Anti-MxA antibody or functional fragment thereof and application thereof

Abstract

The invention 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 invention comprises a heavy chain complementarity determining region and a light chain complementarity determining region, and the antibody or the functional fragment thereof has better specificity and sensitivity to MxA, 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] The present invention relates to the field of biotechnology, and in particular to an anti-MxA antibody or a functional fragment thereof and an application thereof. Background Art

[0002] MxA protein belongs to the dynamin superfamily of large GTPases. It is one of the antiviral proteins produced by host cells induced by type I interferon (IFNα / β) and has a wide range of antiviral effects. The full-length MxA protein contains 662 amino acids and has a molecular weight of 76kD. Its protein structure includes monomeric forms and oligomeric forms such as dimers, trimers, and tetramers. MxA protein usually accumulates in the cytoplasm and endoplasmic reticulum. The membrane compartment of the endoplasmic reticulum provides an interaction platform that promotes viral target recognition. MxA detects viral infection by sensing and capturing nucleocapsid-like structures, and plays an important role in antiviral activity. Therefore, many viruses (including influenza, parainfluenza virus, adenovirus, herpes, Epstein-Barr virus, measles, Coxsackie virus, hepatitis B virus, and Thogoto virus, etc.) will induce cells to produce a large amount of MxA protein after infecting the human body, while bacterial infection cannot induce high expression of MxA protein. In clinical practice, it is necessary to quickly distinguish between bacterial and viral infections to guide the direction of clinical treatment. Detecting the expression of MxA protein can help quickly distinguish viral and bacterial infections. In addition, MxA protein is very sensitive to viruses and even very small amounts of viruses can induce cells to express MxA protein. Detecting the expression of MxA protein can also be used for early diagnosis of viral infections.

[0003] In emergency, rapid discrimination between viral and bacterial infections is of great value for timely and correct medication and thus saving lives. The sensitivity and linear width of detection in immunodiagnosis are often determined by the antibodies or antibody pairs used, so the specificity, sensitivity and linear width of the antibody are particularly important. At present, there are few MxA antibodies reported at home and abroad, and the effectiveness of these antibodies for clinical diagnosis has not been fully confirmed. Therefore, the development of antibodies with high recognition sensitivity for MxA proteins induced by viral infection of the human body has great clinical medical application value. Summary of the invention

[0004] The object of the present invention is to provide an MxA antibody or a functional fragment thereof having high affinity and high specificity for the MxA protein and its use. The antibody or the functional fragment thereof has good affinity for MxA, and the use of the antibody or the functional fragment thereof to detect MxA has good sensitivity and specificity.

[0005] The details are as follows:

[0006] In one aspect, the present invention provides an anti-MxA antibody or a functional fragment thereof, wherein the anti-MxA antibody or a functional fragment thereof has the following complementary determining regions:

[0007] CDR-VL1: RSSQSI-X1-HSNGN-X2-YLE, where X1 is V or T, and X2 is R or T;

[0008] CDR-VL2: K-X1-SN-X2-FS, where X1 is V or H, and X2 is R or A;

[0009] CDR-VL3: F-X1-VSHVP-X2-T, where X1 is Q and X2 is L or W;

[0010] CDR-VH1: T-X1-TL-X2, where X1 is Y or D, and X2 is H;

[0011] CDR-VH2: FI-X1-PTSSY-X2-NYN-X3-KFKD, wherein X1 is N or I, X2 is T or S, and X3 is Q or S;

[0012] CDR-VH3: GS-X1-LR-X2-SMDY, where X1 is Y or W and X2 is P or Y.

[0013] Furthermore, X1 of CDR-VL1 of the complementary determining region 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.

[0014] The CDR segment division described in the present invention adopts the Kabat algorithm.

[0015] "CDR" is used herein to refer to a "complementarity determining region" within an antibody variable sequence. The variable regions of the heavy and light chains each have three CDRs, starting from the N-terminus of the heavy or light chain.

[0016] The antigen binding site may include six CDRs (CDR-VH1, CDR-VH2, CDR-VH3, CDR-VL1, CDR-VL2 and CDR-VL3 in the present invention). A polypeptide comprising a single CDR (e.g., CDR-VH1, CDR-VH2, CDR-VH3, CDR-VL1, CDR-VL2 or CDR-VL3) may be referred to as a "molecular recognition unit". Crystallographic analysis of antigen-antibody complexes has shown that the amino acid residues of 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 be primarily responsible for the specificity of the antigen binding site. In general, the CDR residues are directly and most substantially involved in influencing antigen binding.

[0017] The inventors of the present invention have found that when the mutation sites in each complementary determining region are the amino acid residues, the antibody has a better affinity for the MxA protein.

[0018] In an optional embodiment, X1 in the CDR-VL1 is V;

[0019] In an optional embodiment, X1 in the CDR-VL1 is T;

[0020] In an optional embodiment, X2 in the CDR-VL1 is R;

[0021] In an optional embodiment, X2 in the CDR-VL1 is T;

[0022] In an optional embodiment, X1 in the CDR-VL2 is V;

[0023] In an optional embodiment, X1 in the CDR-VL2 is H;

[0024] In an optional embodiment, X2 in the CDR-VL2 is R;

[0025] In an optional embodiment, X2 in the CDR-VL2 is A;

[0026] In an optional embodiment, X1 in the CDR-VL3 is Q;

[0027] In an optional embodiment, X2 in the CDR-VL3 is L;

[0028] In an optional embodiment, X2 in the CDR-VL3 is W;

[0029] In an optional embodiment, X1 in the CDR-VH1 is Y;

[0030] In an optional embodiment, X1 in the CDR-VH1 is D;

[0031] In an optional embodiment, X2 in the CDR-VH1 is H;

[0032] In an optional embodiment, X1 in the CDR-VH2 is N;

[0033] In an optional embodiment, X1 in the CDR-VH2 is 1;

[0034] In an optional embodiment, X2 in the CDR-VH2 is T;

[0035] In an optional embodiment, X2 in the CDR-VH2 is S;

[0036] In an optional embodiment, X3 in the CDR-VH2 is Q;

[0037] In an optional embodiment, X3 in the CDR-VH2 is S;

[0038] In an optional embodiment, X1 in the CDR-VH3 is Y;

[0039] In an optional embodiment, X1 in the CDR-VH3 is W;

[0040] In an optional embodiment, X2 in the CDR-VH3 is P;

[0041] In an alternative embodiment, X2 is Y in the CDR-VH3.

[0042] Preferably, each complementary determining region is selected from any one of the following mutation combinations:

[0043]

[0044]

[0045]

[0046] Further, the antibody comprises light chain framework regions FR1-L, FR2-L, FR3-L and FR4-L and heavy chain framework regions FR1-H, FR2-H, FR3-H and FR4-H; the heavy chain framework regions FR1-H, FR2-H, FR3-H and FR4-H are selected from SEQ ID NOs: 1-4 in sequence; the light chain framework regions FR1-L, FR2-L, FR3-L and FR4-L are selected from SEQ ID NOs: 5-8 in sequence.

[0047] Furthermore, the antibody also comprises a constant region.

[0048] Preferably, the constant region is selected from the constant region of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE and IgD. Preferably, the species origin of the constant region is cattle, horses, dairy cows, pigs, sheep, goats, rats, mice, dogs, cats, rabbits, camels, donkeys, deer, minks, chickens, ducks, geese, turkeys, fighting cocks or humans.

[0049] Preferably, the functional fragment is selected from any one of VHH, F(ab')2, Fab', Fab, Fv and scFv of the antibody.

[0050] In another aspect, the present invention discloses a conjugate, comprising the anti-MxA antibody or a functional fragment thereof as described above covalently linked to a chemical label or a biological label.

[0051] In another aspect, the present invention discloses a conjugate, wherein the conjugate is formed by coupling the anti-MxA antibody or a functional fragment thereof and / or the conjugate as described above with a solid medium or a semi-solid medium.

[0052] In another aspect, the present invention discloses the use of the anti-MxA antibody or its functional fragment as described above and / or the conjugate as described above and / or the conjugate as described above in a kit for detecting MxA expression.

[0053] Beneficial effects:

[0054] The present invention not only provides a monoclonal antibody (wild type) with stable performance and high affinity binding to MxA protein, but also makes a variety of excellent performance mutations to the wild type, which can achieve specific and efficient binding to MxA in the complex environment of whole blood cell lysate with many interfering factors. The MxA immunochromatographic diagnostic reagent developed based on the antibody can achieve rapid identification of viral infection or bacterial infection, and has important clinical practical value. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 :Linearity verification of 14# and mutation 2-3 antibodies as labeled antibodies in immunochromatographic detection. DETAILED DESCRIPTION

[0056] Specifically, in order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they 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 those generally understood by those of ordinary skill in the art to which the present disclosure belongs. Any methods and materials similar or equivalent to those described herein can be used for the practice or testing of the preparations or unit doses herein. Unless otherwise indicated, the techniques adopted or considered herein are standard methods. Materials, methods and examples are illustrative and non-restrictive only.

[0058] As used herein, the terms "including," "comprising," "having," "may," and variations thereof 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 indicated, the practice of the present invention will employ conventional techniques of cell biology, molecular biology (comprising recombinant technology), microbiology, biochemistry and immunology, which are within the capabilities of those skilled in the art. This technology is fully explained in the literature, such as "Molecular Cloning: A Laboratory Manual", Second Edition (Sambrook et al., 1989); "Oligonucleotide Synthesis" (MJ Gait, 1984); "Animal Cell Culture" (RI Freshney, 1987); "Methods in Enzymology" (Academic Press, Inc.); "Handbook of Experimental Immunology" (DM Weir and CC Black, 1989). ell, ed.); Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, eds., 1987); Current Protocols in Molecular Biology (FM Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction (Mullis et al., eds., 1994); and Current Protocols in Immunology (JE Coligan et al., eds., 1991), each of which is expressly incorporated herein by reference.

[0060] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0061] Example 1 Preparation of monoclonal antibodies

[0062] A. Immunogen Preparation

[0063] The human MxA full-length protein (10-662Aa, P20591) was constructed into a eukaryotic expression vector to obtain an MxA expression plasmid.

[0064] (I) DH10bac transformation

[0065] 1. Take 100ul DH10bac competent cells and place them on ice for 10 minutes until they are completely thawed. Add 50-200ng (about 1ul) plasmid and mix gently, and place them on ice for 30 minutes;

[0066] 2. Heat shock at 42°C for 45 seconds;

[0067] 3. Place on ice for 2-5 minutes;

[0068] 4. Add 700ul LB medium, 37℃, 225rpm, 4h;

[0069] 5. Take 100ul of the culture medium and apply it to the x-gal plate, invert and incubate at 37℃ overnight;

[0070] 6. Take the white colonies and streak them on the x-gal plate, then invert and culture them at 37℃ overnight;

[0071] 7. If the colonies remain white after overnight observation, the transformation is considered successful. Colony PCR can also be performed for further verification.

[0072] (II) Plasmid extraction

[0073] 1. Pick the white clones and place them in 2 ml LB medium (50 ug / ml kanamycin, 7 ug / ml gentamicin, 10 ug / ml tetracycline, 100 ug / ml X-gal, 40 ug / ml IPTG), and culture at 37°C in a shaker at 250-300 rpm for more than 24 hours;

[0074] 2. Extract plasmid using PureLink HiPure Plasmid DNA Purification Kits;

[0075] 3. After extraction, dissolve the plasmid in water and set aside after the concentration is greater than 500ng / ul.

[0076] (III) Fermentation expression

[0077] Shake the 293 cell suspension evenly, use a pipette to aspirate about 1 ml of the cell suspension and add it to a sterile 1.5 ml EP tube to count and observe the cell status. Adjust the cell density to 3.0×106 / mL, inoculate 1L; use PEI (polyethyleneimine) mediated plasmid transfection method to transfect the expression plasmid into 293 cells in suspension culture, add culture medium and feed within 18-20 hours after transfection, and collect cell precipitates by centrifugation at 4000g centrifugal force at 10℃ for 30min after 5 days of continuous fermentation. The cell precipitate is resuspended in PBS and then broken by ultrasonic disruptor. After the cell wall is broken, centrifuge and collect the supernatant for purification of the target protein.

[0078] B. Mouse immunization and antibody detection

[0079] Select 5 6-8 week old SPF female BALB / c mice, mix and emulsify Freund's complete adjuvant and MxA protein at a concentration of 1 mg / mL in equal volumes. Use the emulsified antigen to immunize 6-8 week old SPF female BALB / c mice, and inject 40 μg of antigen protein into each mouse by foot pad injection or back subcutaneous injection. Two weeks after the first immunization, mix the antigen protein with Freund's incomplete adjuvant and emulsify it, and inject 40 μg of antigen protein into each mouse again by foot pad injection or back subcutaneous injection. Two weeks later, collect blood through the tail vein, collect the supernatant by centrifugation, and test the serum titer by ELISA. Immunize once every two weeks and test the serum titer. After two immunizations, the serum titer is as high as 2.0 or above after a million-fold dilution. Screening serum titer 10 6 Lymph nodes or spleens of the above mice were taken to isolate lymphocytes for cell fusion.

[0080] C. Cell fusion, positive hybridoma cell screening and subcloning

[0081] Lymphocytes isolated from immunized mice were fused with cultured SP2 / 0 cells by PEG1500 or electrofusion. The fused cells were cultured in HAT-1640 medium containing 20% ​​FBS serum and screened. After one week, the culture supernatant was used for positive clone screening.

[0082] In the initial screening of antibodies, the ELISA free method was used. First, the ELISA plate was coated with goat anti-mouse IgG, and the culture supernatant was incubated to capture the mouse antibody in the supernatant; then, the MxA protein was labeled with biotin to obtain MxA-biotin. After the potential target antibody was bound to MxA-biotin, avidin-HRP was used in combination with ELISA colorimetric solution to develop the color and screen out highly reactive hybridoma clones. Secondly, the reactivity to positive samples (collecting whole peripheral blood from patients infected with viruses such as PIV, RSV, ADV, and EBV) and negative samples (whole peripheral blood from healthy people without virus infection) was detected. Among them, the hybridoma clones with good reactivity to recombinant MxA protein and positive samples and no obvious reactivity to negative samples were our target clones. Finally, a hybridoma cell line that can secrete MxA monoclonal antibodies and meet the screening requirements was obtained, named 14#, which has good stability.

[0083] D. Production and purification of monoclonal antibodies

[0084] Two groups of 6-8 week old BALB / c mice were selected and intraperitoneally injected with 500 μL paraffin oil to suppress the immune response of mice. One week after the injection, 0.5 ml of 14# hybridoma cells were injected into the peritoneal cavity of the mice. The number of cells was about 1×10 6 After two weeks, ascites was collected. The collected ascites was precipitated with ammonium sulfate and purified with protein G affinity to obtain the target antibody 14#.

[0085] E. Identification of monoclonal antibody subtypes and gene sequence cloning

[0086] The SBA Clonotyping System-HRP kit from Southern Biothech was used to identify the subtypes of the heavy and light chains of the monoclonal antibody according to the instructions. The specific operations are as follows:

[0087] 1. Dilute the capture antibody to 1 μg / mL with coating solution (0.05M pH 9.5 carbonate and bicarbonate buffer), add 100 μL / well to the ELISA plate, and coat overnight at 4°C. Wash the plate three times with PBS buffer (washing solution) containing 0.05% Tween-20.

[0088] 2. Dilute the culture supernatant of the hybridoma cells to be tested with diluent (1% BSA, 0.1% PBST) at a ratio of 1:1, 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) at a ratio of 1:3000 with diluent.

[0089] 3. After washing the plate 3 times with the plate washing solution, add 100 μL of diluted enzyme-labeled antibody to each well and incubate at 37°C for 30 minutes. After washing the plate 3 times again, add the color developing solution. After about 5 minutes (depending on the strength of the reaction), add 2M sulfuric acid to terminate the reaction and read the OD450 absorbance. After identification, the heavy chain subtype of antibody 14# is IgG1, and the light chain is Kappa.

[0090] According to the antibody subtype results, the antibody gene sequence was cloned using a RACE-based method. Hybridoma cells with good growth were collected, and total RNA of hybridoma cells was obtained using a total RNA extraction kit. The mRNA was reverse transcribed into cDNA according to the SMARTerRACE instructions of Takara Company, and the full-length sequence of the target antibody was amplified.

[0091] Example 2 Verification of antibody performance

[0092] 1. ELISA affinity test

[0093] Dilute the goat anti-mouse to 1ug / mL using coating solution (0.05M pH = 9.5 carbonate and bicarbonate buffer), add 100μL per well to a 96-well ELISA plate, and coat overnight at 4°C. Wash the plate three times with PBS buffer containing 0.05% Tween-20. Dilute the antibody to 1ug / mL using diluent (1% BSA, 0.1% PBST), add 100μL per well to a 96-well ELISA plate, and incubate at 37°C for 30 minutes. The MxA protein purified after recombinant expression was biotin-modified using a biotinylation reagent (finished commercial reagent), and dilute the biotin-labeled MxA protein to 180ng / μL with a diluent, and dilute 3 times. Add a specific concentration of MxA protein to the ELISA plate, add 100μL per well, and incubate at 37°C for 30min. Dilute the avidin labeled with horseradish peroxidase at 1:5000 with diluent, take out the ELISA plate and wash it three times, add 100 μL per well to the 96-well ELISA plate, and incubate at 37°C for 30 minutes. Take out the ELISA plate and wash it three times, add the color developing solution, develop the color at room temperature for 3 minutes, add 0.5M sulfuric acid to terminate the reaction, and read the OD450 value on the plate reader.

[0094] The heavy chain sequence of antibody 14# in Example 1 is shown in SEQ ID NO: 11, and the light chain sequence is shown in SEQ ID NO: 12, wherein the light chain variable region is shown in SEQ ID NO: 9, and the amino acid sequences of each complementary determining region on the light chain variable region are as follows:

[0095] CDR-VL1:RSSQSIT(X1)-HSNGNT(X2)-YLE

[0096] CDR-VL2:KV(X1)-SNA(X2)-FS

[0097] CDR-VL3: FQ(X1)-VSHVPL(X2)-T

[0098] The heavy chain variable region is shown in SEQ ID NO: 10, wherein the amino acid sequences of the complementary determining regions on the heavy chain variable region are as follows:

[0099] CDR-VH1:TY(X1)-TLH(X2)

[0100] CDR-VH2:FIN(X1)-PTSSYS(X2)-NYNQ(X3)-KKD

[0101] CDR-VH3:GSW(X1)-LRY(X2)-SMDY

[0102] Based on antibody 14#, mutations were performed at sites related to antibody activity in the complementary determining region, among which X1, X2, and X3 were all mutation sites.

[0103] Table 1 Mutation sites related to antibody activity

[0104]

[0105] Table 2 Antibody activity analysis data

[0106]

[0107]

[0108] As can be seen from the table above, mutation 2 has the best activity effect. Therefore, mutation 2 is used as the backbone sequence to screen other mutation sites with better affinity. Some results are as follows:

[0109] Table 3 Mutation sites related to antibody affinity

[0110]

[0111]

[0112] MxA protein 180ng / mL, measure the absorbance value of each mutant OD450, the results are as follows:

[0113] Table 4 Affinity testing of antibody mutations

[0114] Mutation 2-1 2.3324 Mutation 2-11 2.4586 Mutation 2-21 2.1205 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 Mutation 2-4 2.2738 Mutation 2-14 2.0878 Mutation 2-24 2.1606 Mutation 2-5 2.3682 Mutation 2-15 2.532 Mutation 2-25 2.0901 Mutation 2-6 2.1467 Mutation 2-16 2.0903 Mutation 2-26 2.2977 Mutation 2-7 2.3355 Mutation 2-17 2.4422 Mutation 2-27 1.9948 Mutation 2-8 2.1135 Mutation 2-18 2.1454 Mutation 2-28 1.9608 Mutation 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

[0115] It can be seen from the test results in the above table that the affinity of each mutation is relatively high.

[0116] 2. Stability determination

[0117] In this example, the concentration of the antibody is 1 ug / mL, and the concentration of the MxA protein is 100 ng / mL.

[0118] The antibodies were diluted in a defined buffer (PBS, 0.05% ProClin TM 300) was heat accelerated at 37°C for 7 days and 14 days, and the accelerated antibodies were evaluated by ELISA indirect method, and compared with 4°C to identify the long-term stability of the antibodies. In addition, the test results were displayed as the deviation between the 4°C value and the accelerated value by repeated freezing and thawing at -20°C for 5 times. The measurement results are as follows:

[0119] Table 5 Stability study

[0120]

[0121]

[0122] From the test results in the above table, it can be seen that the properties of mutations 2 to 2-30 are still stable after 14 days of thermal acceleration at 37°C (the slight reactivity change does not affect the performance of the reagent), which ensures the performance of the reagent when the bottle is opened, thereby ensuring the accuracy and stability of the test results.

[0123] Example 3 Application of Antibodies in Immunochromatographic Detection

[0124] In order to verify the effect of the produced MxA antibody in the immunoassay, the present example applies the 14# antibody and its mutations as the marker antibody to the immunochromatography kit. Because the stability and affinity performance of the 14# antibody and the mutations 2 to 2-30 antibodies to the MxA protein are comparable, the present example cannot exhaust the effects of all antibodies, and the present example selects mutations 2-3 for the experiment.

[0125] A. Preparation of chemical membrane template

[0126] 1. Film sticking: Take the backing card, NC film and absorbent paper, cut 30cm, stick the NC film and absorbent paper on the corresponding positions, and balance for 30min to 60min.

[0127] 2. Coating solution preparation: T line is coated with MxA antibody (self-produced) 0.5 mg / mL, C line is coated with DNP polyclonal antibody (from Zhongyuan Huiji Biotechnology Co., Ltd.) 1 mg / mL, and the coating solution is PBS. Calculate and prepare the coating solution according to the use concentration, vortex mix and set aside.

[0128] 3. Membrane drawing: Use a gold spray membrane drawing instrument to chemically coat the membrane. Turn on the instrument and clean the pump tubes to be used at least 15 times. Adjust the parameters of the membrane drawing instrument: the test line liquid output is 0.5 μL / cm, and the ruler card is used to position the marking needle. Pipette 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 discharge 1 μL of liquid to ensure that the marking tip is full and free of air. Place the blank membrane plate in step (1) on the operating table and press it with a magnet to fix it. Click "Start" on the screen to draw the membrane.

[0129] 4. Drying of the membrane plate: Place the membrane plate coated with C-line and T-line in a 45℃ forced air drying oven for 3 days.

[0130] B. 14# and mutation 2-3 antibody labeled fluorescent microspheres

[0131] 1. Activator balance: Take NHS and EDC out of 2-8℃ and -25--10℃ refrigerators respectively and balance at room temperature for 30 minutes.

[0132] 2. Activation of fluorescent microspheres: Take out the fluorescent microspheres from 2-8°C and equilibrate them at room temperature for 10 min. Mix them with an oscillator. Add 400 μL of labeling buffer A05 (from Zhongyuan Huiji Biotechnology Co., Ltd.) and 30 μL of fluorescent microspheres (concentration of 10 mg / mL) into two EP tubes respectively. Mix well and sonicate in a water bath for 5 min.

[0133] 3. Preparation of activator: Weigh 5mg~10mg of NHS, add labeling buffer A05 to dilute to 1mg / mL, take 12μL respectively and add to two 1.5mL EP tubes to mix; weigh 5mg~10mg of EDC, add labeling buffer A05 to dilute to 1mg / mL, take 6μL respectively and add to two 1.5mL EP tubes to mix, place on the blood mixing disk and rotate slowly, and activate at room temperature for 30min.

[0134] 4. Labeled fluorescent microspheres: add samples according to the mass ratio of microspheres: MxA antibody (14# or mutation 2-3) = 300μg: 70μg, microspheres: DNP-BSA = 300μg: 45μg, and mix quickly; then put the EP tube back on the blood mixing plate and react at room temperature for 2.5h.

[0135] 5. Blocking: Block according to the mass ratio, that is, add 60 μL fluorescent blocking agent C02 to 300 μg microspheres, then put the EP tube back on the blood mixing plate and block at room temperature for 1 hour.

[0136] 6. Redissolution: Set the centrifugation parameters to 16000rpm, 15min, and after the supernatant is completely aspirated with a pipette, add 300uL of labeled reconstitution solution D10 (from Zhongyuan Huiji Biotechnology Co., Ltd.) to the EP tube for reconstitution.

[0137] 7. Spraying: 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, and then spray the microspheres with a gold spraying film scribing instrument. After spraying, place the sample in a 45℃ oven for 12-16 hours, then take it out and store it in a room temperature drying cabinet / box, keeping the humidity <30%.

[0138] C. Performance Evaluation

[0139] 1. Linearity experiment

[0140] Experimental preparation: Take out working calibrators C1 to C8, thaw at room temperature and mix thoroughly on a vortex mixer for at least 10 seconds; take out 16 14# antibody reagent cards and mutation 2-3 reagent cards.

[0141] Test: Turn on the immunofluorescence quantitative analyzer Q8pro (from Zhongyuan Huiji Biotechnology Co., Ltd.), open the maintenance page, perform project management, click the corresponding project on the card, set the test time to 8min and the number of T lines to 1, respectively, spot 70μL of each C1-C8 quality control point, repeat twice, and click to start the test.

[0142] Table 6 Linearity test results

[0143]

[0144]

[0145] The results are shown in Table 6 and Figure 1 As shown, 14# and mutation 2-3 both have good linear effects when used as labeled antibodies in MxA immunochromatographic detection.

[0146] 2. Precision experiment

[0147] Experimental preparation: Take out calibrators C3 and C5, thaw them at room temperature and mix thoroughly on a vortex mixer for at least 10 seconds; take out 16 14# antibody reagent cards and mutation 2-3 reagent cards.

[0148] Test: Spot 70 μL of sample at each of the C3 and C5 quality control points, and repeat the test 8 times.

[0149] Table 7 Precision test results

[0150]

[0151]

[0152] The results are shown in Table 7. Both 14# and mutation 2-3, when used as labeled antibodies in the MxA immunochromatographic assay, had good precision, with CVs within 10%.

[0153] Although the embodiments of the present invention have been disclosed as above, they are not limited to the applications listed in the specification and implementation modes. They can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the concept defined by the claims and the scope of equivalents, the present invention is not limited to the specific details and the 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 comprises 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, wherein 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.

2. The anti-MxA antibody or a functional fragment thereof according to claim 1, wherein: X1 of CDR-VL1 of the complementary determining region 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.

3. The anti-MxA antibody or a functional fragment thereof according to claim 1, wherein: In the CDR-VL1, X1 is V; Preferably, X1 in the CDR-VL1 is T; Preferably, X2 in the CDR-VL1 is R; Preferably, X2 in the CDR-VL1 is T; Preferably, in the CDR-VL2, X1 is V; Preferably, in the CDR-VL2, X1 is H; Preferably, X2 in the CDR-VL2 is R; Preferably, X2 in the CDR-VL2 is A; Preferably, in the CDR-VL3, X1 is Q; Preferably, in the CDR-VL3, X2 is L; Preferably, X2 in the CDR-VL3 is W; Preferably, X1 in the CDR-VH1 is Y; Preferably, X1 in the CDR-VH1 is D; Preferably, in the CDR-VH1, X2 is H; Preferably, in the CDR-VH2, X1 is N; Preferably, in the CDR-VH2, X1 is I; Preferably, X2 in the CDR-VH2 is T; Preferably, X2 in the CDR-VH2 is S; Preferably, X3 in the CDR-VH2 is Q; Preferably, X3 in the CDR-VH2 is S; Preferably, X1 in the CDR-VH3 is Y; Preferably, in the CDR-VH3, X1 is W; Preferably, X2 in the CDR-VH3 is P; Preferably, X2 in the CDR-VH3 is Y.

4. The anti-MxA antibody or a functional fragment thereof according to any one of claims 1 to 3, characterized in that: Each complementary determining region is selected from any one of the following mutation combinations:

5. The anti-MxA antibody or a functional fragment thereof according to any one of claims 1 to 4, characterized in that: The antibody or its functional fragment also includes heavy chain framework regions FR1-H, FR2-H, FR3-H and FR4-H whose sequences are shown in SEQ ID NOs: 1-4, and / or light chain framework regions FR1-L, FR2-L, FR3-L and FR4-L whose sequences are shown in SEQ ID NOs: 5-8.

6. The anti-MxA antibody or a functional fragment thereof according to claim 5, wherein: The antibody further comprises a constant region; Preferably, the constant region is selected from the constant region of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE and IgD. Preferably, the species of the constant region is cattle, horses, dairy cows, pigs, sheep, goats, rats, mice, dogs, cats, rabbits, camels, donkeys, deer, minks, chickens, ducks, geese, turkeys, fighting cocks or humans.

7. A combination, characterized in that The conjugate comprises the anti-MxA antibody or a functional fragment thereof according to any one of claims 1 to 6 covalently linked to a chemical label and / or a biological label.

8. A conjugate, characterized in that: The conjugate is formed by coupling the anti-MxA antibody or a functional fragment thereof according to any one of claims 1 to 6 and / or the conjugate according to claim 7 with a solid medium or a semi-solid medium.

9. A carrier, characterized in that It contains a nucleic acid fragment encoding the antibody or a functional fragment thereof as described in any one of claims 1 to 6.

10. Use of the anti-MxA antibody or functional fragment thereof according to any one of claims 1 to 6 and / or the conjugate according to claim 7 and / or the conjugate according to claim 8 in detecting MxA.

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