A creatine kinase isoenzyme (MM) detection kit and its preparation method
By developing a creatine kinase MM detection kit and utilizing a specific component ratio, the problem of low specificity in creatine kinase isoenzyme detection kits was solved, achieving a lower limit of detection, higher specificity, and a wider linear range.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- GUANGZHOU FENGHUA BIOENG
- Filing Date
- 2026-03-10
- Publication Date
- 2026-06-30
AI Technical Summary
Existing creatine kinase isoenzyme detection kits have low specificity and a low linearity range, which cannot meet clinical needs.
A creatine kinase MM detection kit was prepared by using modified magnetic microspheres coated with antibodies containing creatine kinase MM isoenzymes and antibodies labeled with creatine kinase MM isoenzymes coated with alkaline phosphatase, combined with specific ratios of buffer, protectant, complexing agent, stabilizer and preservative.
It lowered the limit of detection for serum creatine kinase isoenzyme MM, improved specificity and repeatability, broadened the linear range, and enhanced the HOOK effect.
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Abstract
Description
Technical Field
[0001] This invention relates to the field of pharmaceutical medicine, and in particular to a creatine kinase isoenzyme (MM) detection kit and its preparation method. Background Technology
[0002] Creatine kinase (CK) is an enzyme found in muscles. Enzymes are proteins that help body cells perform their functions. When muscle cells in the body are damaged, the level of CK increases. This enzyme has three isoenzymes: MM, MB, and BB. Creatine kinase isoenzyme MB is mainly found in the heart muscle. When the heart is damaged for various reasons, this enzyme is released in large quantities into the bloodstream, resulting in elevated levels of creatine kinase isoenzyme MB in the blood. Creatine kinase MB typically rises after a heart attack, myocardial inflammation, cardiac trauma, heart surgery, muscular dystrophy, and other heart-related problems. This indicator is a very sensitive and specific marker of myocardial damage, and it is frequently tested clinically when an acute heart attack is suspected.
[0003] CK-MM antibodies can help determine the extent of muscle damage and detect related diseases in clinical testing. CKMB is a cardiac isoenzyme, mainly distributed in the myocardium; CKBB is a brain isoenzyme, mainly distributed in brain tissue; CKMiMi is a mitochondrial isoenzyme, mainly distributed in the mitochondria of myocardium and skeletal muscle. Among them, CKMM has great diagnostic value for muscle damage and neonatal Duchenne muscular dystrophy.
[0004] CKMM is a creatine kinase isoenzyme containing two M subunits, while CKMB is a creatine kinase isoenzyme containing one M subunit and one B subunit. If the antibody screening specificity in the CKMM detection reagent is not high, cross-reactivity with the M subunit of CKMB will occur. Even if an antibody targeting the M subunit is screened out, some CKMB will still be detected when CKMM and CKMB are detected together, which is not conducive to the specific detection of CKMM. Therefore, current creatine kinase isoenzyme detection kits have low detection specificity and a low linear range, which cannot meet clinical requirements. Summary of the Invention
[0005] To address the shortcomings of existing technologies, this invention provides a creatine kinase isoenzyme (MM) detection kit and its preparation method.
[0006] A creatine kinase isoenzyme (MM) assay kit includes reagent 1 and reagent 2. Reagent 1 includes the following components: Buffer solution 20-50 mmol / L; Creatine kinase isoenzyme B subunit antibody 10-100 mmol / L; 0.2-2 g / L of modified magnetic microspheres coated with antibodies containing creatine kinase MM isoenzyme. Blocking agent 0.5-2 g / L; Protectant 0.5-10 g / L; Complex concentrations: 1-50 mmol / L; Stabilizer 1-10 g / L; Anti-crystallization component 2-5‰; Preservative 0.05-0.1g / L.
[0007] Furthermore, reagent 2 comprises the following components: Buffer solution 20-50 mmol / L; Antibody labeled with 1-20 ug / ml of creatine kinase MM isoenzyme coated with alkaline phosphatase; Protectant 0.5-10 g / L; Surfactant 1-2 g / L; Stabilizer 1-10g / L; Preservative 0.05-0.1g / L.
[0008] Furthermore, the buffer solution is phosphate buffer or Tris buffer; the anti-crystallization component is one or more of Tween-20, Tween-60, Tween-80, Triton X-100, Triton X-450, Span20, and Span80.
[0009] Furthermore, the creatine kinase MM isoenzyme coated antibody-coated modified magnetic microspheres are creatine kinase MM isoenzyme coated antibody-coated modified dextran magnetic microspheres.
[0010] Furthermore, the blocking agent is mouse IgG.
[0011] Furthermore, the protective agent is one or more of BSA, casein, and animal serum.
[0012] Furthermore, the complex is a sodium salt or a potassium salt of EDTA.
[0013] Furthermore, the surfactant is one or more of the following: vinyl ester (9), polyethylene glycol 6000, polyethylene glycol 8000, and polyoxyethylene laurate (10) ether.
[0014] Furthermore, the stabilizers are all selected from bovine serum albumin, trehalose, chitosan, and glycerol.
[0015] Furthermore, the preservative is one or more of proclin 300, sodium azide, and thimerosal.
[0016] Furthermore, the raw materials for preparing the coated modified magnetic microspheres for creatine kinase MM isoenzyme coated antibodies include: 0.5-3 parts of dextran; 0.1-2 parts of iron oxide nanoparticles; 1-3 parts of silane coupling agent; 30-80 parts water; 5-10 copies of antibody-coated creatine kinase MM isoenzyme; 2-9 parts of surfactant; Anti-mouse IgG 1-5 parts.
[0017] Furthermore, the preparation steps of the creatine kinase MM isoenzyme-coated antibody-coated modified magnetic microspheres include: S1: Dissolve dextran in water, adjust the pH to 7-9, and add it to the reaction vessel. Stir until dissolved to obtain solution 1; S2: Add the silane coupling agent to solution 1 prepared in S1, set the reaction conditions, and obtain solution 2; S3: Add the creatine kinase MM isoenzyme-coated antibody, anti-mouse IgG and surfactant to solution 2 in S2, set the reaction conditions, and obtain solution 3; S4: Adjust solution 3 to 1-20ug / ml as needed, and set aside for later use.
[0018] Furthermore, the modification conditions for steps S2 and S3 are both a temperature of 60-120℃ and a time of 5-30 min.
[0019] Furthermore, the preparation method of the reagent kit includes the following steps: Preparation of Reagent 1: Add deionized water to a container, then add buffer, creatine kinase MM isoenzyme coated modified magnetic microspheres, blocking agent, protective agent, complex, stabilizer, anti-crystallization component, and preservative in sequence. Finally, add creatine kinase isoenzyme B subunit antibody, mix well, and bring the volume to the required total volume. Continue stirring until the mixture is homogeneous to obtain Reagent 1.
[0020] Preparation of Reagent 2: Add deionized water to a glass or stainless steel container, then add buffer, creatine kinase MM isoenzyme-labeled antibody coated with alkaline phosphatase, protectant, surfactant, stabilizer, and preservative in sequence. Mix well and bring the volume to the required total volume. Continue stirring until the mixture is homogeneous to obtain Reagent 2.
[0021] The functions of each component in reagent R1 are as follows: (1) Buffer solution: to provide a stable and suitable pH environment for the overall reaction system; (2) Enzyme protectant: one or a combination of mannitol, trehalose, glycine, bovine serum albumin or complex enzyme stabilizer AES, etc., which serve to protect the catalytic efficiency of the enzyme in the reagent; (3) Complexes: can chelate metal ions, reduce the impact on the reaction enzyme, and are beneficial to the accuracy and stability of the reagents; (4) Preservatives: used to prevent the growth of microorganisms in the solution, which is beneficial to the stable preservation of reagents.
[0022] The beneficial effects of this invention are as follows: the kit of this patent lowers the limit of detection of creatine kinase isoenzyme MM in serum, enhances specificity, improves repeatability, broadens the linear range, and enhances the HOOK effect. Detailed Implementation
[0023] To clearly illustrate the technical features of this solution, the following detailed implementation method will be used to describe the solution.
[0024] Example 1: Step 1: Preparation of antibody-coated modified magnetic microspheres containing creatine kinase MM isoenzyme: S1: Dissolve dextran in water, adjust the pH to 7, and add it to the reaction vessel. Stir until dissolved to obtain solution 1; S2: Add 1 part of vinyltrimethoxysilane to solution 1 prepared in S1, and heat at 60°C for 5 min to obtain solution 2; S3: Add the creatine kinase MM isoenzyme-coated antibody, anti-mouse IgG and 2 parts of surfactant polyethylene glycol 8000 to solution 2 in S2, and heat at 600℃ for 5 min to obtain solution 3. S4: Adjust solution 3 to 1ug / ml as needed, and set aside for later use.
[0025] The second step is the preparation of the reagent kit: Preparation of Reagent 1: Add deionized water to a container, then add 20 mmol / L phosphate buffer, 0.2 g / L creatine kinase MM isoenzyme coated modified magnetic microspheres, 0.5 g / L mouse IgG, 0.5 g / L BSA, 1 mmol / L EDTA sodium salt complex, 1 g / L bovine serum albumin stabilizer, 2‰ Tween-20, 0.05 g / L proclin 300 preservative, and finally add 10 mmol / L creatine kinase isoenzyme B subunit antibody. Mix well and bring the volume to the required total volume. Continue stirring until well mixed to obtain Reagent 1.
[0026] Preparation of Reagent 2: Add deionized water to a glass or stainless steel container, then add 20 mmol / L phosphate buffer, 1 μg / ml creatine kinase MM isoenzyme-labeled antibody coated with alkaline phosphatase, 0.5 g / L BSA, 1 g / L polyoxyethylene (9) ester, 1 g / L bovine serum albumin, and 0.05 g / L proclin 300 in sequence. Mix well and bring the volume to the required total volume. Continue stirring until the mixture is homogeneous to obtain Reagent 2.
[0027] Example 2: Step 1: Preparation of antibody-coated modified magnetic microspheres containing creatine kinase MM isoenzyme: S1: Dissolve dextran in water, adjust the pH to 9, and add it to the reaction vessel. Stir until dissolved to obtain solution 1; S2: Add 3 parts of the silane coupling agent vinyltris(β-methoxyethoxy)silane to the solution 1 prepared in S1, and heat at 120°C for 30 min to obtain solution 2; S3: Add the creatine kinase MM isoenzyme-coated antibody, anti-mouse IgG and 9 parts of surfactant polyoxyethylene laurate (10) ether to solution 2 in S2, and heat at 120°C for 30 min to obtain solution 3. S4: Adjust solution 3 to 20ug / ml as needed, and set aside for later use.
[0028] The second step is the preparation of the reagent kit: Preparation of Reagent 1: Add deionized water to a container, then add 20 mmol / L phosphate buffer, 2 g / L creatine kinase MM isoenzyme coated modified magnetic microspheres, 2 g / L mouse IgG, 10 g / L animal serum, 50 mmol / L EDTA sodium salt, 10 g / L trehalose, 5‰ anti-crystallization component Triton X-450, and 0.1 g / L thimerosal. Finally, add 100 mmol / L creatine kinase isoenzyme B subunit antibody, mix well, and bring the volume to the required total volume. Continue stirring until the mixture is homogeneous to obtain Reagent 1.
[0029] Preparation of Reagent 2: Add deionized water to a glass or stainless steel container, then add 50 mmol / L phosphate buffer, 1-20 μg / ml creatine kinase MM isoenzyme-labeled antibody coated with alkaline phosphatase, 10 g / L animal serum as a protective agent, 2 g / L polyethylene glycol 6000 as a surfactant, 10 g / L glycerol, and 0.1 g / L thimerosal as a preservative. Mix well and bring the volume to the required total volume. Continue stirring until the mixture is homogeneous to obtain Reagent 2.
[0030] Example 3: Step 1: Preparation of antibody-coated modified magnetic microspheres containing creatine kinase MM isoenzyme: S1: Dissolve dextran in water, adjust the pH to 8, and add it to the reaction vessel. Stir until dissolved to obtain solution 1; S2: Add 2 parts of the silane coupling agent vinyltrimethoxysilane to solution 1 prepared in S1, and heat at 80°C for 20 minutes to obtain solution 2; S3: Add creatine kinase MM isoenzyme-coated antibody, anti-mouse IgG, 3 parts of surfactant polyoxyethylene (9) ester and 3 parts of polyoxyethylene (10) laurate ether to solution 2 in S2, and heat at 100°C for 15 min to obtain solution 3. S4: Adjust solution 3 to 15ug / ml as needed, and set aside for later use.
[0031] The second step is the preparation of the reagent kit: Preparation of Reagent 1: Add deionized water to a container, then add 30 mmol / L phosphate buffer, 1 g / L creatine kinase MM isoenzyme coated modified magnetic microspheres, 1.5 g / L mouse IgG, 6 g / L casein, 30 mmol / L EDTA potassium salt, 6 g / L chitosan, 4‰ Triton X-100, and 0.07 g / L sodium azide. Finally, add 70 mmol / L creatine kinase isoenzyme B subunit antibody. Mix well and bring the volume to the required total volume. Continue stirring until well mixed to obtain Reagent 1.
[0032] Preparation of Reagent 2: Add deionized water to a glass or stainless steel container, then add 40 mmol / L phosphate buffer, 13 μg / ml creatine kinase MM isoenzyme-labeled antibody coated with alkaline phosphatase, 5 g / L animal serum, 1.4 g / L polyethylene glycol 6000, 5 g / L bovine serum albumin, and 0.08 g / L thimerosal. Mix well and bring the volume to the required total volume. Continue stirring until well mixed to obtain Reagent 2.
[0033] Example 4: Step 1: Preparation of antibody-coated modified magnetic microspheres containing creatine kinase MM isoenzyme: S1: Dissolve dextran in water, adjust the pH to 8, and add it to the reaction vessel. Stir until dissolved to obtain solution 1; S2: Add 1 part vinyltrimethoxysilane and 1 part vinyltriethoxysilane to solution 1 prepared in S1, and heat at 80°C for 20 minutes to obtain solution 2; S3: Add creatine kinase MM isoenzyme-coated antibody, anti-mouse IgG, 3 parts of surfactant polyoxyethylene (9) ester and 3 parts of polyoxyethylene (10) laurate ether to solution 2 in S2, and heat at 100°C for 15 min to obtain solution 3. S4: Adjust solution 3 to 15ug / ml as needed, and set aside for later use.
[0034] The second step is the preparation of the reagent kit: Preparation of Reagent 1: Add deionized water to a container, then add 10 mmol / L phosphate buffer, 20 mmol / L Tris buffer, 1 g / L creatine kinase MM isoenzyme coated modified magnetic microspheres, 1.5 g / L mouse IgG, 6 g / L casein, 30 mmol / L EDTA potassium salt, 6 g / L chitosan, 4‰ Triton X-100, and 0.07 g / L sodium azide. Finally, add 70 mmol / L creatine kinase isoenzyme B subunit antibody. Mix well and bring the volume to the required total volume. Continue stirring until well mixed to obtain Reagent 1.
[0035] Preparation of Reagent 2: Add deionized water to a glass or stainless steel container, then add 40 mmol / L phosphate buffer, 13 μg / ml creatine kinase MM isoenzyme-labeled antibody coated with alkaline phosphatase, 5 g / L animal serum, 1.4 g / L polyethylene glycol 6000, 5 g / L bovine serum albumin, and 0.08 g / L thimerosal. Mix well and bring the volume to the required total volume. Continue stirring until well mixed to obtain Reagent 2.
[0036] Comparative Example 1: Preparation of the reagent kit: Preparation of Reagent 1: Add deionized water to a container, then add 30 mmol / L phosphate buffer, 1 g / L creatine kinase MM isoenzyme coated antibody, 1.5 g / L mouse IgG, 6 g / L casein, 30 mmol / L EDTA potassium salt, 6 g / L chitosan, 4‰ Triton X-100, and 0.07 g / L sodium azide. Finally, add 70 mmol / L creatine kinase isoenzyme B subunit antibody. Mix well and bring the volume to the required total volume. Continue stirring until well mixed to obtain Reagent 1.
[0037] Preparation of Reagent 2: Add deionized water to a glass or stainless steel container, then add 40 mmol / L phosphate buffer, 13 μg / ml creatine kinase MM isoenzyme-labeled antibody coated with alkaline phosphatase, 5 g / L animal serum, 1.4 g / L polyethylene glycol 6000, 5 g / L bovine serum albumin, and 0.08 g / L thimerosal. Mix well and bring the volume to the required total volume. Continue stirring until well mixed to obtain Reagent 2.
[0038] Comparative Example 2: Step 1: Preparation of antibody-coated modified magnetic microspheres containing creatine kinase MM isoenzyme: S1: Adjust the pH of the water to 8 and add it to the reaction vessel to obtain solution 1; S2: Add 2 parts of the silane coupling agent vinyltrimethoxysilane to solution 1 prepared in S1, and heat at 80°C for 20 minutes to obtain solution 2; S3: Add creatine kinase MM isoenzyme-coated antibody, anti-mouse IgG, 3 parts of surfactant polyoxyethylene (9) ester and 3 parts of polyoxyethylene (10) laurate ether to solution 2 in S2, and heat at 100°C for 15 min to obtain solution 3. S4: Adjust solution 3 to 15ug / ml as needed, and set aside for later use.
[0039] The second step is the preparation of the reagent kit: Preparation of Reagent 1: Add deionized water to a container, then add 30 mmol / L phosphate buffer, 1 g / L creatine kinase MM isoenzyme coated modified magnetic microspheres, 1.5 g / L mouse IgG, 6 g / L casein, 30 mmol / L EDTA potassium salt, 6 g / L chitosan, 4‰ Triton X-100, and 0.07 g / L sodium azide. Finally, add 70 mmol / L creatine kinase isoenzyme B subunit antibody. Mix well and bring the volume to the required total volume. Continue stirring until well mixed to obtain Reagent 1.
[0040] Preparation of Reagent 2: Add deionized water to a glass or stainless steel container, then add 40 mmol / L phosphate buffer, 13 μg / ml creatine kinase MM isoenzyme-labeled antibody coated with alkaline phosphatase, 5 g / L animal serum, 1.4 g / L polyethylene glycol 6000, 5 g / L bovine serum albumin, and 0.08 g / L thimerosal. Mix well and bring the volume to the required total volume. Continue stirring until well mixed to obtain Reagent 2.
[0041] Comparative Example 3: Step 1: Preparation of antibody-coated modified magnetic microspheres containing creatine kinase MM isoenzyme: S1: Dissolve dextran in water, adjust the pH to 8, and add it to the reaction vessel. Stir until dissolved to obtain solution 1; S2: Add 2 parts of the silane coupling agent vinyltrimethoxysilane to solution 1 prepared in S1, and heat at 80°C for 20 minutes to obtain solution 2; S3: Add creatine kinase MM isoenzyme-coated antibody, anti-mouse IgG, 3 parts of surfactant polyoxyethylene (9) ester and 3 parts of polyoxyethylene (10) laurate ether to solution 2 in S2, and heat at 100°C for 15 min to obtain solution 3. S4: Adjust solution 3 to 15ug / ml as needed, and set aside for later use.
[0042] The second step is the preparation of the reagent kit: Preparation of Reagent 1: Add deionized water to a container, then add 30 mmol / L phosphate buffer, 1 g / L creatine kinase MM isoenzyme coated modified magnetic microspheres, 1.5 g / L mouse IgG, 6 g / L casein, 30 mmol / L EDTA potassium salt, 6 g / L chitosan, 4‰ Triton X-100, and 0.07 g / L sodium azide. Finally, add 70 mmol / L creatine kinase isoenzyme B subunit antibody. Mix well and bring the volume to the required total volume. Continue stirring until well mixed to obtain Reagent 1.
[0043] Preparation of Reagent 2: Add deionized water to a glass or stainless steel container, then add 40 mmol / L phosphate buffer, 13 μg / ml creatine kinase MM isoenzyme-labeled antibody coated with alkaline phosphatase, 5 g / L animal serum, 1.4 g / L polyethylene glycol 6000, 5 g / L bovine serum albumin, and 0.08 g / L thimerosal. Mix well and bring the volume to the required total volume. Continue stirring until well mixed to obtain Reagent 2.
[0044] Add 50 μL of reagent R1 to the sample to be tested and react for 5 min; then add 50 μL of reagent R2 and react for 5 min. Wash three times with elution buffer, then add chemiluminescence solution and monitor the photon count using a chemiluminescence analyzer. (The sample to be tested is serum.) Examples 1-4 and Comparative Examples 1-3 were tested according to the following testing methods. The experimental results are shown in Table 1 and the instruction manual.
[0045] Table 1 Test Results
[0046] As can be seen from Table 1, the limits of detection of Examples 1-4 of this application are significantly better than those of the comparative examples; the repeatability of Examples 1-4 is also significantly better than that of the comparative examples 1-3; the linear range of Examples 1-4 is wider than that of the comparative examples 1-3; and the hook effect of Examples 1-4 is also better than that of the comparative examples 1-3.
[0047] The results above show that the kit of this patent lowers the detection limit of creatine kinase isoenzyme MM in serum, enhances specificity, improves repeatability, broadens the linear range, and enhances the HOOK effect.
Claims
1. A creatine kinase isoenzyme MM detection kit, characterized by, The kit comprises reagent 1 and reagent 2, the reagent 1 comprises the following components: Buffer 20-50mmol / L; Creatine kinase isozyme B subunit antibody 10-100mmol / L; Coated modified magnetic microspheres of creatine kinase MM isozyme coated antibody 0.2-2g / L; Blocking agent 0.5-2g / L; Protective agent 0.5-10g / L; Complex 1-50mmol / L; Stabilizer 1-10g / L; Anti-crystallization component 2-5‰; Preservative 0.05-0.1g / L.
2. The creatine kinase isoenzyme MM test kit according to claim 1, characterized in that The reagent 2 comprises the following components: Buffer 20-50mmol / L; Coated alkaline phosphatase of creatine kinase MM isozyme marker antibody 1-20ug / ml; Protective agent 0.5-10g / L; Surfactant 1-2g / L; Stabilizer 1-10g / L; Preservative 0.05-0.1g / L.
3. The creatine kinase isoenzyme MM test kit according to claim 2, characterized in that The buffer is phosphate buffer or Tris buffer; the anti-crystallization component is one or several of Tween-20, Tween-60, Tween-80, Triton X-100, Triton X-450, Span20, Span80.
4. The creatine kinase isoenzyme MM test kit according to claim 2, characterized in that The coated modified magnetic microspheres of creatine kinase MM isozyme coated antibody are coated modified dextran magnetic microspheres of creatine kinase MM isozyme coated antibody; the blocking agent is mouse IgG.
5. The creatine kinase isoenzyme MM test kit according to claim 1, characterized in that The complex is sodium EDTA or potassium EDTA; the surfactant is one or several of polyoxyethylene (9) laurate, polyethylene glycol 6000, polyethylene glycol 8000, polyoxyethylene (10) lauryl ether.
6. The creatine kinase isozyme MM detection kit according to claim 2, characterized in that: The stabilizer is one of bovine serum albumin, trehalose, chitosan, glycerol; the preservative is one or several of proclin300, sodium azide and thiomersal.
7. The creatine kinase isoenzyme MM test kit according to claim 2, characterized in that The raw materials for preparing the coated modified magnetic microspheres of creatine kinase MM isozyme coated antibody comprise: Dextran 0.5-3 parts; Ferrosoferric oxide nanoparticles 0.1-2 parts; Silane coupling agent 1-3 parts; Water 30-80 parts; Creatine kinase MM isozyme coated antibody 5-10 parts; Surfactant 2-9 parts; Anti-mouse IgG 1-5 parts.
8. The creatine kinase isoenzyme MM test kit according to claim 2, characterized in that The preparation steps of the coated modified magnetic microspheres of creatine kinase MM isozyme coated antibody comprise: S1: Dissolve dextran in water, adjust PH to 7-9, and pour into a reaction kettle, stir until dissolved to obtain solution 1; S2: Pour the silane coupling agent into the prepared solution 1 of S1, set the reaction conditions to obtain solution 2; S3: Pour the creatine kinase MM isozyme coated antibody, anti-mouse IgG and surfactant into the solution 2 in S2, set the reaction conditions to obtain solution 3; S4: Adjust solution 3 to 1-20ug / ml as needed.
9. The creatine kinase isoenzyme MM test kit according to claim 2, characterized in that The modification conditions of steps S2 and S3 are both temperature of 60-120℃ and time of 5-30min.
10. A method for preparing a creatine kinase isoenzyme MM detection kit, characterized by, The preparation method of the kit comprises the following steps: Preparation of Reagent 1: Add deionized water to a container, then add buffer, creatine kinase MM isoenzyme coated modified magnetic microspheres, blocking agent, protective agent, complex, stabilizer, anti-crystallization component, and preservative in sequence. Finally, add creatine kinase isoenzyme B subunit antibody, mix well, and bring the volume to the required total volume. Continue stirring until the mixture is homogeneous to obtain Reagent 1. Preparation of Reagent 2: Add deionized water to a glass or stainless steel container, then add buffer, creatine kinase MM isoenzyme-labeled antibody coated with alkaline phosphatase, protectant, surfactant, stabilizer, and preservative in sequence. Mix well and bring the volume to the required total volume. Continue stirring until the mixture is homogeneous to obtain Reagent 2.