A cardiac myosin binding protein C specific antibody composition and kit

Abstract

The present invention discloses a cardiac myosin binding protein C-specific antibody composition and a kit thereof, wherein the composition comprises a first antibody and a second antibody, and the composition specifically recognizes cardiac myosin binding protein C, and the amino acid sequence of the recognition site is shown in SEQ ID No. 5. The kit comprises magnetic microspheres coupled with the first antibody, fluorescent microspheres coupled with the second antibody, a calibrator, and a diluent. The cardiac myosin binding protein C antibody provided by the present invention has good specificity and high affinity with cardiac myosin binding protein C, avoids cross-talk with skeletal muscle myosin, and can be used to prepare fluorescent immunoassay products for detecting cardiac myosin binding protein C. The cardiac myosin binding protein C detection kit provided by the present invention has the advantages of simple equipment, simple operation, and easy promotion.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a cardiac myosin binding protein C specific antibody composition and a kit thereof. Background Art

[0002] Cardiac myosin-binding protein C (cMyBP-C) is a key structural protein of cardiomyocyte thick filaments. With a molecular weight of approximately 140 kDa, it consists of 1273 amino acids, including three type III fibronectin domains (Fn3) and eight immunoglobulin-like domains (Ig). Its N-terminal C0 domain (amino acids 1-155) is cardiac-specific and shares low homology with the skeletal muscle version (MyBPC1), making it an ideal target for antibody design.

[0003] After acute myocardial infarction (AMI), cMyBP-C is dephosphorylated and proteolytically cleaved, releasing a 30-40 kDa N-terminal fragment (C0C1F) into the bloodstream. Blood concentrations rise within 3 hours, peak at 6 hours, and return to baseline by 12 hours, earlier than the 6-12 hour window for troponin (cTn). 2 Myocardial necrosis can be detected with as little as 0.07 mg, compared to 3-9 mg for cTn, resulting in a tenfold increase in sensitivity. However, cMyBP-C has a short half-life in the blood (<12 hours) and coexists with both the intact protein and fragments, posing challenges to the affinity, epitope specificity, and sensitivity of detection antibodies.

[0004] Chinese patent publication number CN119192364A discloses specific antibodies for detecting human cardiac myosin binding protein C. The disclosed heavy chain variable region (VH-CDR1 / 2 / 3) sequences (SEQ ID NOs: 2-4, 8) and light chain variable region (VL-CDR1 / 2 / 3) sequences (SEQ ID NOs: 5-7, 9) specifically bind to linear and conformational epitopes of cMyBP-C, with a cross-reactivity rate of less than 5% with skeletal muscle. Recombinant antibody fragments, including single-chain Fv (scFv), Fab fragments, and bispecific antibodies, were obtained through phage display library screening, but their stability is poor. CDR grafting of murine antibodies (e.g., IgG1κ) reduces immunogenicity, but this sometimes results in a 30-50% decrease in affinity. Tagged fusion proteins (e.g., His-Myc tag) were expressed in Escherichia coli BL21(DE3) and purified by Ni-NTA chromatography, but the inclusion body renaturation efficiency was only 30-60%. Insect cells / CHO cells express full-length cMyBP-C, and the glycosylation modification is closer to the native conformation, but the cost is high and the yield is low (<5 mg / L).

[0005] Chinese patent publication number CN118731367A discloses a cardiac myosin binding protein C detection kit, which has a sensitivity bottleneck problem: the detection limit of cMyBP-C using traditional ELISA is only 1 ng / mL, which is difficult to meet the needs of ultra-early AMI diagnosis.

[0006] Chinese patent publication number CN111004323A discloses methods and processes for preparing cardiac myosin-binding protein C antibodies and detection methods. However, the problem of insufficient specificity is that when skeletal muscle is injured, anti-C1C2 antibodies cross-react with MyBPC1, causing false positives (incidence rate of approximately 12%).

[0007] Chinese patent publication number CN118731367A discloses a cardiac myosin binding protein C detection kit, which has the problem of complex operation: the electroluminescence method (ECLIA) requires special equipment and the reagent cost is 300% higher than ELISA. Summary of the Invention

[0008] In order to solve the defects of the prior art, the present invention provides a cardiac myosin binding protein C specific antibody composition and a kit thereof.

[0009] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0010] The first object of the present invention is to provide a cardiac myosin binding protein C-specific antibody composition, comprising a first antibody and a second antibody, wherein the first antibody and the second antibody both comprise a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region both comprise a complementary region determining region and a framework region; and the complementary region determining region of the heavy chain variable region and the light chain variable region both comprise CDR1, CDR2, and CDR3;

[0011] The amino acid sequence of CDR1 of the heavy chain variable region of the first antibody is shown at positions 25 to 32 of SEQ ID No. 1; the amino acid sequence of CDR2 of the heavy chain variable region of the first antibody is shown at positions 50 to 56 of SEQ ID No. 1; and the amino acid sequence of CDR3 of the heavy chain variable region of the first antibody is shown at positions 93 to 103 of SEQ ID No. 1;

[0012] The amino acid sequence of CDR1 of the light chain variable region of the first antibody is shown at positions 27 to 32 of SEQ ID No. 2; the amino acid sequence of CDR2 of the light chain variable region of the first antibody is shown at positions 50 to 52 of SEQ ID No. 2; and the amino acid sequence of CDR3 of the light chain variable region of the first antibody is shown at positions 89 to 100 of SEQ ID No. 2;

[0013] The amino acid sequence of CDR1 of the heavy chain variable region of the second antibody is shown at positions 25 to 32 of SEQ ID No. 3; the amino acid sequence of CDR2 of the heavy chain variable region of the second antibody is shown at positions 50 to 56 of SEQ ID No. 3; and the amino acid sequence of CDR3 of the heavy chain variable region of the second antibody is shown at positions 93 to 106 of SEQ ID No. 3;

[0014] The amino acid sequence of CDR1 of the light chain variable region of the second antibody is shown at positions 27-32 of SEQ ID No. 4; the amino acid sequence of CDR2 of the light chain variable region of the second antibody is shown at positions 50-52 of SEQ ID No. 4; and the amino acid sequence of CDR3 of the light chain variable region of the second antibody is shown at positions 89-100 of SEQ ID No. 4.

[0015] Preferably, the composition specifically recognizes cardiac myosin binding protein C, and the amino acid sequence of the recognition site is shown in SEQ ID No.5.

[0016] The second object of the present invention is to provide a cardiac myosin binding protein C fluorescence detection kit, comprising:

[0017] Capture antibodies coupled to a solid phase carrier include magnetic microspheres coupled to a primary antibody or a secondary antibody;

[0018] Fluorescently labeled detection antibodies, including fluorescent microspheres coupled to secondary antibodies or primary antibodies;

[0019] Calibrators, including cardiac myosin binding protein C standard with a concentration gradient of 0–6900 pg / mL;

[0020] Diluents include PBS buffer, pH 7.4.

[0021] When the first antibody acts as a capture antibody, the second antibody acts as a detection antibody. When the second antibody acts as a capture antibody, the first antibody acts as a detection antibody.

[0022] Preferably, the magnetic microspheres are carboxyl-modified polystyrene magnetic beads.

[0023] Preferably, the fluorescent microspheres are latex microspheres labeled with phycoerythrin.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] The cardiac myosin binding protein C antibody provided by the present invention has good specificity and high affinity for cardiac myosin binding protein C, avoids cross-reactivity with skeletal muscle myosin, and can be used to prepare fluorescent immunoassay products for detecting cardiac myosin binding protein C. The cardiac myosin binding protein C detection kit provided by the present invention has the advantages of simple equipment, simple operation, and easy promotion. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Schematic diagram of the operation of the fluorescent immunoassay method of the present invention;

[0027] Figure 2 Schematic diagram of the fluorescent Aleax 350 fluorophore on magnetic beads under a fluorescence microscope;

[0028] Figure 3 Schematic diagram of fluorescent microspheres on magnetic beads under a fluorescence microscope;

[0029] Figure 4 Comparison of fluorescent microsphere intensities at different antigen concentrations;

[0030] Figure 5 Calibration curve of the fluorescent immunoassay method. DETAILED DESCRIPTION

[0031] The preferred embodiments of the present invention are described below with reference to the accompanying drawings. It should be understood that the preferred embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

[0032] Example 1: Antibody preparation.

[0033] Phase I: Animal immunization and titer verification.

[0034] 1. Immunogen Preparation:

[0035] Antigen: Human cardiac myosin binding protein C fragment (1-280aa)-His tag protein;

[0036] Purity ≥ 90%, concentration > 0.5 mg / mL, stored in PBS buffer;

[0037] Immunized animals: New Zealand white rabbit (1, pre-immunization titer>1:512K).

[0038] 2. Immunization plan:

[0039] First immunization: 200 μg antigen + complete Freund's adjuvant (CFA), subcutaneous injection at multiple points;

[0040] Booster immunization: 100 μg antigen + Freund's incomplete adjuvant (IFA) once every 2 weeks, for a total of 3 times.

[0041] 3. Potency test:

[0042] Serum ELISA test: titer must be ≥1:512,000 (OD positive / negative ≥2.1).

[0043] Phase II: Monoclonal antibody preparation and screening.

[0044] 1. B cell enrichment and high-throughput expression: Isolate rabbit peripheral blood mononuclear cells (PBMCs) and enrich antigen-specific B cells (MBC+PBCs); use BGE technology to express antibody supernatant at high throughput.

[0045] 2. Positive clone screening:

[0046] Initial screening: ELISA to detect antigen binding activity (standard: positive well OD / negative well OD ≥ 2.1);

[0047] Validation: Cross-reaction test between positive supernatant and natural sample (mouse / human serum);

[0048] Six antibodies were selected based on binding activity and natural sample specificity.

[0049] Phase III: Antibody expression and purification.

[0050] 1. Transient transfection expression:

[0051] System: HEK293 cells, serum-free medium;

[0052] Scale: 6 antibodies were expressed in parallel, with a yield of ≥1 mg per antibody.

[0053] 2. Purification and quality inspection:

[0054] Purification: Protein A affinity chromatography (buffer: PBS, pH 7.4);

[0055] Quality control: purity ≥90% (SDS-PAGE); concentration >1 mg / mL (UV spectrophotometry).

[0056] Phase IV: Antibody pairing and validation.

[0057] 1. Pairing design:

[0058] Combination: 6 rabbit monoclonal antibodies, paired in pairs;

[0059] Methods: Double antibody sandwich ELISA / fluorescence immunoassay;

[0060] Standard: Signal value (S / N) ≥ 10 and no cross-reaction.

[0061] 1. Antibody pairing test.

[0062] Prepare the ELISA plate for coating: Dilute the antibody to 1 μg / mL in 0.01M PBS, add 100 μL / well to the ELISA plate, and coat overnight at 4°C. Wash three times with 300 μL / well of wash buffer (1g casein dissolved in 500 mL of 0.01M PBS buffer and 0.1-2‰ Triton X-405). Add 200 μL of blocking buffer (5% skim milk powder) and incubate at 37°C for 2 hours. Aspirate the blocking buffer and dry overnight.

[0063] To prepare the biotinylated antibody: Add 0.5 mg of cardiac myosin binding protein C monoclonal antibody to 5 mL of 0.01 M PBS (pH 7.4). Vortex to mix thoroughly, then concentrate using a Millipore Amicon Ultra centrifuge tube to a concentration of >5 mg / mL. Place the concentrated cardiac myosin binding protein C monoclonal antibody in a dialysis bag and add 5 μL of a 5 mg / mL solution of BNHS in DMF. Incubate at 2-8°C for 2 h. Then, dialyze against at least 2 L of 0.1 M MES buffer (pH 5.0) for 24 h, changing the buffer four times to separate free BNHS. Collect the dialyzed labeled material, add 5% BSA solution to a final BSA concentration of 1%, then add an equal volume of glycerol and store at -20°C.

[0064] Prepare HRP-labeled streptavidin: Dissolve 10 mg of HRP in 1 mL of distilled water, add 0.2 mL of 0.1 M NaIO₄ solution, and incubate at room temperature in the dark for 30 minutes. Remove excess NaIO₄ by dialyzing (3 changes of 1 L each against PBS at 4°C). Dissolve 5 mg of streptavidin in 1 mL of carbonate buffer (pH 9.5). Add the activated HRP solution, mix thoroughly, and react with slow stirring at 4°C for 2 hours. Add 0.1 mL of 2 mg / mL NaBH₄ solution and reduce at 4°C for 30 minutes. Dialyze (3 changes of 1 L each against PBS at 4°C). Collect the dialyzed labeled product and add 5% BSA solution to a final BSA concentration of 1%. Then, add an equal volume of glycerol and store at -20°C.

[0065] Detection: Prepare a 1 μg / mL solution of recombinant cardiac myosin binding protein C. Dilute the biotinylated antibody to 0.5 mg / mL in 0.01 M PBS. Dilute SA-HRP to 0.5 mg / mL in 0.01 M PBS. Add 20 μL of sample, 50 μL of biotinylated antibody, and 50 μL of SA-HRP to the sample and incubate at 37°C for 1 hour. Wash three times with wash buffer. Add horseradish peroxidase substrate (TMB) and perform detection (see Tables 1-3 for results).

[0066] Table 1: Detection results of 1 μg / mL cardiac myosin binding protein C.

[0067]

[0068] Table 2: Detection results of 0.5 μg / mL cardiac myosin binding protein C.

[0069]

[0070] Table 3: Blank test results.

[0071]

[0072] 2. Antibody titer testing

[0073] Prepare antigen-coated ELISA plates: Dilute cardiac myosin binding protein C antigen to 1 μg / mL in 0.01 M PBS, add 100 μL / well to the ELISA plate, and coat overnight at 4°C. Wash three times with 300 μL / well of wash buffer (1 g casein dissolved in 500 mL of 0.01 M PBS buffer and 0.1-2‰ Triton X-405). Add 200 μL of blocking buffer (5% skim milk powder) and incubate at 37°C for 2 hours. Aspirate the blocking buffer and dry overnight.

[0074] Prepare goat anti-rabbit HRP: Dissolve 10 mg of HRP in 1 mL of distilled water, add 0.2 mL of 0.1 M NaIO4 solution, and incubate at room temperature in the dark for 30 minutes. Remove excess NaIO4 by dialyzing (3 changes of 1 L each against PBS at 4°C). Dissolve 5 mg of goat anti-rabbit antibody in 1 mL of carbonate buffer (pH 9.5). Add the activated HRP solution, mix thoroughly, and incubate at 4°C with slow stirring for 2 hours. Add 0.1 mL of 2 mg / mL NaBH4 solution and reduce the solution at 4°C for 30 minutes. Dialyze (3 changes of 1 L each against PBS at 4°C). Collect the dialyzed labeled material and add 5% BSA solution to a final BSA concentration of 1%. Then, add an equal volume of glycerol and store at -20°C.

[0075] Prepare antibody dilution solution: dilute the antibody to 1 μg / mL in the first well, and then dilute it 3-fold to 9 concentrations.

[0076] Table 4: Comparison of titers of six antibody strains.

[0077]

[0078] Example 2: This example provides a method for detecting cardiac myosin binding protein C based on Aleax 350.

[0079] Detection principle Figure 1As shown, the preparation steps are as follows:

[0080] Prepare the first antibody-coated magnetic microspheres: Weigh 1 mg of magnetic microspheres (average particle size 2.8 μm, purchased from JSR, 10% solids content) and suspend them in 1 mL of 0.1 M MES buffer. After magnetic adsorption for 5–10 minutes, discard the supernatant and repeat the wash steps 3–5 times. Add 1 mL of the above-mentioned 0.1 M MES buffer and vortex to mix thoroughly. Add 100 μg of the first cardiac myosin binding protein C monoclonal antibody (SD1C12) to a magnetic microsphere:antibody ratio of 10:1, vortex to mix thoroughly, and incubate at 37°C for 2 hours. Weigh 100 mg / mL of 1-(3-dimethylaminopropyl)-3-ethyldiimide hydrochloride (EDC) solution and add it at a volume ratio of 10:1 (magnetic microspheres:EDC solution). Vortex to mix thoroughly and incubate at 37°C for 4 hours. ) Block the beads overnight with 200 μL of 1% BSA in PBS (0.02 M). Add 1 mL of washing solution (1 g casein dissolved in 500 mL of 0.01 M PBS buffer and 0.1-2‰ Triton X-405) to the blocked magnetic bead suspension. Apply magnet to the beads, remove the supernatant, and repeat the washing steps 3-5 times. Place the prepared magnetic beads in 1 mL of washing solution and store at 2-8°C.

[0081] Prepare biotinylated monoclonal antibody solution: Add 0.5 mg of cardiac myosin binding protein C monoclonal antibody (1M1D11) to 5 mL of 0.01 M PBS (pH 7.4). Vortex to mix thoroughly, then concentrate using a Millipore Amicon Ultra centrifuge tube to a concentration of >5 mg / mL. Place the concentrated cardiac myosin binding protein C monoclonal antibody in a dialysis bag and add 5 μL of a 5 mg / mL solution of BNHS in DMF. Incubate at 2-8°C for 2 h. Then, dialyze against at least 2 L of 0.1 M MES buffer (pH 5.0) for 24 h, changing the buffer four times to separate free BNHS. Collect the dialyzed labeled material, add 5% BSA solution to a final BSA concentration of 1%, then add an equal volume of glycerol and store at -20°C.

[0082] The amino acid sequence of CDR1 of the heavy chain variable region of the cardiac myosin binding protein C monoclonal antibody (1M1D11) is shown at positions 25 to 32 of SEQ ID No. 1; the amino acid sequence of CDR2 of the heavy chain variable region is shown at positions 50 to 56 of SEQ ID No. 1; and the amino acid sequence of CDR3 of the heavy chain variable region is shown at positions 93 to 103 of SEQ ID No. 1.

[0083] The amino acid sequence of CDR1 of the light chain variable region of the cardiac myosin binding protein C monoclonal antibody (1M1D11) is shown at positions 27-32 of SEQ ID No. 2; the amino acid sequence of CDR2 of the light chain variable region of the first antibody is shown at positions 50-52 of SEQ ID No. 2; and the amino acid sequence of CDR3 of the light chain variable region of the first antibody is shown at positions 89-100 of SEQ ID No. 2.

[0084] The amino acid sequence of CDR1 of the heavy chain variable region of the cardiac myosin binding protein C monoclonal antibody (SD1C12) is shown at positions 25 to 32 of SEQ ID No. 3; the amino acid sequence of CDR2 of the heavy chain variable region is shown at positions 50 to 56 of SEQ ID No. 3; and the amino acid sequence of CDR3 of the heavy chain variable region is shown at positions 93 to 106 of SEQ ID No. 3.

[0085] The amino acid sequence of CDR1 of the light chain variable region of the cardiac myosin binding protein C monoclonal antibody (SD1C12) is shown at positions 27 to 32 of SEQ ID No. 4; the amino acid sequence of CDR2 of the light chain variable region is shown at positions 50 to 52 of SEQ ID No. 4; and the amino acid sequence of CDR3 of the light chain variable region is shown at positions 89 to 100 of SEQ ID No. 4.

[0086] Prepare a fluorescein-labeled streptavidin solution: Add 0.5 mg of streptavidin solution (from the source leaf) to 5 mL of 0.01 M PBS (pH 7.4). Vortex to mix thoroughly, then concentrate using a Millipore Amicon Ultra centrifuge tube to a concentration of >5 mg / mL. Place the concentrated streptavidin in a dialysis bag and add 5 μL of 5 mg / mL Aleax 350 solution in DMF. Incubate at 2-8°C for 2 h. Then, dialyze against at least 2 L of 0.1 M MES buffer (pH 5.0) for 24 h, changing the buffer four times to separate free BNHS. Collect the dialyzed labeled product and add 5% BSA solution to a final BSA concentration of 1%. Then, add an equal volume of glycerol and store at -20°C.

[0087] Preparation of calibrators: cardiac myosin binding protein C recombinant protein was diluted to 5 ng / mL, 2.5 ng / mL, 1.25 ng / mL, 0.625 ng / mL, 0.3125 ng / mL, and 0 ng / mL using 0.05 M MES solution containing 4% casein.

[0088] Preparation of cleaning solution: The cleaning solution of the present invention is a PBST solution with a pH value of 7.0-9.0 and a concentration of 0.01 mol / L, containing 0.05% by mass of Triton X-405.

[0089] The kit consists of a magnetic microsphere suspension coated with an antibody against cardiac myosin binding protein C, a biotin-labeled antibody against cardiac myosin binding protein C, streptavidin labeled with fluorescein, and a calibrator. The kit is used in conjunction with a self-developed portable fluorimeter to measure cardiac myosin binding protein C concentration in samples.

[0090] Specifically, the instrument was used to measure the concentration of cardiac myosin binding protein C in the sample to be tested according to the following steps: 10 μL of the above-mentioned immunomagnetic beads were mixed with the treated serum sample and incubated at 37°C and 150 r / min for 45 minutes. 200 μL (1:200 dilution) of avidinized cardiac myosin binding protein C antibody was added and the incubation was continued at 37°C and 150 r / min for 45 minutes. 100 μL (1:100 dilution) of streptavidin labeled with fluorescein was added and the incubation was continued at 37°C and 150 r / min for 45 minutes. After the incubation was completed, the sample was washed with a cleaning solution, using 400 μL of cleaning solution each time. Finally, the fluorescence intensity was measured using a fluorescence detection instrument. The use of fluorescein had a low sensitivity and could not be detected. The fluorescence signal could be seen under a fluorescence microscope ( Figure 2 、 Figure 3 ).

[0091] Example 3: This example provides a method for detecting cardiac myosin binding protein C based on fluorescent microspheres.

[0092] Detection principle Figure 1 As shown, the preparation steps are as follows:

[0093] Prepare the first antibody-coated magnetic microspheres: Measure 1 mg of magnetic microspheres (average particle size 2.8 μm, purchased from JSR, 10% solids content) and suspend them in 1 mL of 0.1 M MES buffer. After magnetic adsorption for 5–10 minutes, discard the supernatant and repeat the wash steps 3–5 times. Add 1 mL of the above-mentioned 0.1 M MES buffer and vortex to mix thoroughly. Add 100 μg of the first cardiac myosin binding protein C monoclonal antibody (SD1C12) to a magnetic microsphere:antibody ratio of 10:1. Vortex to mix thoroughly and incubate at 37°C for 2 hours. Prepare a 100 mg / mL 1-(3-dimethylaminopropyl)-3-ethyldiimide hydrochloride (EDC) solution and add the EDC solution at a volume ratio of 10:1 (magnetic microspheres:EDC solution). Vortex to mix thoroughly and incubate at 37°C for 4 hours. Block the beads overnight with 200 μL of 1% BSA in PBS (0.02 M). Add 1 mL of washing solution (1 g casein dissolved in 500 mL of 0.01 M PBS buffer and 0.1-2‰ Triton X-405) to the blocked magnetic bead suspension. Apply magnet to the beads, remove the supernatant, and repeat the washing steps 3-5 times. Place the prepared magnetic beads in 1 mL of washing solution and store at 2-8°C.

[0094] Prepare biotinylated monoclonal antibody solution: Add 0.5 mg of cardiac myosin binding protein C monoclonal antibody (1M1D11) to 5 mL of 0.01 M PBS (pH 7.4). Vortex to mix thoroughly, then concentrate using a Millipore Amicon Ultra centrifuge tube to a concentration of >5 mg / mL. Place the concentrated cardiac myosin binding protein C monoclonal antibody in a dialysis bag and add 5 μL of a 5 mg / mL solution of BNHS in DMF. Incubate at 2-8°C for 2 h. Then, dialyze against at least 2 L of 0.1 M MES buffer (pH 5.0) for 24 h, changing the buffer four times to separate free BNHS. Collect the dialyzed labeled material, add 5% BSA solution to a final BSA concentration of 1%, then add an equal volume of glycerol and store at -20°C.

[0095] Prepare a streptavidin solution for fluorescent microspheres: Use 300 nm time-resolved fluorescent microspheres modified with carboxyl groups. Suspend 1 mg of microspheres in 1 mL of 0.1 M MES buffer and centrifuge at 20,000 rpm for 20 minutes. Remove the supernatant and add 1 mL of 0.1 M MES buffer. Disperse the microspheres using pipette tip. Ultrasonicate the solution in water for 5 minutes. Then, add 250 μg of streptavidin solution and 100 μL of 100 mg / mL 1-(3-dimethylaminopropyl)-3-ethyldiimide hydrochloride (EDC) solution. Incubate at 37°C and 150 rpm for 30-60 minutes. Block the microspheres with 200 μL of 0.02 M 1% BSA in PBS overnight. Centrifuge at 20,000 rpm for 20 minutes. Remove the supernatant and add 1 mL of cleaning solution (1 g casein dissolved in 500 mL of 0.01 M PBS buffer and 0.1-2‰ Triton X-405). Disperse the solution by pipetting with a pipette. Then, ultrasonicate the solution in a water bath for 5 minutes. Store at 2-8°C after dispersion.

[0096] To prepare fluorescent microsphere-linked antibodies: Dilute 100 μg of biotinylated antibody to 0.1 mg / mL in PBST (0.02 M, pH 7.4, 0.05%-2% Triton X-405). Add 100 μg of streptavidin-labeled microspheres and incubate at 37°C and 150 rpm for 30-60 min. Centrifuge at 20,000 rpm for 20 min. Remove the supernatant and add 1 mL of washing solution (1 g casein dissolved in 500 mL of 0.01 M PBS buffer and 0.1-2‰ Triton X-405). Disperse the mixture using a pipette, then sonicate in a water-sealable solution for 5 min. Store at 2-8°C after dispersion.

[0097] Preparation of calibrators: cardiac myosin binding protein C recombinant protein was diluted to 5 ng / mL, 2.5 ng / mL, 1.25 ng / mL, 0.625 ng / mL, 0.3125 ng / mL, and 0 ng / mL using 0.05 M MES solution containing 4% casein.

[0098] Preparation of cleaning solution: The cleaning solution of the present invention is a PBST solution with a pH value of 7.0-9.0 and a concentration of 0.01 mol / L, containing 0.05% by mass of Triton X-405.

[0099] The kit consists of a suspension of magnetic microspheres coated with an antibody against cardiac myosin binding protein C, fluorescent microspheres linked to the antibody against cardiac myosin binding protein C, and a calibrator. The kit is used with a self-developed portable fluorometer to measure cardiac myosin binding protein C concentration in samples.

[0100] Specifically, the following steps were used to measure the concentration of cardiac myosin binding protein C in the sample to be tested using an instrument: 10 μL of the above-mentioned immunomagnetic beads were mixed with the treated serum sample and incubated at 37°C and 150 r / min for 45 minutes. 200 μL (1:20 dilution) of cardiac myosin binding protein C antibody linked to fluorescent microspheres was added and incubated at 37°C and 150 r / min for another 45 minutes. After the incubation, the sample was washed with a cleaning solution, using 400 μL of cleaning solution each time. Finally, the fluorescence intensity was measured using a fluorescence detection instrument. The results were obvious, and the fluorescence intensity was visible to the naked eye ( Figure 2 、 Figure 3 ).

[0101] Example 4: This example provides a comparison of the intensity of fluorescent microspheres with different antigen concentrations and the establishment of a calibration curve for the fluorescent immunoassay method.

[0102] Preparation of antigen solutions of different concentrations: Cardiac myosin binding protein C recombinant protein was diluted with 0.05M MES solution to 5 ng / mL, 2.5 ng / mL, 1.25 ng / mL, 0.625 ng / mL, 0.312 ng / mL, 0 ng / mL, 0, 0.312, 0.625, 1.25, 2.5, and 5 ng / mL.

[0103] Sandwich reaction: Place 10 μL of magnetic beads in six EP tubes, wash the beads four times with PBST buffer, add the above antigen diluent, and incubate at 37°C, 150 rpm for 1 hour. Wash the beads four times with PBST buffer. Incubate with 400 μL of 8 μg / mL biotinylated detection antibody at room temperature for 30 minutes, then wash four times with PBST. Add 10 μg / mL streptavidin fluorescent microspheres and incubate for 30 minutes, then wash four times with PBST.

[0104] Fluorescence excitation photography: Add the coupled magnetic beads to new buffer, place them in a 96-well plate, and use a portable fluorescence instrument with a 365 nm wavelength to excite and photograph. The fluorescence intensity is proportional to the antigen concentration ( Figure 4 ).

[0105] Fluorescence spectrum scanning: Place the 96-well plate into the fluorescence enzyme analyzer and scan to draw the spectrum curve of different concentrations of antigen. The area under the emission peak curve is proportional to the antigen concentration ( Figure 5 ).

[0106] Example 5: This example provides a process for drawing a standard curve.

[0107] Recombinant cardiac myosin binding protein C was diluted to 6.9 ng / mL, 3.6 ng / mL, 0.82 ng / mL, 0.43 ng / mL, 0.049 ng / mL, and 0 ng / mL using 0.05 M MES solution containing 4% casein. 10 μL of the aforementioned immunomagnetic beads was mixed with the calibrator and incubated at 37°C and 150 rpm for 45 min. 200 μL (1:20 dilution) of cardiac myosin binding protein C antibody linked to fluorescent microspheres was added, and the cells were incubated at 37°C and 150 rpm for another 45 min. After incubation, the cells were washed with 400 μL of wash buffer each time. Fluorescence intensity was measured using a fluorescence detector, and a four-parameter curve was plotted (see Table 5 for results).

[0108] Table 5: Calibration curve detection results.

[0109]

[0110] Example 6: This example provides a test process for the detection limit.

[0111] Recombinant cardiac myosin binding protein C was diluted to 20 pg / mL, 10 pg / mL, 5 pg / mL, and 2.5 pg / mL using 0.05 M MES solution containing 4% casein. 10 μL of the aforementioned immunomagnetic beads was mixed with the calibrator and incubated at 37°C and 150 rpm for 45 minutes. Ten replicates were performed for each concentration. 200 μL (1:20 dilution) of cardiac myosin binding protein C antibody linked to fluorescent microspheres was added, and incubation continued at 37°C and 150 rpm for 45 minutes. After incubation, the cells were washed with 400 μL of wash buffer each time. Finally, fluorescence intensity was measured using a fluorescence detector. Actual test results were calculated using the calibration curve to account for deviations (see Table 6 for test results).

[0112] Table 6: Detection limit test results.

[0113]

[0114] The results in Table 2 show that when the concentration is greater than 5 pg / mL, the relative deviation is less than 20%, and the detection limit of this method is 5 pg / mL.

[0115] Example 7: This example shows the cross-detection results of skeletal muscle myosin.

[0116] Three myosin-positive samples were collected and aliquoted into two vials at 200 μL per vial. 10 μL of 0.01 M PBS was added to the control group, and 10 μL of 1 mg / mL skeletal muscle cardiac myosin binding protein C recombinant protein was added to the crossover group, resulting in a skeletal muscle cardiac myosin binding protein C concentration of 10 μg / mL in the crossover group. 10 μL of the aforementioned immunomagnetic beads were mixed with the sample and incubated at 37°C and 150 rpm for 45 minutes. Three replicates were performed for each concentration. 200 μL (1:20 dilution) of fluorescent microsphere-linked cardiac myosin binding protein C antibody was added, and incubation continued at 37°C and 150 rpm for another 45 minutes. After incubation, the samples were washed with 400 μL of wash buffer each time. Finally, fluorescence intensity was measured using a fluorescence detector. The actual test results were calculated using a calibration curve, and the average was taken to calculate the relative deviation between the control and crossover samples (see Table 7).

[0117] Table 7: Skeletal muscle myosin crossover results test.

[0118]

[0119] The results in Table 3 show that the deviations of the test results of the three groups of samples were all within ±10%. The test kit prepared using the antibody of the present invention did not have a significant cross-reaction with skeletal muscle myosin.

[0120] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art will be able to modify the technical solutions described in the aforementioned embodiments or substitute equivalents for some of the technical features. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A cardiac myosin binding protein C-specific antibody composition, characterized in that: The invention comprises a first antibody and a second antibody, wherein the first antibody and the second antibody each comprise a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region each comprise a complementary region determining region and a framework region, and the complementary region determining region of the heavy chain variable region and the light chain variable region each comprise a CDR1, a CDR2 and a CDR3; The amino acid sequence of CDR1 of the heavy chain variable region of the first antibody is shown at positions 25 to 32 of SEQ ID No. 1; the amino acid sequence of CDR2 of the heavy chain variable region of the first antibody is shown at positions 50 to 56 of SEQ ID No. 1; and the amino acid sequence of CDR3 of the heavy chain variable region of the first antibody is shown at positions 93 to 103 of SEQ ID No. 1; The amino acid sequence of CDR1 of the light chain variable region of the first antibody is shown at positions 27 to 32 of SEQ ID No. 2; the amino acid sequence of CDR2 of the light chain variable region of the first antibody is shown at positions 50 to 52 of SEQ ID No. 2; and the amino acid sequence of CDR3 of the light chain variable region of the first antibody is shown at positions 89 to 100 of SEQ ID No. 2; The amino acid sequence of CDR1 of the heavy chain variable region of the second antibody is shown at positions 25 to 32 of SEQ ID No. 3; the amino acid sequence of CDR2 of the heavy chain variable region of the second antibody is shown at positions 50 to 56 of SEQ ID No. 3; and the amino acid sequence of CDR3 of the heavy chain variable region of the second antibody is shown at positions 93 to 106 of SEQ ID No. 3; The amino acid sequence of CDR1 of the light chain variable region of the second antibody is shown at positions 27-32 of SEQ ID No. 4; the amino acid sequence of CDR2 of the light chain variable region of the second antibody is shown at positions 50-52 of SEQ ID No. 4; and the amino acid sequence of CDR3 of the light chain variable region of the second antibody is shown at positions 89-100 of SEQ ID No.

4.

2. A fluorescent detection kit for cardiac myosin binding protein C, characterized in that: include: A capture antibody coupled to a solid phase carrier, comprising a magnetic microsphere coupled to the first antibody or the second antibody according to claim 1; A fluorescently labeled detection antibody comprising fluorescent microspheres coupled to the second antibody or the first antibody as claimed in claim 1; Calibrators, including cardiac myosin binding protein C standard with a concentration gradient of 0–6900 pg / mL; diluents, including PBS buffer, pH 7.4; When the first antibody serves as the capture antibody, the second antibody serves as the detection antibody; when the second antibody serves as the capture antibody, the first antibody serves as the detection antibody.

3. A cardiac myosin binding protein C fluorescence detection kit according to claim 2, characterized in that, The magnetic microspheres are carboxyl-modified polystyrene magnetic beads.

4. A cardiac myosin binding protein C fluorescence detection kit according to claim 2, characterized in that, The fluorescent microspheres are latex microspheres labeled with phycoerythrin.

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