MxA monoclonal paired antibodies and uses thereof

Abstract

The application relates to the technical field of monoclonal antibodies, in particular to a MxA monoclonal paired antibody and application, which comprises MxA mAb 01 and MxA mAb 02, the heavy chain sequence of MxA mAb 01 is shown as SEQ ID NO:1, and the light chain sequence is shown as SEQ ID NO:2; the heavy chain sequence of MxA mAb 02 is shown as SEQ ID NO:3, and the light chain sequence is shown as SEQ ID NO:4. The MxA monoclonal paired antibody has high specificity and high sensitivity, can keep stability under various experimental conditions, is not easily interfered by external factors, and thus the accuracy and reliability of detection results are guaranteed. The paired antibody is suitable for biochemical immunoturbidimetric detection, immunochromatographic detection and ELISA and multiple detection fields, and can realize rapid, efficient and high-sensitivity clinical detection of MxA antigen.

Description

Technical Field

[0001] This invention relates to the field of monoclonal antibody technology, specifically to an MxA monoclonal paired antibody and its applications. Background Technology

[0002] MxA protein (Myxovirus resistance protein A), also known as MX1 protein, is a core antiviral effector protein induced by type I and type III interferon in humans. It belongs to the dynein-like GTPase family and is widely involved in the body's innate immune antiviral response. MxA protein can bind to the viral nucleocapsid and inhibit viral genome transport and transcriptional replication, making it a key biomarker and functional protein for activating the body's antiviral immune system. Furthermore, numerous studies have confirmed that the expression level of MxA protein is closely related to the degree of viral infection and the activity of autoimmune inflammatory diseases. It can serve as a core biomarker for early screening of viral infections, diagnosis of inflammatory diseases, and prognostic assessment, possessing extremely high application value in clinical in vitro diagnostics, biological research detection, and disease mechanism research.

[0003] Currently, the detection of MxA protein mainly relies on antibody-specific immunoassay techniques, including ELISA, chemiluminescent immunoassay, Western blotting, immunohistochemistry, and flow cytometry. Paired antibodies are the core raw material for achieving efficient and accurate quantitative detection of MxA protein, directly determining the sensitivity, specificity, and repeatability of the detection method. Most commercially available MxA antibodies and a small number of paired antibody products are ordinary polyclonal antibodies or conventional monoclonal antibodies, which generally have many technical shortcomings and cannot meet the needs of high-precision clinical testing and research quantification. These shortcomings are mainly reflected in the following aspects: (1) Insufficient specificity: Existing MxA antibodies are prone to non-specific binding with M family homologous protein MxB, resulting in high background noise and false positive results, and cannot accurately distinguish the specific expression level of MxA protein.

[0004] (2) The sensitivity is low. The antigen affinity of conventional paired antibodies is weak and the ability to capture low-abundance MxA protein is poor. It is difficult to achieve accurate detection of low-concentration samples in the early stage of viral infection and cannot meet the needs of early diagnosis.

[0005] (3) Poor antibody pairing adaptability. Most commercially available antibodies are single-recognition epitopes. The synergistic binding efficiency of paired combinations is low, the detection linear range is narrow, the batch-to-batch differences are large, and the detection repeatability and stability are poor.

[0006] (4) Limited applicability: Most existing antibodies are only applicable to a single detection scenario and cannot meet the diversified needs of scientific research qualitative detection and clinical quantitative diagnosis. In addition, some antibodies have poor specificity and low purity and are easily affected by sample matrix interference.

[0007] In summary, the current lack of a set of high-specificity, high-affinity, low-cross-reactivity, and multi-scenario-compatible MxA paired antibodies greatly limits the large-scale application of MxA proteins in early diagnosis of viral infections, assessment of inflammatory diseases, and research on immune mechanisms. Summary of the Invention

[0008] To address the aforementioned problems in existing technologies, this invention provides an MxA monoclonal paired antibody and its applications. This invention effectively solves the technical challenges of poor specificity, low sensitivity, and insufficient stability in existing antibodies, providing high-quality core raw materials for efficient detection of MxA protein, development of clinical diagnostic reagents, and basic scientific research, possessing significant clinical value and industrialization prospects.

[0009] To achieve the above objectives, the present invention provides the following technical solution: A monoclonal paired antibody of MxA includes MxA mAb 01 and MxA mAb 02, wherein the heavy chain sequence of MxA mAb 01 is shown in SEQ ID NO:1 and the light chain sequence is shown in SEQ ID NO:2; the heavy chain sequence of MxA mAb 02 is shown in SEQ ID NO:3 and the light chain sequence is shown in SEQ ID NO:4.

[0010] The method for preparing MxA monoclonal paired antibody of the present invention includes the following steps: (1) Immunization animals: Female Balb / c mice aged 6-8 weeks were selected and immunized according to the pre-established immunization protocol. The antigen enters the peripheral immune organs through blood circulation or lymphatic circulation, stimulates the corresponding B lymphocyte clones, activates and proliferates them, and differentiates them into sensitized B lymphocytes.

[0011] (2) Cell fusion: Mice were euthanized by enucleation and bloodletting. The spleen was aseptically removed and crushed in a petri dish to prepare a spleen cell suspension. The prepared syngeneic myeloma cells were mixed with mouse spleen cells in a certain proportion, and polyethylene glycol, a fusion promoter, was added. Under the action of polyethylene glycol, various lymphocytes could fuse with myeloma cells to form hybridoma cells.

[0012] (3) Selective culture: Selected hybridoma cells were screened using HAT selective culture medium.

[0013] (4) Screening and Cloning of Hybridoma Positive Clones: Only a small number of hybridoma cells grown in HAT medium secrete monoclonal antibodies of the desired specificity; therefore, screening and cloning are necessary. The limiting dilution method is typically used for hybridoma cell cloning. Sensitive, rapid, and specific immunological methods are employed to screen for positive hybridoma cells capable of producing the desired monoclonal antibodies, and clonal amplification is then performed. After comprehensive identification of the immunoglobulin type, subclass, specificity, affinity, epitope recognizing the antigen, and molecular weight of the secreted monoclonal antibodies, they are promptly cryopreserved.

[0014] (5) Purification of monoclonal antibodies: Affinity purification method was used to purify MxA monoclonal paired antibodies MxA mAb 01 and MxAmAb 02.

[0015] The MxA monoclonal paired antibody of the present invention can be used to prepare MxA qualitative detection kits and MxA quantitative detection kits.

[0016] The preparation method of the MxA qualitative detection kit includes the following steps: (1) Labeling antibody: Take 0.05 mg of colored microspheres into a centrifuge tube, add 1 mL of 25 mM pH 6.5 MES buffer, and vortex to mix; add 5 μL of 100 mg / mL NHS, vortex to mix, then add 5 μL of 100 mg / mL EDC, vortex to mix, and incubate at room temperature for 30 min; centrifuge the activated microspheres at 15000 rpm for 20 min, remove the supernatant, resuspend the microspheres with 1 mL of 25 mM pH 6.5 MES buffer, and repeat 3 times; add 0.05 mg of MxA mAb 01, vortex to mix, and incubate at room temperature for 2 h; add 100 μL of 10% (w / v) BSA, and incubate at room temperature for 30 min; centrifuge at 15000 rpm for 15 min, remove the supernatant, resuspend the microspheres with 1 mL of 50 mM pH 7.4 PBS, repeat 3 times, and store at 2-8℃ for later use.

[0017] (2) Preparation of nitrocellulose membrane: The second antibody MxA mAb 02 with a concentration of 1 mg / mL was applied to the nitrocellulose membrane as a detection line with a coating amount of 1 μL / cm, and the sheep-mouse IgG antibody with a concentration of 1 mg / mL was applied to the nitrocellulose membrane as a quality control line with a coating amount of 1 μL / cm. The membrane was dried overnight at 45°C.

[0018] (3) Preparation of conjugate pads: The antibody complex labeled with colored microspheres was sprayed onto a glass cellulose membrane at a coating amount of 6 μL / cm and dried overnight at 45°C to prepare conjugate pads.

[0019] (4) Test strip assembly: attach the coated nitrocellulose membrane to the support base plate, attach the conjugate pad to one end of the marking line, place the sample pad (glass cellulose membrane) on the conjugate pad, and attach the absorbent pad to one end of the control line.

[0020] The preparation method of the MxA quantitative detection kit includes the following steps: (1) Labeling antibody: Take 0.05 mg of time-resolved fluorescent microspheres into a centrifuge tube, add 1 mL of 25 mM pH 6.5 MES buffer, and vortex to mix; add 5 μL of 100 mg / mL NHS, vortex to mix, then add 5 μL of 100 mg / mL EDC, vortex to mix, and incubate at room temperature in the dark for 30 min; centrifuge the activated microspheres at 15000 rpm for 20 min, remove the supernatant, resuspend the microspheres with 1 mL of 25 mM pH 6.5 MES buffer, and repeat 3 times; add 0.05 mg of MxA mAb 01, vortex to mix, and incubate at room temperature in the dark for 2 h; add 100 μL of 10% (w / v) BSA, and incubate at room temperature for 30 min; centrifuge at 15000 rpm for 15 min, remove the supernatant, resuspend the microspheres with 1 mL of 50 mM pH 7.4 PBS, repeat 3 times, and store at 2-8℃ for later use.

[0021] (2) Preparation of nitrocellulose membrane: The second antibody MxA mAb 02 with a concentration of 1 mg / mL was applied to the nitrocellulose membrane as a detection line with a coating amount of 1 μL / cm, and the sheep-mouse IgG antibody with a concentration of 1 mg / mL was applied to the nitrocellulose membrane as a quality control line with a coating amount of 1 μL / cm. The membrane was dried overnight at 45°C.

[0022] (3) Preparation of conjugate pad: Time-resolved fluorescent microsphere-labeled antibody complex was sprayed onto a glass cellulose membrane at a coating amount of 6 μL / cm and dried overnight at 45°C to prepare a conjugate pad.

[0023] (4) Test strip assembly: attach the coated nitrocellulose membrane to the support base plate, attach the conjugate pad to one end of the marking line, place the sample pad (glass cellulose membrane) on the conjugate pad, and attach the absorbent pad to one end of the control line.

[0024] Compared with the prior art, the beneficial effects of the present invention are: (1) The MxA monoclonal paired antibody of the present invention has high specificity and high sensitivity, can maintain stability under various experimental conditions, and is not easily affected by external factors, thereby ensuring the accuracy and reliability of the detection results.

[0025] (2) The MxA monoclonal paired antibody of the present invention is applicable to multiple detection fields such as biochemical immunoturbidimetric detection, immunochromatographic detection and ELISA, and can realize rapid, efficient and highly sensitive clinical detection of MxA antigen. Attached Figure Description

[0026] Figure 1 The results of SDS-PAGE analysis of purified MxA mAb 01 antibody are shown.

[0027] Figure 2 The results of SDS-PAGE analysis of purified MxA mAb 02 antibody are shown.

[0028] Figure 3 Quantitative detection results for MxA calibrators. Detailed Implementation

[0029] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0030] Example 1: Preparation of MxA monoclonal paired antibody (1) Immunized animals The immunization regimen is selected based on the characteristics of the antigen. For soluble antigens with weak immunogenicity, adjuvants are generally added. Commonly used adjuvants are Freund's complete adjuvant and Freund's incomplete adjuvant. The antigen and adjuvant should be mixed in equal volumes and ground into a water-in-oil emulsion.

[0031] The initial immunization was performed by subcutaneous injection of 50 μg antigen with Freund's complete adjuvant at multiple sites; the second immunization was performed 3 weeks later (same dose of antigen with Freund's incomplete adjuvant); the third immunization was performed by intraperitoneal injection without adjuvant, and the titer was tested 7 days later; the final booster immunization was performed (50 μg antigen intraperitoneal injection), and spleen cells were harvested for fusion 3 days later.

[0032] (2) Cell fusion Myeloma cell suspension: Select a good myeloma cell line, isolate myeloma cells from tumors growing in vivo, and prepare a cell suspension; Spleen cell suspension: Mice that received a booster 3 days prior underwent orbital bloodletting, and the serum was separated and frozen for later use. The mice were euthanized, and the cells were immersed in 75% ethanol for 3-5 minutes. The spleen was aseptically removed and washed in a petri dish containing 5 mL of incomplete culture medium. The surrounding connective tissue was removed, and the spleen was transferred to a steel mesh in another petri dish containing 5 mL of incomplete culture medium. It was first cut into 3-5 small pieces with scissors, then ground with a syringe core. The spleen cell suspension was transferred to a 50 mL centrifuge tube, 50 mL of incomplete culture medium was added, and the tube was centrifuged at 1000 rpm for 5 minutes. The supernatant was discarded, and the cells were washed and centrifuged again using the same method. The precipitated cells were then resuspended in 10 mL of incomplete culture medium, and the viable cell count was recorded. 0.5-2 × 10⁶ cells were obtained from one mouse. 8 One spleen cell.

[0033] Feeder Cells: After euthanizing, disinfecting, and fixing the mice, use sterile scissors to lift the abdominal skin from the posterior abdomen to expose the peritoneum. Disinfect the peritoneum with an alcohol swab. Inject 10 mL of incomplete culture medium into the peritoneal cavity using a syringe, taking care to avoid perforation of the intestine. Hold the syringe firmly with your right hand, leaving the needle in the peritoneal cavity, and gently massage the abdomen with an alcohol swab in your left hand for 1 minute. Then aspirate the injected culture medium. Centrifuge at 1000 rpm for 10 minutes and discard the supernatant. Resuspend the pelleted cells in 5 mL of HAT medium. Based on the cell count, add more HAT medium to bring the cell concentration to 2 × 10⁶ cells / mL. 5 / mL, for later use.

[0034] Cell fusion: Mix the prepared myeloma cells and mouse spleen cells at a ratio of 1:5, add 20 mL of PRMI-1640 solution; centrifuge at 1000 r / min for 10 min, discard the supernatant, and aspirate as much supernatant as possible; gently tap the bottom of the centrifuge tube with your finger to disperse the precipitated cells, and place the centrifuge tube in a 37℃ water bath; slowly add 1 mL of 50% PEG pre-warmed at 37℃ into the centrifuge tube over about 45 seconds, stirring gently while adding, and let stand at 37℃ for 5 min; add 20 mL of incomplete culture medium (pre-warmed at 37℃) over 5 min, adding 1 mL in the first minute, 2 mL in the second minute, 5 mL in the third minute, and 6 mL each in the fourth and fifth minutes, while gently rotating the centrifuge tube. The medium should be added along the tube wall, not directly onto the precipitated cells, to prevent dislodging the newly fused cells. Then add PRMI-1640 solution to 50 mL to terminate the PEG effect; centrifuge at 800 r / min for 10 min, discard the supernatant, gently suspend the precipitated cells in the required volume of HAT culture medium, and seed 0.10-0.15 mL per well in a 96-well culture plate. After seeding, place the culture plate in a 37℃ 5% CO2 incubator for culture.

[0035] (3) Selective culture: After inoculating 96-well plates for 5 days, replace half of the medium with HAT medium. After 7-10 days, replace HAT medium with HT medium. After 14 days, ordinary complete medium can be used.

[0036] (4) Screening and cloning of hybridoma-positive clones Coating: The antigen was coated with Na2CO3-NaHCO3 buffer at pH 9.6. 20 μg of antigen was added to each microplate, and the coating volume per well was 50 μL. Coating was carried out at 37°C for 2 h.

[0037] Blocking: Discard the antigen, blot dry the liquid in the wells of the ELISA plate, and block with 1% BSA at 37°C for 2 hours.

[0038] Primary antibody: After blocking, discard the liquid in the well, pat dry, add 100 μL of the supernatant of the cells to be tested to each well, and incubate at 37°C for 1 h.

[0039] Secondary antibody: After incubation with primary antibody, discard the liquid in the wells, blot dry, and wash three times with PBS buffer (pH 7.4) for 5 minutes each time. Dilute the secondary antibody according to the instructions, add 100 μL to each well, and incubate at 37°C for 1 hour.

[0040] Color development: After incubation with the secondary antibody, discard the liquid in the wells, pat dry, add the colorimetric reagent, and stop the reaction with the stop solution when the color is the deepest. Read the OD value of each well with a microplate reader, select the positive wells, expand the culture and freeze them.

[0041] (5) Purification of monoclonal antibodies MxA monoclonal paired antibodies were purified using affinity purification. Staphylococcal A protein was cross-linked with a carrier, and an affinity chromatography column was prepared to bind the antibodies before elution. The antibodies were then concentrated to 5 mg / mL. The purified monoclonal paired antibodies are shown in SEQ ID NO:1 for the heavy chain sequence and SEQ ID NO:2 for the light chain sequence; and in SEQ ID NO:3 for the heavy chain sequence and SEQ ID NO:4 for the light chain sequence.

[0042] Example 2

[0043] Antibody concentration was analyzed by SDS-PAGE electrophoresis. Results are as follows: Figure 1-2 As shown.

[0044] SDS-PAGE electrophoresis experimental procedure: First, prepare the separating gel and stacking gel, pour the gels in sequence, and remove the comb after they solidify; mix the protein sample with SDS loading buffer at a ratio of 4:1, heat in a boiling water bath for 5-10 minutes to denature, and then cool; then load the gel into the electrophoresis tank, fill it with buffer, add the prepared sample to the sample wells, and electrophoresis at a constant voltage of 80V until the indicator enters the separating gel, then adjust to 120V until the indicator is close to the bottom of the gel; finally, remove the gel, stain it with Coomassie Brilliant Blue staining solution with shaking for more than 30 minutes, change the destaining solution and shake several times until the bands are clear and the background is transparent.

[0045] Example 3: Preparation of MxA Qualitative Detection Kit from MxA Monoclonal Paired Antibodies in Example 1 (1) Antibody labeling: Take 0.05 mg of colored microspheres (Nanjing Liding Medical Technology Co., Ltd., catalog number: LD202002) into a centrifuge tube, add 1 mL of 25 mM pH 6.5 MES buffer, and vortex to mix; add 5 μL of 100 mg / mL NHS, vortex to mix, then add 5 μL of 100 mg / mL EDC, vortex to mix, and incubate at room temperature in the dark for 30 min; centrifuge the activated microspheres at 15000 rpm for 20 min, remove the supernatant, and resuspend the microspheres with 1 mL of 25 mM pH 6.5 MES buffer, repeat 3 times; add 0.05 mg of MxA mAb 01 from Example 1, vortex to mix, and incubate at room temperature in the dark for 2 h; add 100 μL of 10% (w / v) BSA, and incubate at room temperature for 30 min; centrifuge at 15000 rpm for 15 min, remove the supernatant, and resuspend the microspheres with 1 mL of 50 mM pH 6.5 MES buffer. 7.4 Resuspend the microspheres in PBS, repeat 3 times, and store at 2-8℃ for later use.

[0046] (2) Preparation of nitrocellulose membrane: The second antibody MxA mAb 02 with a concentration of 1 mg / mL was applied to the nitrocellulose membrane as a detection line with a coating amount of 1 μL / cm, and the sheep-mouse IgG antibody with a concentration of 1 mg / mL was applied to the nitrocellulose membrane as a quality control line with a coating amount of 1 μL / cm. The membrane was dried overnight at 45°C.

[0047] (3) Preparation of conjugate pads: The antibody complex labeled with colored microspheres was sprayed onto a glass cellulose membrane at a coating amount of 6 μL / cm and dried overnight at 45°C to prepare conjugate pads.

[0048] (4) Test strip assembly: attach the coated nitrocellulose membrane to the support base plate, attach the conjugate pad to one end of the marking line, place the sample pad (glass cellulose membrane) on the conjugate pad, and attach the absorbent pad to one end of the control line.

[0049] Example 4: Preparation of MxA quantitative detection kit using MxA monoclonal paired antibody from Example 1 (1) Antibody labeling: Take 0.05 mg of time-resolved fluorescent microspheres (Nanjing Liding Medical Technology Co., Ltd., catalog number: LD302017) into a centrifuge tube, add 1 mL of 25 mM pH 6.5 MES buffer, and vortex to mix; add 5 μL of 100 mg / mL NHS, vortex to mix, then add 5 μL of 100 mg / mL EDC, vortex to mix, and incubate at room temperature in the dark for 30 min; centrifuge the activated microspheres at 15000 rpm for 20 min, remove the supernatant, and resuspend the microspheres in 1 mL of 25 mM pH 6.5 MES buffer, repeat 3 times; add 0.05 mg of MxA mAb 01 from Example 1, vortex to mix, and incubate at room temperature in the dark for 2 h; add 100 μL of 10% (w / v) BSA, and incubate at room temperature for 30 min; centrifuge at 15000 rpm for 15 min, remove the supernatant, and resuspend the microspheres in 1 mL of 50 mM pH 6.5 MES buffer. 7.4 Resuspend the microspheres in PBS, repeat 3 times, and store at 2-8℃ for later use.

[0050] (2) Preparation of nitrocellulose membrane: The second antibody MxA mAb 02 with a concentration of 1 mg / mL was applied to the nitrocellulose membrane as a detection line with a coating amount of 1 μL / cm, and the sheep-mouse IgG antibody with a concentration of 1 mg / mL was applied to the nitrocellulose membrane as a quality control line with a coating amount of 1 μL / cm. The membrane was dried overnight at 45°C.

[0051] (3) Preparation of conjugate pad: Time-resolved fluorescent microsphere-labeled antibody complex was sprayed onto a glass cellulose membrane at a coating amount of 6 μL / cm and dried overnight at 45°C to prepare a conjugate pad.

[0052] (4) Test strip assembly: attach the coated nitrocellulose membrane to the support base plate, attach the conjugate pad to one end of the marking line, place the sample pad (glass cellulose membrane) on the conjugate pad, and attach the absorbent pad to one end of the control line.

[0053] Testing instrument: Lambert AFS1520 fluorescence immunoassay analyzer.

[0054] To highlight the beneficial effects of the present invention, the following comparative experiments are provided.

[0055] Comparative Example 1: An MxA qualitative detection kit was prepared using MxA mAb from Merk. (1) Antibody labeling: Take 0.05 mg of colored microspheres (Nanjing Liding Medical Technology Co., Ltd., catalog number: LD202002) into a centrifuge tube, add 1 mL of 25 mM pH 6.5 MES buffer, and vortex to mix; add 5 μL of 100 mg / mL NHS, vortex to mix, then add 5 μL of 100 mg / mL EDC, vortex to mix, and incubate at room temperature in the dark for 30 min; centrifuge the activated microspheres at 15000 rpm for 20 min, remove the supernatant, and resuspend the microspheres with 1 mL of 25 mM pH 6.5 MES buffer, repeat 3 times; add 0.05 mg of MxA mAb (Merk, MABF938), vortex to mix, and incubate at room temperature for 2 h; add 100 μL of 10% (w / v) BSA, and incubate at room temperature for 30 min; centrifuge at 15000 rpm for 15 min, remove the supernatant, and resuspend the microspheres with 1 mL of 50 mM pH 6.5 MES buffer. 7.4 Resuspend the microspheres in PBS, repeat 3 times, and store at 2-8℃ for later use.

[0056] (2) Preparation of nitrocellulose membrane: MxA mAb (Merk, ZRB2592) with a concentration of 1 mg / mL was applied to the nitrocellulose membrane as a detection line at a coating amount of 1 μL / cm, and sheep-rat IgG antibody with a concentration of 1 mg / mL was applied to the nitrocellulose membrane as a quality control line at a coating amount of 1 μL / cm. The membrane was dried overnight at 45°C.

[0057] (3) Preparation of conjugate pads: The antibody complex labeled with colored microspheres was sprayed onto a glass cellulose membrane at a coating amount of 6 μL / cm and dried overnight at 45°C to prepare conjugate pads.

[0058] (4) Test strip assembly: attach the coated nitrocellulose membrane to the support base plate, attach the conjugate pad to one end of the marking line, place the sample pad (glass cellulose membrane) on the conjugate pad, and attach the absorbent pad to one end of the control line.

[0059] Comparative Example 2: An MxA quantitative detection kit was prepared using MxA mAb from Merk. (1) Antibody labeling: Take 0.05 mg of time-resolved fluorescent microspheres (Nanjing Liding Medical Technology Co., Ltd., catalog number: LD302017) into a centrifuge tube, add 1 mL of 25 mM pH 6.5 MES buffer, and vortex to mix; add 5 μL of 100 mg / mL NHS, vortex to mix, then add 5 μL of 100 mg / mL EDC, vortex to mix, and incubate at room temperature in the dark for 30 min; centrifuge the activated microspheres at 15000 rpm for 20 min, remove the supernatant, and resuspend the microspheres with 1 mL of 25 mM pH 6.5 MES buffer, repeat 3 times; add 0.05 mg of MxA mAb (Merk, MABF938), vortex to mix, and incubate at room temperature in the dark for 2 h; add 100 μL of 10% (w / v) BSA, and incubate at room temperature for 30 min; centrifuge at 15000 rpm for 15 min, remove the supernatant, and resuspend the microspheres with 1 mL of 25 mM pH 6.5 MES buffer, repeat 3 times; add 0.05 mg of MxA mAb (Merk, MABF938), vortex to mix, and incubate at room temperature in the dark for 2 h; add 100 μL of 10% (w / v) BSA, and incubate at room temperature for 30 min; centrifuge at 15000 rpm for 15 min, remove the supernatant, and resuspend the microspheres with 1 mL of 25 mM pH 6.5 MES buffer, repeat 3 times. Resuspend the microspheres in 50mM pH 7.4 PBS, repeat 3 times, and store at 2-8℃ for later use.

[0060] (2) Preparation of nitrocellulose membrane: MxA mAb (Merk, ZRB2592) with a concentration of 1 mg / mL was applied to the nitrocellulose membrane as a detection line at a coating amount of 1 μL / cm, and sheep-rat IgG antibody with a concentration of 1 mg / mL was applied to the nitrocellulose membrane as a quality control line at a coating amount of 1 μL / cm. The membrane was dried overnight at 45°C.

[0061] (3) Preparation of conjugate pad: Time-resolved fluorescent microsphere-labeled antibody complex was sprayed onto a glass cellulose membrane at a coating amount of 6 μL / cm and dried overnight at 45°C to prepare a conjugate pad.

[0062] (4) Test strip assembly: attach the coated nitrocellulose membrane to the support base plate, attach the conjugate pad to one end of the marking line, place the sample pad (glass cellulose membrane) on the conjugate pad, and attach the absorbent pad to one end of the control line.

[0063] Testing instrument: Lambert AFS1520 fluorescence immunoassay analyzer.

[0064] Table 1 shows the qualitative detection results of MxA calibrators. The data shows that the colorimetric signal (T value) of Example 3 significantly increased with increasing MxA concentration, and the T values ​​at the same concentration were all higher than those of Comparative Example 1. This indicates that the proposed method has higher detection sensitivity and stronger signal response capability, effectively improving the performance of MxA detection and possessing good application prospects.

[0065] Table 1 Qualitative Testing of MxA Calibrators

[0066] Note: The T value is obtained from the card reader. The larger the value, the more obvious the color rendering.

[0067] Table 2 and Figure 3 For the quantitative detection results of MxA calibrators, the T / C values ​​of both Example 4 and Comparative Example 2 increased with increasing concentration, and both showed a good linear relationship; among them, the linear correlation coefficient R of Example 4 was [missing value]. 2 =0.9949, which is higher than 0.9920 of Comparative Example 2, and the T / C value is higher at all concentrations, indicating that the quantitative accuracy, signal response intensity and detection sensitivity of the proposed method are superior.

[0068] Table 2 Quantitative Detection of MxA Calibrators

[0069] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. An MxA monoclonal paired antibody, characterized in that: Includes MxA mAb01 and MxA mAb02, the heavy chain sequence of MxA mAb01 is shown in SEQ ID NO:1, and the light chain sequence is shown in SEQ ID NO:2; the heavy chain sequence of MxA mAb02 is shown in SEQ ID NO:3, and its light chain sequence is shown in SEQ ID NO:

4.

2. The use of the MxA monoclonal paired antibody as described in claim 1 in the preparation of an immunochromatographic qualitative detection kit for detecting MxA protein.

3. The application of the MxA monoclonal paired antibody according to claim 2 in the preparation of an immunochromatographic qualitative detection kit for detecting MxA protein, characterized in that, The preparation method of the MxA qualitative detection kit includes the following steps: (1) Labeling antibody: Take 0.05 mg of colored microspheres into a centrifuge tube, add 1 mL of 25 mM pH 6.5 MES buffer, and vortex to mix; add 5 μL of 100 mg / mL NHS, vortex to mix, then add 5 μL of 100 mg / mL EDC, vortex to mix, and incubate at room temperature for 30 min; centrifuge the activated microspheres at 15000 rpm for 20 min, remove the supernatant, resuspend the microspheres in 1 mL of 25 mM pH 6.5 MES buffer, and repeat 3 times; add 0.05 mg of MxA mAb 01, vortex to mix, and incubate at room temperature for 2 h; add 100 μL of 10% BSA, and incubate at room temperature for 30 min; centrifuge at 15000 rpm for 15 min, remove the supernatant, resuspend the microspheres in 1 mL of 50 mM pH 7.4 PBS, repeat 3 times, and store at 2-8℃ for later use; (2) Preparation of nitrocellulose membrane: The second antibody MxA mAb 02 with a concentration of 1 mg / mL was stripped onto the nitrocellulose membrane as a detection line at a coating amount of 1 μL / cm, and the sheep-mouse IgG antibody with a concentration of 1 mg / mL was stripped onto the nitrocellulose membrane as a quality control line at a coating amount of 1 μL / cm. The membrane was dried overnight at 45°C. (3) Preparation of conjugate pads: The antibody complex labeled with colored microspheres was sprayed onto a glass cellulose membrane at a coating amount of 6 μL / cm and dried overnight at 45°C to prepare conjugate pads; (4) Test strip assembly: attach the coated nitrocellulose membrane to the support base plate, attach the conjugate pad to one end of the marking line, place the sample pad on the conjugate pad, and attach the absorbent pad to one end of the control line.

4. The use of the MxA monoclonal paired antibody as described in claim 1 in the preparation of an immunochromatographic quantitative detection kit for detecting MxA protein.

5. The application of the MxA monoclonal paired antibody according to claim 4 in the preparation of an immunochromatographic quantitative detection kit for detecting MxA protein, characterized in that, The preparation method of the MxA quantitative detection kit includes the following steps: (1) Antibody labeling: Take 0.05 mg of time-resolved fluorescent microspheres into a centrifuge tube, add 1 mL of 25 mM pH 6.5 MES buffer, and vortex to mix; add 5 μL of 100 mg / mL NHS, vortex to mix, then add 5 μL of 100 mg / mL EDC, vortex to mix, and incubate at room temperature in the dark for 30 min; centrifuge the activated microspheres at 15000 rpm for 20 min, remove the supernatant, resuspend the microspheres in 1 mL of 25 mM pH 6.5 MES buffer, and repeat 3 times; add 0.05 mg of MxA mAb 01, vortex to mix, and incubate at room temperature in the dark for 2 h; add 100 μL of 10% BSA, and incubate at room temperature for 30 min; centrifuge at 15000 rpm for 15 min, remove the supernatant, resuspend the microspheres in 1 mL of 50 mM pH 7.4 PBS, repeat 3 times, and store at 2-8℃ for later use; (2) Preparation of nitrocellulose membrane: The second antibody MxA mAb 02 with a concentration of 1 mg / mL was stripped onto the nitrocellulose membrane as a detection line at a coating amount of 1 μL / cm, and the sheep-mouse IgG antibody with a concentration of 1 mg / mL was stripped onto the nitrocellulose membrane as a quality control line at a coating amount of 1 μL / cm. The membrane was dried overnight at 45°C. (3) Preparation of conjugate pad: Time-resolved fluorescent microsphere-labeled antibody complex was sprayed onto glass cellulose membrane at a coating amount of 6 μL / cm and dried overnight at 45°C to prepare conjugate pad; (4) Test strip assembly: attach the coated nitrocellulose membrane to the support base plate, attach the conjugate pad to one end of the marking line, place the sample pad on the conjugate pad, and attach the absorbent pad to one end of the control line.

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