CK-MB monoclonal paired antibody and application thereof

Abstract

The application relates to the technical field of monoclonal antibodies, in particular to a CK-MB monoclonal paired antibody and application, wherein the heavy chain sequence of a first antibody is shown as SEQ ID NO:1, and the light chain sequence is shown as SEQ ID NO:2; the heavy chain sequence of a second antibody is shown as SEQ ID NO:3, and the light chain sequence is shown as SEQ ID NO:4. The CK-MB monoclonal paired antibody has high specificity and high sensitivity, can maintain stability under various experimental conditions, is not easily interfered by external factors, and guarantees the accuracy and reliability of detection results. The CK-MB monoclonal paired antibody can be used in the fields of biochemical immunoturbidimetric detection, immunochromatographic detection and ELISA, and can realize rapid, efficient and high-sensitivity clinical detection of CK-MB antigen.

Description

Technical Field

[0001] This invention relates to the field of monoclonal antibody technology, specifically to a CK-MB monoclonal paired antibody and its applications. Background Technology

[0002] Creatine kinase (CK), a key enzyme in cellular energy metabolism, plays a crucial role in intracellular energy transport, muscle contraction, and adenosine triphosphate (ATP) regeneration, making it an important molecule for maintaining energy homeostasis. CK isoenzymes are dimers formed by different combinations of the M and B subunits. Three main isoenzyme forms exist intracellularly: CK-MM, CK-MB, and CK-BB. Among these, CK-MB, primarily derived from myocardial tissue, has become an important biochemical marker related to myocardial function in clinical practice.

[0003] In clinical applications, CK-MB is one of the earliest blood biochemical markers used to assess myocardial injury. Its dynamic changes follow a clear temporal pattern: it typically begins to rise 3-8 hours after the onset of myocardial injury, reaches its peak at 9-30 hours, and gradually returns to normal levels after 48-72 hours. However, it is important to note that CK-MB is not a specific marker for cardiomyocytes. Besides myocardial tissue, skeletal muscle also contains a certain amount of CK-MB. Therefore, damage to skeletal muscle can also lead to elevated CK-MB levels in the blood. Based on this characteristic, in the clinical diagnosis of acute myocardial infarction (AMI), CK-MB needs to be combined with other more specific myocardial injury markers such as cardiac troponin I (cTnI) for a more accurate diagnostic assessment.

[0004] Currently, the main methods used in laboratories to detect CK-MB include immunochromatography (colloidal gold), two-dimensional flow immunoassay, chemiluminescent enzyme-linked immunosorbent assay (ELISA), and immunoturbidimetric assay. However, these methods all depend on high-quality CK-MB-specific antibodies, and existing detection technologies still have significant shortcomings in terms of specificity, sensitivity, and accuracy, making it difficult to fully meet the clinical needs for early and accurate diagnosis of myocardial injury.

[0005] Therefore, developing CK-MB-specific antibodies with high specificity and affinity is crucial for significantly improving the accuracy and sensitivity of CK-MB detection, providing more reliable technical support for clinical diagnosis of myocardial injury. Simultaneously, establishing efficient antibody preparation methods (such as using hybridoma technology to prepare monoclonal antibodies) can effectively overcome the problems of complex production processes, high production costs, and poor batch stability in existing technologies, opening up new pathways for the industrial production of CK-MB detection reagents and further promoting the clinical translation and application of myocardial injury diagnostic technology. Summary of the Invention

[0006] The purpose of this invention is to provide a CK-MB monoclonal paired antibody and its application.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] A CK-MB monoclonal paired antibody, wherein the heavy chain sequence of the first antibody is shown in SEQ ID NO:1 and its light chain sequence is shown in SEQ ID NO:2; the heavy chain sequence of the second antibody is shown in SEQ ID NO:3 and its light chain sequence is shown in SEQ ID NO:4.

[0009] The CK-MB monoclonal paired antibody described in this invention can be used to prepare a latex immunoturbidimetric assay kit. The kit includes reagent R1 and reagent R2.

[0010] The reagent R1 is 15mM pH 6.8 Tris buffer, NaCl 2g / L, PEG20000 5g / L, BSA 1w%, Proclin 300 0.08% (v / v);

[0011] The preparation method of the reagent R2 is as follows:

[0012] (1) Coating the first antibody CK-MB mAb01: Take 0.05 mL of PS microspheres, add 2.5 mL of 0.025 M pH=6.5 MES buffer and mix, then add 5 μL of 0.1 g / mL EDC solution (prepared and used immediately), mix and stir at room temperature for 15 min; add 0.05 mg of CK-MB mAb01 to the reaction system and stir at room temperature for 1 h; add 10 μL of blocking agent (blocking agent is 1% BSA and 0.2% glycine aqueous solution) to the reaction system and stir for 1 h; centrifuge at 8000 rpm for 15 min, resuspend in 2.5 mL of pH 7.0 PBS solution, and sonicate for 10 min;

[0013] (2) Coating the second antibody CK-MB mAb02: Take 0.05 mL of PS microspheres, add 2.5 mL of 0.025 M pH=6.5 MES buffer and mix, then add 5 μL of 0.1 g / mL EDC solution (prepared and used immediately), mix and stir at room temperature for 15 min; add 0.05 mg of CK-MB mAb02 to the reaction system and stir at room temperature for 1 h; add 10 μL of blocking agent (blocking agent is 1% BSA and 0.2% glycine aqueous solution) to the reaction system and stir for 1 h; centrifuge at 8000 rpm for 15 min, resuspend in 2.5 mL of pH 7.0 PBS solution, and sonicate for 10 min;

[0014] (3) Mix the two microsphere antibody-labeled complexes and store at 2-8℃ for later use.

[0015] The CK-MB monoclonal paired antibody described in this invention can be used to prepare an immunochromatographic detection kit. The preparation method is as follows:

[0016] (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 LEDC, 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, repeating 3 times; add 0.05 mg of CK-MB 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, and resuspend the microspheres in 1 mL of 50 mM pH 7.4 MES buffer. Resuspend the microspheres in PBS, repeat 3 times, and store at 2-8℃ for later use.

[0017] (2) Preparation of nitrocellulose membrane: The second antibody CK-MB mAb02 with a concentration of 1 mg / mL was stripped onto 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 stripped onto 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 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;

[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 CK-MB monoclonal paired antibody described in this invention can also be used to prepare ELISA detection kits.

[0021] Compared with the prior art, the beneficial effects of the present invention are:

[0022] (1) The CK-MB monoclonal paired antibody of the present invention has high specificity and high sensitivity, can maintain stability under various experimental conditions, is not easily affected by external factors, and ensures the accuracy and reliability of the detection results.

[0023] (2) The CK-MB monoclonal paired antibody of the present invention can be used in multiple detection fields such as biochemical immunoturbidimetric detection, immunochromatographic detection and ELISA, realizing rapid, efficient and high-sensitivity clinical detection of CK-MB antigen. Attached Figure Description

[0024] Figure 1 The SDS-PAGE results are for CK-MB mAb01 purified.

[0025] Figure 2 This is the SEC-HPLC chromatogram of CK-MB mAb01 after purification.

[0026] Figure 3 The SDS-PAGE results are for CKM-MB mAb02 purified.

[0027] Figure 4 The image shows the SEC-HPLC chromatogram of purified CK-MB mAb02. Detailed Implementation

[0028] 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.

[0029] Example 1: Preparation of CK-MB monoclonal paired antibody

[0030] (1) Immunized animals

[0031] 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.

[0032] 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.

[0033] (2) Cell fusion

[0034] Myeloma cell suspension: Select a good myeloma cell line, isolate myeloma cells from tumors growing in vivo, and prepare a cell suspension;

[0035] Spleen cell suspension: Mice that received a booster 3 days prior were subjected to orbital bloodletting, and the serum was separated and frozen for later use. The mice were euthanized and immersed in 75% alcohol 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 mixture was centrifuged at 1000 rpm for 5 minutes. The supernatant was discarded, and the mixture was 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.

[0036] 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.

[0037] 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; within 5 min, add 20 mL of incomplete complete culture medium (pre-warmed at 37℃), 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 as you add the medium. 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.

[0038] (3) Selective culture

[0039] Five days after inoculating 96-well plates, replace half of the medium with HAT medium. After 7-10 days, replace the HAT medium with HT medium. After the 14th day, ordinary complete medium can be used.

[0040] (4) Screening and cloning of hybridoma-positive clones

[0041] 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.

[0042] 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.

[0043] 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.

[0044] Secondary antibody: After incubation with primary antibody, discard the liquid in the wells, blot dry, and wash three times with PBST buffer (pH 7.4) for 5 min each time. Dilute the secondary antibody according to the instructions, add 100 μL to each well, and incubate at 37°C for 1 h.

[0045] 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.

[0046] (5) Purification of monoclonal antibodies

[0047] CK-MB 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 and SEQ ID NO:2 for the light chain; and in SEQ ID NO:3 for the heavy chain and SEQ ID NO:4 for the light chain.

[0048] Example 2

[0049] The concentration of the primary antibody CK-MB mAb01 was analyzed by SDS-PAEG electrophoresis and SEC-HPLC, respectively. The results are as follows: Figure 1 and Figure 2 As shown. The experimental steps are as follows:

[0050] SDS-PAGE electrophoresis:

[0051] A. Prepare the separating adhesive and the concentrating adhesive, pour them into the adhesive in sequence, and then remove the comb after solidification.

[0052] B. Mix the protein sample with SDS loading buffer at a ratio of 4:1, and denature in a boiling water bath for 5-10 minutes before cooling.

[0053] C. Load the gel into the electrophoresis tank, fill with buffer and add the sample. Electrophore 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.

[0054] D. Remove the gel, stain with Coomassie Brilliant Blue staining solution with shaking for more than 30 minutes, and change the destaining solution several times with shaking until the bands are clear and the background is transparent.

[0055] SEC-HPLC method:

[0056] A. Equilibrate the column with phosphate buffer (mobile phase) until baseline is stable.

[0057] B. The sample is filtered through a 0.22 μm filter membrane to avoid particulate contamination of the chromatographic column.

[0058] C. Set the HPLC parameters (flow rate 0.5-1.0 mL / min, column temperature 30℃), and inject 25 μL of sample after the system stabilizes.

[0059] D. After the operation is complete, flush the column with the mobile phase and seal with pure solvent. During the operation, it is necessary to degas the mobile phase, prevent sudden increases in column pressure, and avoid air bubbles in the system.

[0060] Example 3

[0061] The concentration of the secondary antibody CK-MB mAb02 was analyzed by SDS-PAEG electrophoresis and SEC-HPLC, respectively. The results are as follows: Figure 3-4 As shown. The experimental steps are as follows:

[0062] SDS-PAGE electrophoresis:

[0063] A. Prepare separating and concentrating adhesives, pour them into the adhesives in sequence, allow them to solidify, and then remove the comb.

[0064] B. Mix the protein sample with SDS loading buffer at a 4:1 ratio, denature in a boiling water bath for 5-10 minutes, and then cool.

[0065] C. Load the gel into the electrophoresis tank, fill with buffer and add the sample. Electrophore 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.

[0066] D. Remove the gel, stain with Coomassie Brilliant Blue staining solution with shaking for more than 30 minutes, and change the destaining solution several times with shaking until the bands are clear and the background is transparent.

[0067] SEC-HPLC method:

[0068] A. Equilibrate the column with phosphate buffer (mobile phase) until baseline stability is achieved;

[0069] B. The sample is filtered through a 0.22μm filter membrane to avoid particulate contamination of the chromatographic column;

[0070] C. Set the HPLC parameters (flow rate 0.5-1.0 mL / min, column temperature 30℃), and inject 25 μL of sample after the system stabilizes;

[0071] D. After the operation is complete, flush the column with the mobile phase and seal with pure solvent. During the operation, it is necessary to degas the mobile phase, prevent sudden increases in column pressure, and avoid air bubbles in the system.

[0072] Example 4: Preparation of CK-MB Monoclonal Paired Antibodies for CK-MB Detection Kit (Latex Immunoturbidimetric Assay)

[0073] Reagent R1: 15mM pH 6.8 Tris buffer, NaCl 2g / L, PEG20000 5g / L, BSA 1w%, Proclin300 0.08% (v / v).

[0074] Preparation of reagent R2:

[0075] (1) Coating the first antibody CK-MB mAb01: Take 0.05 mL of PS microspheres (Nanjing Liding Medical Technology Co., Ltd., catalog number: LD102026), add 2.5 mL of 0.025 M pH=6.5 MES buffer, mix, add 5 μL of 0.1 g / mL EDC solution (prepared and used immediately), mix and stir at room temperature for 15 min; add 0.05 mg of CK-MB mAb01 prepared in Example 1 to the reaction system, stir at room temperature for 1 h; add 10 μL of blocking agent (blocking agent is 1% BSA and 0.2% glycine aqueous solution) to the reaction system, stir for 1 h; centrifuge at 8000 rpm for 15 min, resuspend in 2.5 mL of pH 7.0 PBS solution, and sonicate for 10 min.

[0076] (2) Coating the second antibody CK-MB mAb02: Take 0.05 mL of PS microspheres, add 2.5 mL of 0.025 M pH=6.5 MES buffer, mix, add 5 μL of 0.1 g / mL EDC solution (prepared and used immediately), mix and stir at room temperature for 15 min; add 0.05 mg of CK-MB mAb02 prepared in Example 1 to the reaction system, stir at room temperature for 1 h; add 10 μL of blocking agent (blocking agent is 1% BSA and 0.2% glycine aqueous solution) to the reaction system, stir for 1 h; centrifuge at 8000 rpm for 15 min, resuspend in 2.5 mL of pH 7.0 PBS solution, and sonicate for 10 min.

[0077] (3) Mix the two microsphere antibody-labeled complexes and store at 2-8℃ for later use.

[0078] Testing instrument: Hitachi 7180 fully automated biochemical analyzer.

[0079] Parameters: Sample volume 20μL, reagent R1 180μL, reagent R2 60μL, fitting method Spline, main wavelength 600nm, secondary wavelength none.

[0080] Example 5: Preparation of CK-MB Monoclonal Paired Antibodies for CK-MB Detection Kit (Fluorescent Immunochromatography)

[0081] (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 CK-MB 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.

[0082] (2) Preparation of nitrocellulose membrane: The second antibody CK-MB mAb02 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.

[0083] (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.

[0084] (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.

[0085] Testing instrument: Lambert AFS1520 fluorescence immunoassay analyzer.

[0086] To highlight the beneficial effects of the present invention, the following comparative experiments are provided.

[0087] Comparative Example 1: A CK-MB detection kit (latex immunoturbidimetric assay) was prepared using recombinant CK-MB monoclonal antibody from Aladdin Company.

[0088] Reagent R1: 15mM pH 6.8 Tris buffer, NaCl 2g / L, PEG20000 5g / L, BSA 1w%, Proclin300 0.08% (v / v).

[0089] Preparation of reagent R2:

[0090] (1) Coating the first antibody CK-MB mAb01: Take 0.05 mL of PS microspheres, add 2.5 mL of 0.025 M pH=6.5 MES buffer, mix, add 5 μL of 0.1 g / mL EDC solution (prepared and used immediately), mix and stir at room temperature for 15 min; add 0.05 mg of CK-MB mAb (Aladdin Company, catalog number: Ab169527) prepared in Example 1 to the reaction system, stir at room temperature for 1 h; add 10 μL of blocking agent (blocking agent is 1% BSA and 0.2% glycine aqueous solution) to the reaction system, stir for 1 h; centrifuge at 8000 rpm for 15 min, resuspend in 2.5 mL of pH 7.0 PBS solution, and sonicate for 10 min.

[0091] (2) Coating the second antibody CK-MB mAb02: Take 0.05 mL of PS microspheres, add 2.5 mL of 0.025 M pH=6.5 MES buffer, mix, add 5 μL of 0.1 g / mL EDC solution (prepared and used immediately), mix and stir at room temperature for 15 min; add 0.05 mg of CK-MB mAb (Aladdin Company, catalog number: Ab170211) prepared in Example 1 to the reaction system, stir at room temperature for 1 h; add 10 μL of blocking agent (blocking agent is 1% BSA and 0.2% glycine aqueous solution) to the reaction system, stir for 1 h; centrifuge at 8000 rpm for 15 min, resuspend in 2.5 mL of pH 7.0 PBS solution, and sonicate for 10 min.

[0092] (3) Mix the two microsphere antibody-labeled complexes and store at 2-8℃ for later use.

[0093] Testing instrument: Hitachi 7180 fully automated biochemical analyzer.

[0094] Parameters: Sample volume 20μL, reagent R1 180μL, reagent R2 60μL, fitting method Spline, main wavelength 600nm, secondary wavelength none.

[0095] Comparative Example 2: A CK-MB detection kit (fluorescent immunochromatography) was prepared using recombinant CK-MB monoclonal antibody from Aladdin Company.

[0096] (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 LEDC, 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, repeating 3 times; add 0.05 mg of CK-MB mAb (aladdin, catalog number: Ab169527) 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 7.4 MES buffer. Resuspend the microspheres in PBS, repeat 3 times, and store at 2-8℃ for later use.

[0097] (2) Preparation of nitrocellulose membrane: The second antibody CK-MB mAb (Aladdin Company, catalog number: Ab170211) 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. The sheep rat 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.

[0098] (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.

[0099] (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.

[0100] Testing instrument: Lambert AFS1520 fluorescence immunoassay analyzer.

[0101] Based on the clinical sample test data of the CK-MB detection kit (latex immunoturbidimetric assay) in Table 1, it can be concluded that: at the three concentration gradients of low (L), medium (M), and high (H), the coefficients of variation (CV) of Example 4 were 1.6%, 1.2%, and 2.7%, respectively, significantly lower than those of Comparative Example 1 (4.2%, 3.8%, and 3.2%). This indicates that the detection results of Example 4 exhibit excellent intra-batch repeatability and its precision better meets the high-precision clinical testing requirements. Further analysis of the standard deviation (SD) shows that Example 4 at the three concentration levels were 10.2870, 43.7748, and 203.7031, respectively, all lower than those of Comparative Example 1 (24.1249, 115.0403, and 214.6933), indicating that the detection data of Example 4 has lower dispersion and greater analytical stability. Looking at the average absorbance change (ΔAbs), Example 4 at low, medium, and high concentrations were 644.4, 3582.7, and 7603.6, respectively, compared to Comparative Example 1. The differences between Example 4 and 580.3, 3055.5, and 6695.1 are more significant, demonstrating that Example 4 has higher sensitivity to antigen-antibody responses to different concentrations of CK-MB, more comprehensive linear range coverage, and better dose-response relationship.

[0102] Table 1. CK-MB Detection Kit (Latex Immunoturbidimetric Assay) for Clinical Sample Testing

[0103]

[0104] Table 2 shows the clinical sample test data of the CK-MB detection kit (fluorescence immunochromatography): At the three concentration gradients of low (L), medium (M), and high (H), the coefficients of variation (CV) of Example 5 were 4.2%, 4.0%, and 4.4%, respectively, significantly lower than those of Comparative Example 2 (7.6%, 7.5%, and 7.3%), indicating that the detection results of Example 5 have good repeatability and outstanding precision. The standard deviations (SD) of Example 5 at the three concentration levels were 0.0090, 0.0247, and 0.0505, respectively, much smaller than those of Comparative Example 2 (0.0146, 0.0421, and 0.0757), indicating that the detection data of Example 5 have smaller fluctuations and stronger analytical stability. From T / C (detection line / Analysis of the average value of the control line signal ratio showed that the values ​​of Example 5 at low, medium, and high concentrations were 0.21577, 0.61562, and 1.1466, respectively. Compared with Comparative Example 2 (0.19218, 0.56148, and 1.04193), the differences were more significant, demonstrating that the different concentrations of CK-MB in Example 5 had higher immunoreactivity sensitivity, more comprehensive linear range coverage, and greater advantages in quantitative detection performance.

[0105] Table 2. CK-MB Detection Kit (Fluorescence Immunochromatography) for Clinical Sample Testing

[0106]

[0107] 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. A CK-MB monoclonal paired antibody, characterized in that: The heavy chain sequence of the first antibody CK-MB mAb01 is shown in SEQ ID NO:1, and its light chain sequence is shown in SEQ ID NO:2; the heavy chain sequence of the second antibody CK-MB 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 CK-MB monoclonal paired antibody as described in claim 1 in the preparation of a latex immunoturbidimetric assay kit for detecting creatine kinase isoenzyme-MB (CK-MB).

3. The application according to claim 2, characterized in that: The kit includes reagent R1 and reagent R2; The reagent R1 is 15mM pH 6.8 Tris buffer, NaCl 2g / L, PEG20000 5g / L, BSA 1w%, Proclin 300 0.08%; The preparation method of the reagent R2 is as follows: (1) Coating the first antibody CK-MB mAb01: Take 0.05 mL of PS microspheres, add 2.5 mL of 0.025 M pH=6.5 MES buffer and mix, then add 5 μL of 0.1 g / mL EDC solution and mix and stir at room temperature for 15 min; add 0.05 mg of CK-MB mAb01 to the reaction system and stir at room temperature for 1 h; add 10 μL of blocking agent to the reaction system and stir for 1 h; centrifuge at 8000 rpm for 15 min, resuspend in 2.5 mL of pH 7.0 PBS solution, and sonicate for 10 min; (2) Coating the second antibody CK-MB mAb02: Take 0.05 mL of PS microspheres, add 2.5 mL of 0.025 M pH=6.5 MES buffer, mix, add 5 μL of 0.1 g / mL EDC solution, mix and stir at room temperature for 15 min; add 0.05 mg of CK-MB mAb02 to the reaction system, stir at room temperature for 1 h; add 10 μL of blocking agent to the reaction system, stir for 1 h; centrifuge at 8000 rpm for 15 min, resuspend in 2.5 mL of pH 7.0 PBS solution, and sonicate for 10 min; (3) Mix the two microsphere antibody-labeled complexes and store at 2-8℃ for later use.

4. The use of the CK-MB monoclonal paired antibody as described in claim 1 in the preparation of an immunochromatographic assay kit for detecting CK-MB.

5. The application according to claim 4, characterized in that, The reagent kit is prepared as follows: (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 CK-MB 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 CK-MB mAb02 with a concentration of 1 mg / mL was stripped onto the nitrocellulose membrane as a detection line with a coating amount of 1 μL / cm, and the goat anti-mouse IgG antibody with a concentration of 1 mg / mL was stripped onto the nitrocellulose membrane as a quality control line with 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.

6. The use of the CK-MB monoclonal paired antibody as described in claim 1 in the preparation of an ELISA detection kit for detecting CK-MB.

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