL1 is T, X2 of CDR-VL2 is A, X1 of CDR-VL3 is Q, X2 of CDR-VH1 is H, X1 of CDR-VH2 is N, and X2 of CDR-VH3 is Y.

[0007] The CDR segmentation described in this invention uses the Kabat algorithm.

[0008] 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.

[0009] 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, or CDR-VL3) can be termed 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.

[0010] The inventors of this invention have discovered that when the mutation sites in each complementarity-determining region are the aforementioned amino acid residues, the antibody has a better affinity for the MxA protein.

[0011] In an optional embodiment, X1 in CDR-VL1 is V; In an optional embodiment, X1 in CDR-VL1 is T; In an optional embodiment, X2 in CDR-VL1 is R; In an optional embodiment, X2 in CDR-VL1 is T; In an optional embodiment, X1 in CDR-VL2 is V; In an optional embodiment, X1 in CDR-VL2 is H; In an optional embodiment, X2 in CDR-VL2 is R; In an optional embodiment, X2 in CDR-VL2 is A; In an optional embodiment, X1 in the CDR-VL3 is Q; In an optional embodiment, X2 in CDR-VL3 is L; In an optional embodiment, X2 in the CDR-VL3 is W; In an optional embodiment, X1 in CDR-VH1 is Y; In an optional embodiment, X1 in CDR-VH1 is D; In an optional embodiment, X2 in CDR-VH1 is H; In an optional embodiment, X1 in CDR-VH2 is N; In an optional embodiment, X1 in CDR-VH2 is I; In an optional embodiment, X2 in CDR-VH2 is T; In an optional embodiment, X2 in CDR-VH2 is S; In an optional embodiment, X3 in CDR-VH2 is Q; In an optional embodiment, X3 in CDR-VH2 is S; In an optional embodiment, X1 in CDR-VH3 is Y; In an optional embodiment, X1 in CDR-VH3 is W; In an optional embodiment, X2 in CDR-VH3 is P; In an optional embodiment, X2 in the CDR-VH3 is Y.

[0012] Preferably, each complementarity-determining region is selected from any one of the following mutation combinations: Mutation 2 T V L Y S / Q W Mutation 2-1 R V L Y T / Q Y Mutation 2-2 R H L D T / S W Mutation 2-3 T V L Y T / Q W Mutations 2-4 R H L D T / Q Y Mutations 2-5 T H W Y T / S W Mutations 2-6 T H W Y S / S Y Mutations 2-7 R H L Y S / Q Y Mutations 2-8 T H L Y T / Q W Mutations 2-9 R V W D T / S Y Mutation 2-10 R H W D S / S Y Mutation 2-11 T V L D S / S W Mutation 2-12 T H L D S / S Y Mutation 2-13 R H L Y S / S W Mutation 2-14 T V L D T / S Y Mutation 2-15 T V L D S / Q Y Mutation 2-16 R H W Y T / Q W Mutation 2-17 R V W Y S / S W Mutation 2-18 R V L D S / S Y Mutation 2-19 T H W D S / Q W Mutation 2-20 R V L Y T / S W Mutation 2-21 R V W Y S / Q Y Mutation 2-22 T V W D T / Q W Mutation 2-23 T V W D S / S Y Mutation 2-24 T H W Y T / Q Y Mutation 2-25 T V W Y S / Q W Mutation 2-26 T H W D T / S Y Mutation 2-27 R H L D S / Q W Mutation 2-28 R V W D S / Q W Mutation 2-29 T V W Y T / S Y Mutation 2-30 T H L Y T / S W Further, the antibody includes light chain backbone regions FR1-L, FR2-L, FR3-L and FR4-L and heavy chain backbone regions FR1-H, FR2-H, FR3-H and FR4-H; the heavy chain backbone regions FR1-H, FR2-H, FR3-H and FR4-H are selected from SEQ ID NO:1-4 in sequence; the light chain backbone regions FR1-L, FR2-L, FR3-L and FR4-L are selected from SEQ ID NO:5-8 in sequence.

[0013] Furthermore, the antibody also contains a constant region.

[0014] Preferably, the constant region is selected from the constant regions of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE and IgD. Preferably, 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.

[0015] Preferably, the functional fragment is selected from any one of VHH, F(ab')2, Fab', Fab, Fv and scFv of the antibody.

[0016] On the other hand, the present invention discloses a conjugate comprising an anti-MxA antibody or a functional fragment thereof as described above, covalently linked to a chemical label or a biolabel.

[0017] On the other hand, the present invention discloses a conjugate formed by conjugating an anti-MxA antibody or its functional fragment as described above and / or a conjugate as described above with a solid or semi-solid medium.

[0018] On the other hand, the present invention discloses the use of the anti-MxA antibody or its functional fragment and / or the conjugate and / or the conjugate as described above in a kit for detecting MxA expression.

[0019] Beneficial effects: This invention not only provides a stable monoclonal antibody (wild-type) with high affinity for the MxA protein, but also introduces several high-performance mutations into this wild-type antibody, enabling it to achieve specific and efficient binding to MxA even in the complex environment of whole blood cell lysates where numerous interfering factors exist. The MxA immunochromatographic diagnostic reagent developed based on this antibody can rapidly identify viral or bacterial infections, possessing significant clinical value. Attached Figure Description

[0020] Figure 1 Linearity validation of antibodies 14# and mutation 2-3 as labeled antibodies in immunochromatographic assays. Detailed Implementation

[0021] 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.

[0022] 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.

[0023] 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.

[0024] 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); and *Animal Cell Culture*. The following references are explicitly incorporated herein by reference: *Cell Culture* (R.Freshney, ed., 1987); *Methods in Enzymology* (Academic Press, Inc.); *Handbook of Experimental Immunology* (D.M.Weir and C.C.Blackwell, eds.); *Gene Transfer Vectors for Mammalian Cells* (J.M. Miller and M.C.C. Alos, eds., 1987); *Current Protocols in Molecular Biology* (F.M.A. Susubel et al., eds., 1987); *PCR: The Polymerase Chain Reaction* (Mullis et al., eds., 1994); and *Current Protocols in Immunology* (J.E. C. Olgan et al., eds., 1991).

[0025] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0026] Example 1: Preparation of Monoclonal Antibodies A. Immunogen preparation The full-length human MxA protein (10-662Aa, P20591) was constructed into a eukaryotic expression vector to obtain the MxA expression plasmid. (I) DH10bac Conversion 1. Take 100 μL of DH10bac competent cells and place on ice for 10 min until completely thawed. Add 50-200 ng (approximately 1 μL) of plasmid, mix gently, and place on ice for 30 min. 2.42℃ heat shock for 45 s; 3. Place on ice for 2-5 minutes; 4. Add 700 μL of LB medium, incubate at 37°C and 225 rpm for 4 hours; 5. Spread 100 μL onto an x-gal plate and incubate inverted at 37°C overnight; 6. Streak white colonies on an X-gal plate and incubate inverted at 37°C overnight; 7. If the colonies remain white after overnight observation, the transformation can be considered successful. Alternatively, colony PCR can be performed for further verification.

[0027] (II) Plasmid Extraction 1. Pick white clones and place them in 2 ml LB medium (50 ug / ml kanamycin, 7 ug / ml gentamicin, 10 ug / ml tetracycline, X-gal 100 ug / ml, IPTG 40 ug / ml), and incubate at 37℃ on a shaker at 250-300 rpm for at least 24 h; 2. Extract plasmids using PureLink HiPure Plasmid DNA Purification Kits; 3. After extraction, dissolve the plasmid in water and measure the concentration to be greater than 500 ng / ul before use. (III) Fermentation Expression Shake the 293 cell suspension well, pipette approximately 1 ml of the cell suspension into a sterile 1.5 ml EP tube, count the cells, and observe their state. Adjust the cell density to 3.0 × 10⁻⁶. 6 Cells were inoculated at 1L per mL. The expression plasmid was transfected into 293 cells in suspension culture using PEI (polyethyleneimine)-mediated transfection. Culture medium and feed were added within 18-20 hours post-transfection, and fermentation continued for 5 days. The cell pellet was collected by centrifugation at 10°C for 30 min at 4000g. The cell pellet was resuspended in PBS and then sonicated to disrupt the cell walls. After disruption, the supernatant was collected by centrifugation for protein purification. B. Mouse immunization and antibody detection Five 6-8 week old SPF-grade female BALB / c mice were selected. Freund's complete adjuvant was mixed with an equal volume of MxA protein at a concentration of 1 mg / mL and emulsified. The emulsified antigen was used to immunize the 6-8 week old SPF-grade female BALB / c mice, with each mouse receiving 40 μg of antigen protein via paw or subcutaneous injection in the back. Two weeks after the initial immunization, the antigen protein was mixed with Freund's incomplete adjuvant and emulsified, and each mouse received 40 μg of antigen protein again via paw or subcutaneous injection in the back. Two weeks later, blood was collected via tail vein, the supernatant was collected by centrifugation, and serum titer was determined by ELISA. Immunization was repeated every two weeks, and serum titer was measured. After two immunizations, the serum titer, after a million-fold dilution, was higher than 2.0. A serum titer of 10 was selected for screening. 6 Lymphocytes were isolated from the lymph nodes or spleen of the mice mentioned above for cell fusion. C. Cell fusion and screening and subcloning of positive hybridoma cells 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. The culture supernatant was collected one week later for screening positive clones. In the initial antibody screening stage, a free ELISA method was used. First, goat anti-mouse IgG was coated onto the ELISA plate, and the culture supernatant was incubated to capture the mouse antibody. Then, biotin-labeled MxA protein was used to obtain MxA-biotin. After the potential target antibody bound to MxA-biotin, avidin-HRP was used in conjunction with ELISA chromogenic solution for color development, screening for highly reactive hybridoma clones. Next, the reactivity to positive samples (peripheral blood from patients infected with PIV, RSV, ADV, EBV, etc.) and negative samples (peripheral blood from healthy individuals without viral infection) was tested. Hybridoma clones showing good reactivity to recombinant MxA protein and positive samples, but no significant reactivity to negative samples, were our target clones. Finally, a hybridoma cell line capable of secreting MxA monoclonal antibodies and meeting the screening requirements was obtained, named #14, which exhibited good stability. D. Production and purification of monoclonal antibodies 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 14# hybridoma cells (approximately 1 × 10⁻⁶ cells) were 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 14#. E. Identification of monoclonal antibody subtypes and cloning of gene sequences The heavy and light chain isotypes of monoclonal antibodies were identified using the Southern Biothech SBA Clonotyping System-HRP kit, following the manufacturer's instructions. The specific procedures were as follows: 1. Dilute the capture antibody to 1 μg / mL with coating buffer (0.05 M pH 9.5 carbonate and bicarbonate buffer), add 100 μL / well to the microplate, and coat overnight at 4°C. Wash the plate three times with PBS buffer containing 0.05% Tween-20 (wash buffer). 2. Dilute the culture supernatant of the hybridoma cells to be tested 1:1 with diluent (1% BSA, 0.1% PBST), add 100 μL / well to the ELISA plate, and incubate at 37°C for 30 minutes. Dilute the corresponding enzyme-labeled antibodies (Ig-HRP, IgG1-HRP, IgG2a-HRP, IgG2b-HRP, IgG3-HRP, IgM-HRP, kappa-HRP, lamda-HRP) 1:3000 with diluent. 3. After washing the plate three times with washing buffer, add 100 μL of diluted enzyme-labeled antibody to each well and incubate at 37°C for 30 minutes. After washing the plate three more times, add the chromogenic buffer and incubate for approximately 5 minutes (depending on the reaction strength). Then, add 2M sulfuric acid to stop the reaction and read the OD450 absorbance. Identification showed that the heavy chain subtype of antibody #14 was IgG1, and the light chain was Kappa.

[0028] Based on the antibody subtype results, the antibody gene sequence was cloned using a RACE-based method. Hybridoma cells in good growth condition were collected, and total RNA was obtained from the hybridoma cells using a total RNA extraction kit. The mRNA was reverse transcribed into cDNA according to the Takara SMARTer RACE instruction manual, and the full-length sequence of the target antibody was amplified.

[0029] Example 2: Validation of antibody performance 1. ELISA Affinity Test Goat anti-mouse antibody was diluted to 1 μg / mL using coating buffer (0.05 M pH 9.5 carbonate and bicarbonate buffer), and 100 μL was added to each well of a 96-well microplate. The plates were incubated overnight at 4°C. The plates were washed three times with PBS buffer containing 0.05% Tween-20. The antibody was diluted to 1 μg / mL using dilution buffer (1% BSA, 0.1% PBST), and 100 μL was added to each well of a 96-well microplate. The plates were incubated at 37°C for 30 minutes. The purified MxA protein obtained after recombinant expression was biotinylated using a commercially available biotinylation reagent. The biotinylated MxA protein was diluted to 180 ng / μL using dilution buffer in a 3-fold serial dilution. Specific concentrations of MxA protein were added to each well (100 μL), and the plates were incubated at 37°C for 30 minutes. Dilute the horseradish peroxidase-labeled avidin 1:5000 with diluent. Wash the ELISA plate three times. Add 100 μL to each well of the 96-well ELISA plate and incubate at 37°C for 30 minutes. Wash the plate three times again, add the chromogenic buffer, and incubate at room temperature for 3 minutes. Stop the reaction by adding 0.5 M sulfuric acid and read the OD450 value using a plate reader. The antibody 14# heavy chain sequence in Example 1 is shown in SEQ ID NO:11, and the light chain sequence is shown in SEQ ID NO:12. The variable region of the light chain is shown in SEQ ID NO:9, and the amino acid sequences of the complementarity-determining regions on the variable region of the light chain are as follows: CDR-VL1:RSSQSIT(X1)-HSNGNT(X2)-YLE CDR-VL2:KV(X1)-SNA(X2)-FS CDR-VL3: FQ(X1)-VSHVPL(X2)-T Its heavy chain variable region is shown in SEQ ID NO:10, wherein the amino acid sequences of each complementarity-determining region on the heavy chain variable region are as follows: CDR-VH1:TY(X1)-TLH(X2) CDR-VH2: FIN(X1)-PTSSYS(X2)-NYNQ(X3)-KFKD CDR-VH3:GSW(X1)-LRP(X2)-SMDY Based on antibody 14#, mutations were made at sites related to antibody activity in the complementarity-determining region, where X1, X2, and X3 are mutation sites. Table 1 Mutation sites related to antibody activity WT T A Q H N Y Mutation 1 T Y T A Y D Mutation 2 V R Q H N P Mutation 3 V N L C R V Mutation 4 T I R N W D Mutation 5 V Y T A S P Table 2 Antibody activity analysis data 180 2.25745 1.865 2.4034 1.9656 1.99865 2.06875 60 2.09865 1.4253 2.21145 1.6161 1.6288 1.5854 20 1.5479 1.05635 1.83585 1.08555 1.07835 0.99765 6.7 0.9169 0.6143 1.0615 0.57025 0.55925 0.5181 2.2 0.4515 0.2789 0.52865 0.25755 0.26865 0.2242 0.7 0.1958 0.13305 0.20755 0.14055 0.1298 0.13335 0.0 0.1181 0.09435 0.13735 0.09785 0.08905 0.0899 As can be seen from the table above, mutation 2 exhibits the best activity. Therefore, using mutation 2 as the backbone sequence, other mutation sites with good affinity were screened, and some results are shown below: Table 3 Mutation sites related to antibody affinity Mutation 2 T V L Y S / Q W Mutation 2-1 R V L Y T / Q Y Mutation 2-2 R H L D T / S W Mutation 2-3 T V L Y T / Q W Mutations 2-4 R H L D T / Q Y Mutations 2-5 T H W Y T / S W Mutations 2-6 T H W Y S / S Y Mutations 2-7 R H L Y S / Q Y Mutations 2-8 T H L Y T / Q W Mutations 2-9 R V W D T / S Y Mutation 2-10 R H W D S / S Y Mutation 2-11 T V L D S / S W Mutation 2-12 T H L D S / S Y Mutation 2-13 R H L Y S / S W Mutation 2-14 T V L D T / S Y Mutation 2-15 T V L D S / Q Y Mutation 2-16 R H W Y T / Q W Mutation 2-17 R V W Y S / S W Mutation 2-18 R V L D S / S Y Mutation 2-19 T H W D S / Q W Mutation 2-20 R V L Y T / S W Mutation 2-21 R V W Y S / Q Y Mutation 2-22 T V W D T / Q W Mutation 2-23 T V W D S / S Y Mutation 2-24 T H W Y T / Q Y Mutation 2-25 T V W Y S / Q W Mutation 2-26 T H W D T / S Y Mutation 2-27 R H L D S / Q W Mutation 2-28 R V W D S / Q W Mutation 2-29 T V W Y T / S Y Mutation 2-30 T H L Y T / S W MxA protein concentration of 180 ng / mL was measured, and the absorbance at OD450 for each mutation was measured. The results are as follows: Table 4. Affinity test for antibody mutations Mutation 2-2 2.4744 Mutation 2-12 2.1573 Mutation 2-22 2.4586 Mutation 2-3 2.6172 Mutation 2-13 1.9427 Mutation 2-23 2.1229 Mutations 2-4 2.2738 Mutation 2-14 2.0878 Mutation 2-24 2.1606 Mutations 2-5 2.3682 Mutation 2-15 2.532 Mutation 2-25 2.0901 Mutations 2-6 2.1467 Mutation 2-16 2.0903 Mutation 2-26 2.2977 Mutations 2-7 2.3355 Mutation 2-17 2.4422 Mutation 2-27 1.9948 Mutations 2-8 2.1135 Mutation 2-18 2.1454 Mutation 2-28 1.9608 Mutations 2-9 2.3170 Mutation 2-19 2.3004 Mutation 2-29 2.079 Mutation 2-10 2.0838 Mutation 2-20 2.1454 Mutation 2-30 2.1276 The experimental results in the table above show that all mutations have high affinity.

[0030] 2. Stability test In this embodiment, the antibody concentration is 1 ug / mL and the MxA protein concentration is 100 ng / mL.

[0031] The antibody was subjected to accelerated thermal treatment at 37°C for 7 and 14 days in a predetermined buffer (PBS, 0.05% ProClin™ 300). The accelerated antibody was evaluated using an indirect ELISA method, with a control at 4°C, to assess its long-term stability. Additionally, the antibody underwent five freeze-thaw cycles at -20°C, and the results are shown as the deviation between the values ​​at 4°C and the accelerated values. The measurement results are as follows: Table 5 Stability Study WT 10% -1% -2% Mutation 2 8% -5% -5% Mutation 2-1 -1% 3% -1% Mutation 2-2 -3% 0% 1% Mutation 2-3 -4% 2% -1% Mutations 2-4 1% 2% 6% Mutations 2-5 -11% 1% 5% Mutations 2-6 -3% 10% -7% Mutations 2-7 10% 7% -1% Mutations 2-8 5% -12% 1% Mutations 2-9 9% 8% -4% Mutation 2-10 6% 12% -10% Mutation 2-11 8% 15% -16% Mutation 2-12 9% 4% -14% Mutation 2-13 16% 10% -11% Mutation 2-14 -2% 1% 9% Mutation 2-15 1% 0% 7% Mutation 2-16 -1% -7% 8% Mutation 2-17 -2% -3% 14% Mutation 2-18 2% 2% 10% Mutation 2-19 0% 0% 11% Mutation 2-20 3% 0% 9% Mutation 2-21 2% 4% -1% Mutation 2-22 11% 7% -10% Mutation 2-23 4% 8% 3% Mutation 2-24 11% 5% 1% Mutation 2-25 7% 4% 4% Mutation 2-26 11% 3% -2% Mutation 2-27 -6% -10% 5% Mutation 2-28 -5% -2% -3% Mutation 2-29 -6% -4% -2% Mutation 2-30 -4% -6% 3% The experimental results in the table above show that mutation 2 to mutation 2-30 remained stable after 14 days of accelerated heating at 37℃ (slight changes in reactivity did not affect reagent performance), ensuring the performance of the reagents after opening and thus guaranteeing the accuracy and stability of the test results.

[0032] Example 3: Application of Antibodies in Immunochromatographic Detection To verify the effectiveness of the produced MxA antibody in immunoassays, this example uses antibody 14# and its mutations as labeling antibodies in an immunochromatographic kit. Because antibody 14# and antibodies with mutations 2 to 2-30 have comparable stability and affinity for the MxA protein, this example cannot exhaustively represent the effects of all antibodies; therefore, mutations 2-3 were selected for this experiment.

[0033] A. Preparation of the film template 1. Applying the film: Take the backing card, NC film, and absorbent paper, cut 30cm, and apply the NC film and absorbent paper to the corresponding positions. Allow to rest for 30 to 60 minutes.

[0034] 2. Coating solution preparation: T line is coated with MxA antibody (self-produced) 0.5mg / mL, C line is coated with DNP polyclonal antibody (from Zhongyuan Huiji Biotechnology Co., Ltd.) 1mg / mL. PBS is used as the coating solution. Calculate and prepare the coating solution according to the concentration to be used, and vortex mix well for later use.

[0035] 3. Dicing: Use a gold-sprayed die-cutting instrument to etch the film. Turn on the instrument and clean the pump tubes to be used at least 15 times. Adjust the parameters of the die-cutting instrument: the liquid output of the detection line is 0.5 μL / cm, and the scale card is used to position the scribe needle. Backflow 35 μL of T-line coating liquid into the pump tube and 70 μL of C-line coating liquid into the pump tube. Select all pump tubes and drain 1 μL of liquid to ensure that the scribe head is full and free of air. Place the blank film plate from step (1) on the operating table and press it down with a magnet to fix it. Click "Start" on the screen to etch the film.

[0036] 4. Drying the membrane: Place the membrane with C-line and T-line coated in a 45℃ forced-air drying oven for 3 days.

[0037] B, 14# and mutant 2-3 antibody-labeled fluorescent microspheres 1. Activator equilibration: Take NHS and EDC out of the refrigerators at 2~8℃ and -25~-10℃ respectively, and equilibrate at room temperature for 30 minutes.

[0038] 2. Activation of fluorescent microspheres: After removing the fluorescent microspheres from 2~8℃, equilibrate at room temperature for 10 min, mix with a shaker, add 400 μL of labeling buffer A05 (from Zhongyuan Huiji Biotechnology Co., Ltd.) and 30 μL of fluorescent microspheres (concentration of 10 mg / mL) to two EP tubes respectively, mix well, and sonicate in a water bath for 5 min.

[0039] 3. Preparation of activator: Weigh 5-10 mg of NHS, add labeling buffer A05 to dilute to 1 mg / mL, and add 12 μL to two 1.5 mL EP tubes and mix well; Weigh 5-10 mg of EDC, add labeling buffer A05 to dilute to 1 mg / mL, and add 6 μL to two 1.5 mL EP tubes and mix well. Place the tubes on a blood mixing plate and rotate slowly. Activate at room temperature for 30 min.

[0040] 4. Labeling fluorescent microspheres: Add samples at the following mass ratios: microspheres: MxA antibody (14# or mutation 2-3) = 300 μg: 70 μg, microspheres: DNP-BSA = 300 μg: 45 μg. Mix quickly. Then place the EP tube back onto the blood mixing tray and react at room temperature for 2.5 h.

[0041] 5. Blocking: Block according to the mass ratio, that is, add 60μL of fluorescent blocking agent CO2 to 300μg microspheres, then place the EP tube back on the blood mixing plate and block at room temperature for 1 hour.

[0042] 6. Reconstitution: Set the centrifugation parameters to 16000 rpm and 15 min. After pipetting away the supernatant, add 300 μL of labeled reconstitution solution D10 (from Zhongyuan Huiji Biotechnology Co., Ltd.) to the EP tube for reconstitution.

[0043] 7. Spraying the microspheres: Take out the marked microspheres. Mix the microspheres according to the volume ratio: MxA-microspheres: DNP-BSA microspheres: 0.5% amaranth red: MxA gold spraying solution = 20:5:2:123. Vortex for 30 seconds to mix thoroughly, then spray the microspheres using a gold spraying scribing instrument. After spraying, place the sample in a 45℃ oven for 12-16 hours, then remove and store in a room temperature desiccant / box, maintaining a humidity of <30%.

[0044] C. Performance Evaluation 1. Linear Experiment Experimental preparation: Take out working calibrators C1~C8, thaw them at room temperature, and mix them thoroughly on a vortex mixer for at least 10 seconds; take out 16 #14 antibody reagent cards and mutation 2-3 reagent cards.

[0045] Test: Power on the Q8pro immunofluorescence quantitative analyzer (from Zhongyuan Huiji Biotechnology Co., Ltd.), open the maintenance page, manage the project, click on the corresponding project on the card, set the test time to 8 minutes and the number of T lines to 1, spot 70 μL of each of the C1-C8 quality control points, repeat twice, and click Start Test.

[0046] Table 6. Results of Linear Experiments

[0047] The results are shown in Table 6 and Figure 1 As shown, both 14# and mutations 2-3, when used as labeling antibodies in MxA immunochromatographic assays, exhibited good linearity.

[0048] 2. Precision Experiment Experimental preparation: Take out calibrators C3 and C5, thaw them at room temperature, and mix them thoroughly on a vortex mixer for at least 10 seconds; take out 16 #14 antibody reagent cards and mutation 2-3 reagent cards.

[0049] Test: 70 μL of sample was spotted at each of the C3 and C5 quality control points, and each test was repeated 8 times.

[0050] Table 7 Precision Experiment Results

[0051] The results are shown in Table 7. Both 14# and mutation 2-3, used as labeling antibodies in MxA immunochromatographic assay, showed good precision with CVs below 10%.

[0052] 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 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-MxA antibody or a functional fragment thereof, characterized in that, The antibody or its functional fragment includes the following complementarity-determining regions: CDR-VL1: RSSQSIVHSNGNTYLE; CDR-VL2: KVSNRFS; CDR-VL3: FQVSHVPLT; CDR-VH1: TYTLH; CDR-VH2:FINPTSSYTNYNQKFKD; CDR-VH3: GSWLRPSMDY.

2. The anti-MxA antibody or its functional fragment as described in claim 1, characterized in that, The antibody or its functional fragment further includes heavy chain backbone regions FR1-H, FR2-H, FR3-H and FR4-H as shown in sequence as SEQ ID NO:1-4, and / or light chain backbone regions FR1-L, FR2-L, FR3-L and FR4-L as shown in sequence as SEQ ID NO:5-8.

3. The anti-MxA antibody or its functional fragment as described in claim 2, characterized in that, The antibody also includes a constant region.

4. The anti-MxA antibody or its functional fragment as described in claim 3, characterized in that, The constant region is selected from the constant regions of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE and IgD.

5. The anti-MxA antibody or its functional fragment as described in claim 4, 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.

6. A composite, characterized in that, The conjugate comprises an anti-MxA antibody or a functional fragment thereof as described in any one of claims 1-5, covalently linked to a chemical label and / or a biolabel.

7. A coupling, characterized in that, The conjugate is formed by conjugating the anti-MxA antibody or its functional fragment as described in any one of claims 1-5 and / or the conjugate as described in claim 6 with a solid or semi-solid medium.

8. A carrier, characterized in that, It contains a nucleic acid fragment encoding an antibody or a functional fragment thereof as described in any one of claims 1-5.

9. The use of the anti-MxA antibody or its functional fragment as described in any one of claims 1-5 and / or the conjugate as described in claim 6 and / or the conjugate as described in claim 7 in the preparation of reagents for detecting MxA.

Images