A latex enhanced immunoturbidimetric assay kit for highly specific determination of CK-MM and its preparation method

Abstract

The present invention relates to the field of immunoassay technology, and particularly relates to a latex-enhanced immunoturbidimetric assay kit for highly specific determination of CK-MM and a preparation method thereof. The kit provided by the present invention adds a sample cleaning solution component for processing dried blood filter paper, solving the problem that dried blood filter paper cannot be rapidly analyzed by an automatic biochemical analyzer. Through the cooperation of each reagent component, the kit provided by the present invention reduces the interference of external factors, improves the reaction specificity between the analyte and the antibody, and can significantly improve the screening speed of neonatal progressive muscular dystrophy.

Description

Technical Field

[0001] The present invention relates to the technical field of kit detection, and particularly relates to a latex enhanced immunoturbidimetric assay kit for highly specific determination of CK-MM and a preparation method thereof. Background Art

[0002] Creatine kinase (CK) has a dimer structure and has two subunits, M and B, so it can form three isoenzymes, namely CK-MM, CK-MB, and CK-BB. Most of the CK in skeletal muscle is CK-MM (97%-99%), and a very small part is CK-MB (1%-3%); 78% of CK in the myocardium is CK-MM, and 22% is CK-MB; all of the CK in the brain tissue is CK-BB, and most of the gastrointestinal and bladder smooth muscles are CK-BB (>90%), and the rest are CK-MM and CK-MB. Almost all of the CK in normal human serum is CK-MM. Progressive muscular dystrophy is mainly dominated by muscular CK-MM. Therefore, the determination of CK-MM activity is a relatively specific indicator for diagnosing skeletal muscle diseases.

[0003] As an important and relatively specific indicator for skeletal muscle diseases, the detection of CK-MM in dried blood filter paper, serum, and urine is of great significance. There are many factors that affect the CK-MM detection value, such as age, gender, race, region, and exercise status. The classic method for determining CK isoenzymes is electrophoresis. Currently, the commonly used detection methods mainly include biochemical methods, such as colorimetry, ultraviolet spectrophotometry, and fluorescence methods. One of the more commonly used methods is the enzyme-coupled assay method, but this method is easily interfered by hemolysis and other enzyme substances in the serum, and the operation is cumbersome; the content of CK-MM is low, and the sensitivity requirements for the kit are relatively high. Currently, the detection specificity of related creatine kinase isoenzyme detection kits is not high, and the detection linear range is relatively low, which cannot meet the clinical requirements. Summary of the Invention

[0004] The purpose of the present invention is to provide a latex enhanced immunoturbidimetric assay kit for highly specific determination of CK-MM and a preparation method thereof; the kit provided by the present invention is suitable for the detection of multiple samples, including dried blood filter paper, serum, and urine; the kit provided by the present invention is easy to operate and can be automated.

[0005] In the first aspect, the present invention provides a creatine kinase isoenzyme detection kit, and the creatine kinase isoenzyme detection kit includes reagent R1 and reagent R2; the reagent R1 contains 1.0 - 5.0 g / L of non-ionic surfactant and CK-MB specific antibody.

[0006] In the creatine kinase isoenzyme detection kit provided by the present invention, the reagent R1 includes:

[0007] Buffer: one of 5 - 50 mmol / L Tris-HCL buffer (pH = 7.00 - 9.00), HEPES buffer (pH = 6.80 - 8.20), MOPS buffer (pH = 6.50 - 7.90), Tricine buffer (pH = 6.00 - 8.00), MES buffer (pH = 5.00 - 8.00), glycine buffer (pH = 7.00 - 8.00);

[0008] Preservative: one of 0.1 - 1.0 g / L Proclin-300, NaN3, thimerosal;

[0009] Matrix: purified water;

[0010] Metal ion chelator: 0.5 - 2.0 g / L disodium EDTA;

[0011] Stabilizer: one of 5 - 30 g / L sucrose, trehalose, mannitol;

[0012] Protective agent: one of 0.5 - 5 g / L NaCl, KCl;

[0013] Surfactant: 1.0 - 5.0 g / L non-ionic surfactant, such as TWEEN series (TWEEN-20, TWEEN-80), Triton series (Triton X-100), PEG series (PEG2000, PEG6000, PEG20000); among them, Triton series is used for anti-interference and preventing non-specific adsorption of miscellaneous proteins from interfering with the reaction; PEG series is used for promoting antigen-antibody reaction.

[0014] CK-MB specific antibody (commercially available): concentration 1 - 5 μg / mL; after adding to R1, CK-MB is pre-reacted by CK-MB antibody to prevent affecting the determination of CK-MM, thus significantly improving the detection sensitivity and detection range; for cost reduction, polyclonal antibody can be used for CK-MB specific antibody.

[0015] Specifically, reagent R1 includes: one of 5 - 50 mmol / L Tris-HCL buffer, HEPES buffer, MOPS buffer, Tricine buffer, MES buffer, glycine buffer; 0.5 - 2 g / L disodium EDTA; one of 5 - 40 g / L sucrose, trehalose, mannitol; 0.5 - 5.0 g / L NaCl or KCl; 1.0 - 5.0 g / L non-ionic surfactant; CK-MB specific antibody; pH is 7.5 - 8.0.

[0016] More specifically, the reagent R1 includes: 10 - 50 mmol / L Tris-HCL buffer, 0.2 g / L NaN3, purified water, 0.5 - 1 g / L disodium EDTA, 20 - 40 g / L sucrose, 2.0 - 5.0 g / L NaCl, 0.5 - 1 g / L TWEEN-20, 0.5 - 1 g / L PEG2000, 3.0 μg / mL CK-MB specific antibody; with a pH of 7.5 - 8.0.

[0017] In the creatine kinase isoenzyme detection kit provided by the present invention, the reagent R2 contains 1.0 - 2.5 g / L non-ionic surfactant; 3.0 - 8.0 μg / mL CK-MM specific antibody, 0.02 - 0.3% latex microspheres.

[0018] In the creatine kinase isoenzyme detection kit provided by the present invention, the reagent R2 includes:

[0019] Buffer: one of 5 - 50 mmol / L Tris-HCL buffer (pH = 7.00 - 9.00), HEPES buffer (pH = 6.80 - 8.20), MOPS buffer (pH = 6.50 - 7.90), Tricine buffer (pH = 6.00 - 8.00), MES buffer (pH = 5.00 - 8.00), glycine buffer (pH = 7.00 - 8.00);

[0020] Preservative: one of 0.1 - 1.0 g / L Proclin-300, NaN3, thimerosal;

[0021] Matrix: purified water;

[0022] Metal ion chelator: 0.5 - 2.0 g / L disodium EDTA;

[0023] Stabilizer: one of 5 - 30 g / L sucrose, trehalose, mannitol;

[0024] Protective agent: one of 0.5 - 5 g / L NaCl, KCl.

[0025] Surfactant: 1.0 - 2.5 g / L non-ionic surfactant, such as TWEEN series (TWEEN-20, TWEEN-80), Triton series (Triton X-100), polyols (glycerol): for the purpose of uniform dispersion to prevent aggregation and precipitation of antibody microsphere conjugates; among them, the Triton series is used for anti-interference and to prevent non-specific adsorption of heteroproteins from interfering with the reaction.

[0026] CK-MM specific antibody (commercially available) (monoclonal antibody: high sensitivity): concentration 1 - 10 μg / mL;

[0027] Latex microspheres (commercially available): concentration 0.02% - 0.3%; particle size selected from 50 - 350 nm; single type of microspheres, all carboxyl microspheres (the smaller the particle size, the more antibodies bound).

[0028] Specifically, in the creatine kinase isoenzyme detection kit provided by the present invention, the reagent R2 includes: 10 - 50 mmol / L Tris-HCL buffer, 0.2 g / L NaN3, purified water, 0.5 - 1.0 g / L disodium EDTA, 20 - 40 g / L sucrose, 2.0 - 5.0 g / L NaCl, 0.5 - 1.0 g / L TWEEN-20, 0.5 - 1 g / L PEG2000, 0.5 g / L glycerol; pH is 7.5 - 8.0; 3.0 - 8.0 μg / mL CK-MM specific antibody, 0.2 - 0.3% latex microspheres.

[0029] In the creatine kinase isoenzyme detection kit provided by the present invention, the creatine kinase isoenzyme detection kit further includes a sample cleaning solution, and the sample cleaning solution contains ATP.

[0030] In the creatine kinase isoenzyme detection kit provided by the present invention, the sample cleaning solution includes:

[0031] Buffer: one of 5 - 50 mmol / L Tris-HCL buffer (pH = 7.00 - 9.00), HEPES buffer (pH = 6.80 - 8.20), MOPS buffer (pH = 6.50 - 7.90), Tricine buffer (pH = 6.00 - 8.00), MES buffer (pH = 5.00 - 8.00), glycine buffer (pH = 7.00 - 8.00);

[0032] Preservative: one of 0.1 - 0.5 g / L Proclin-300, NaN3, thimerosal;

[0033] Matrix: purified water;

[0034] Metal ion chelator: 0.2 - 1.0 g / L disodium EDTA;

[0035] Protectant: one of 0.5 - 5 g / L NaCl, KCl;

[0036] Surfactant: 0.5 - 1.5 g / L non-ionic surfactant, such as TWEEN series (TWEEN-20, TWEEN-80), Triton series (Triton X-100), PEG series (PEG2000, PEG6000, PEG20000); among them, Triton series is used for anti-interference and preventing non-specific adsorption of miscellaneous proteins from interfering with the reaction; PEG series is used to promote antigen-antibody reaction.

[0037] Others: 20 - 100 g / L BSA, 2 - 10 mmol / L ATP, and ATP is used to resist the interference of adenylate kinase (AK).

[0038] Specifically, the sample cleaning solution includes one of 5 - 50 mmol / L Tris-HCL buffer, HEPES buffer, MOPS buffer, Tricine buffer, MES buffer, glycine buffer; 0.2 - 1 g / L disodium EDTA; 0.5 - 5.0 g / L NaCl or KCl; 0.5 - 1.5 g / L non-ionic surfactant; 20 - 100 g / L BSA, 2 - 10 mmol / L ATP; with a pH of 7.5 - 8.0.

[0039] More specifically, in the creatine kinase isoenzyme detection kit provided by the present invention, the sample cleaning solution includes 10 - 50 mmol / L Tris-HCL buffer, 0.2 g / L NaN3, purified water, 0.5 - 1.0 g / L disodium EDTA, 0.5 - 2.0 g / L NaCl, 0.5 - 1.0 g / L TWEEN-20, 0.5 - 1 g / L PEG2000, 50 - 100 g / L BSA, 5 - 8 mmol / L ATP; with a pH of 7.5 - 8.0.

[0040] According to the understanding of those skilled in the art, the present invention claims the application of the above creatine kinase isoenzyme detection kit in improving the detection sensitivity of CK-MM in a sample or detecting trace CK-MM in a sample.

[0041] Specifically, the samples that can be detected by the creatine kinase isoenzyme detection kit provided by the present invention are dried blood filter paper, serum or urine.

[0042] Second aspect, the present invention provides a method for detecting creatine kinase isoenzyme in dry blood samples. In this method, the components of the dry blood sample eluate are 10 - 50 mmol / L Tris-HCL buffer, 0.2 g / L NaN3, purified water, 0.5 - 1.0 g / L disodium EDTA, 0.5 - 2.0 g / L NaCl, 0.5 - 1.0 g / L TWEEN-20, 0.5 - 1 g / L PEG2000, 50 - 100 g / L BSA, 5 - 8 mmol / L ATP; the pH is 7.5 - 8.0.

[0043] In the method provided by the present invention, a sample eluate is added to the dry blood filter paper sample, incubated with shaking at room temperature, and the sample is eluted for standby; the above-mentioned creatine kinase isoenzyme detection kit is used to detect the sample; the detection instrument is an automatic biochemical analyzer.

[0044] The beneficial effects of the present invention are as follows:

[0045] (1) The present invention provides a kit for detecting blood samples in dry blood filter paper. In the kit of the present invention, a sample cleaning component for treating dry blood filter paper is added, and ATP is added to the sample cleaning component to help specifically elute creatine kinase isoenzyme.

[0046] (2) The present invention combines a monoclonal antibody with a small particle size carboxyl latex microsphere to develop a highly specific and sensitive CK-MM detection kit for simultaneously detecting creatine kinase isoenzyme in dry blood filter paper, serum and urine. Compared with the prior art, this kit has the characteristics of high detection sensitivity, high accuracy, strong specificity, good repeatability, wide linear range, etc.; it can be applied to the screening of neonatal progressive muscular dystrophy and the diagnosis of skeletal muscle diseases.

[0047] (3) The present invention can be applied to dry blood filter paper samples, and is not affected by the release of AK from red blood cells during hemolysis, making the screening of neonatal progressive muscular dystrophy popular. The sample cleaning solution provided by the present invention elutes the blood sample in the dry blood filter paper and is used for kit detection, solving the problem that dry blood filter paper cannot be rapidly analyzed by an automatic biochemical analyzer, reducing the interference of external factors at the same time, improving the reaction specificity between the analyte and the antibody, and improving the screening speed of neonatal progressive muscular dystrophy.

[0048] (4) A non-ionic surfactant is added to the kit of the present invention, which effectively resists the interference of chyle and hemolysis, promotes the antigen-antibody reaction. At the same time, the CK-MB polyclonal antibody in the R1 kit blocks the interference of the B subunit on the detection of the M subunit in advance, further improving the detection sensitivity, reaction specificity and linear range, fully avoiding the appearance of false results, and improving the accuracy and reliability of the detection results. Brief Description of the Drawings

[0049] Figure 1 It is a correlation diagram of the detection results of the kits of Example 1 and Comparative Example 1 of the present invention.

[0050] Figure 2 It is a correlation diagram of the detection results of the kits of Example 1 and Comparative Example 2 of the present invention.

[0051] Figure 3 It is a correlation diagram of the detection results of the kits of Example 1 and Comparative Example 3 of the present invention.

[0052] Figure 4 It is a correlation diagram of the detection results of the kits of Example 2 and Comparative Example 3 of the present invention.

[0053] Figure 5 It is a correlation diagram of the detection results of the kits of Example 3 and Comparative Example 3 of the present invention. Detailed Description of the Invention

[0054] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

[0055] Example 1

[0056] This example provides a creatine kinase isoenzyme detection kit, and its composition is as follows.

[0057] The composition of the CK-MM kit includes: sample eluent, R1 reagent, and R2 reagent.

[0058] (1) The composition of the sample cleaning solution includes: 50 mmol / L Tris-HCL buffer, pH = 7.5 - 8.0, 0.2 g / L NaN3, purified water, 1.0 g / L EDTA disodium, 5.0 g / L NaCl, 1.0 g / L TWEEN-20, 1.0 g / L PEG2000, 100 g / L BSA, 8 mmol / L ATP;

[0059] (2) The composition of the R1 reagent includes: 50 mmol / L Tris-HCL buffer, pH = 7.5 - 8.0, 0.2 g / L NaN3, purified water, 1.0 g / L EDTA disodium, 40 g / L sucrose, 5.0 g / L NaCl, 1.0 g / L TWEEN-20, 1.0 g / L PEG2000, 5.0 μg / mL CK-MB specific antibody;

[0060] (3) The composition of Reagent R2 includes: 50 mmol / L Tris-HCL buffer solution, pH = 7.5 - 8.0, 0.2 g / L NaN3, purified water, 1.0 g / L disodium EDTA, 40 g / L sucrose, 5.0 g / L NaCl, 1.0 g / L TWEEN-20, 1.0 g / L PEG2000, 0.5 g / L glycerol, 8.0 μg / mL CK-MM specific antibody; 0.3% latex microspheres.

[0061] Example 2

[0062] This example provides a creatine kinase isoenzyme detection kit, and its composition is as follows.

[0063] The composition of the CK-MM kit includes: sample eluent, Reagent R1, Reagent R2.

[0064] (1) The composition of the sample cleaning solution includes: 30 mmol / L Tris-HCL buffer solution, pH = 7.5 - 8.0, 0.2 g / L NaN3, purified water, 1.0 g / L disodium EDTA, 2.0 g / L NaCl, 1.0 g / L TWEEN-20, 0.5 g / L PEG2000, 50 g / L BSA, 5 mmol / L ATP;

[0065] (2) The composition of Reagent R1 includes: 30 mmol / L Tris-HCL buffer solution, pH = 7.5 - 8.0, 0.2 g / L NaN3, purified water, 1.0 g / L disodium EDTA, 20 g / L sucrose, 2.0 g / L NaCl, 1.0 g / L TWEEN-20, 0.5 g / L PEG2000, 5.0 μg / mL CK-MB specific antibody;

[0066] (3) The composition of Reagent R2 includes: 30 mmol / L Tris-HCL buffer solution, pH = 7.5 - 8.0, 0.2 g / L NaN3, purified water, 1.0 g / L disodium EDTA, 20 g / L sucrose, 2.0 g / L NaCl, 1.0 g / L TWEEN-20, 0.5 g / L PEG2000, 0.5 g / L glycerol, 5.0 μg / mL CK-MM specific antibody; 0.3% latex microspheres.

[0067] Example 3

[0068] This example provides a creatine kinase isoenzyme detection kit, and its composition is as follows.

[0069] The composition of the CK-MM kit includes: sample eluent, Reagent R1, Reagent R2.

[0070] (1) The composition of the sample cleaning solution includes: 10 mmol / L Tris-HCL buffer, pH = 7.5 - 8.0, 0.2 g / L NaN3, purified water, 0.5 g / L disodium EDTA, 2.0 g / L NaCl, 0.5 g / L TWEEN-20, 0.5 g / L PEG2000, 50 g / L BSA, 5 mmol / L ATP;

[0071] (2) The composition of reagent R1 includes: 10 mmol / L Tris-HCL buffer, pH = 7.5 - 8.0, 0.2 g / L NaN3, purified water, 0.5 g / L disodium EDTA, 20 g / L sucrose, 2.0 g / L NaCl, 0.5 g / L TWEEN-20, 0.5 g / L PEG2000, 3.0 μg / mL CK-MB specific antibody;

[0072] (3) The composition of reagent R2 includes: 10 mmol / L Tris-HCL buffer, pH = 7.5 - 8.0, 0.2 g / L NaN3, purified water, 0.5 g / L disodium EDTA, 20 g / L sucrose, 2.0 g / L NaCl, 0.5 g / L TWEEN-20, 0.5 g / L PEG2000, 0.5 g / L glycerol, 3.0 μg / mL CK-MM specific antibody; 0.2% latex microspheres.

[0073] Comparative Example 1

[0074] This example provides a creatine kinase isoenzyme detection kit, and its composition components are as follows.

[0075] The composition of the CK-MM kit includes: sample eluent, reagent R1, reagent R2.

[0076] (1) The composition of the sample cleaning solution includes: 10 mmol / L Tris-HCL buffer, pH = 7.5 - 8.0, 0.2 g / L NaN3, purified water, 0.5 g / L disodium EDTA, 2.0 g / L NaCl, 0.5 g / L TWEEN-20, 0.5 g / L PEG2000, 50 g / L BSA;

[0077] (2) The composition of reagent R1 includes: 10 mmol / L Tris-HCL buffer, pH = 7.5 - 8.0, 0.2 g / L NaN3, purified water, 0.5 g / L disodium EDTA, 20 g / L sucrose, 2.0 g / L NaCl, 0.5 g / L TWEEN-20, 0.5 g / L PEG2000, 3.0 μg / mL CK-MB specific antibody;

[0078] (3) The composition of the R2 reagent includes: 10 mmol / L Tris-HCL buffer, pH = 7.5 - 8.0, 0.2 g / L NaN3, purified water, 0.5 g / L disodium EDTA, 20 g / L sucrose, 2.0 g / L NaCl, 0.5 g / L TWEEN-20, 0.5 g / L PEG2000, 0.5 g / L glycerol, 3.0 μg / mL CK-MM specific antibody; 0.2% latex microspheres.

[0079] Comparative Example 2

[0080] This example provides a creatine kinase isoenzyme detection kit, and its composition components are as follows.

[0081] The composition of the CK-MM kit includes: sample eluent, R1 reagent, and R2 reagent.

[0082] (1) The composition of the sample cleaning solution includes: 10 mmol / L Tris-HCL buffer, pH = 7.5 - 8.0, 0.2 g / L NaN3, purified water, 0.5 g / L disodium EDTA, 2.0 g / L NaCl, 0.5 g / L TWEEN-20, 0.5 g / L PEG2000, 50 g / L BSA;

[0083] (2) The composition of the R1 reagent includes: 10 mmol / L Tris-HCL buffer, pH = 7.5 - 8.0, 0.2 g / L NaN3, purified water, 0.5 g / L disodium EDTA, 20 g / L sucrose, 2.0 g / L NaCl, 0.5 g / L TWEEN-20, 0.5 g / L PEG2000;

[0084] (3) The composition of the R2 reagent includes: 10 mmol / L Tris-HCL buffer, pH = 7.5 - 8.0, 0.2 g / L NaN3, purified water, 0.5 g / L disodium EDTA, 20 g / L sucrose, 2.0 g / L NaCl, 0.5 g / L TWEEN-20, 0.5 g / L PEG2000, 0.5 g / L glycerol, 3.0 μg / mL CK-MM specific antibody; 0.2% latex microspheres;

[0085] Comparative Example 3

[0086] This example provides a creatine kinase isoenzyme detection kit, and its composition components are as follows.

[0087] The composition of the CK-MM kit includes: sample eluent, R1 reagent, and R2 reagent.

[0088] (1) The composition of the sample cleaning solution includes: 10 mmol / L Tris-HCL buffer solution, pH = 7.5 - 8.0, 0.2 g / L NaN3, purified water, 0.5 g / L disodium EDTA, 2.0 g / L NaCl, 50 g / L BSA, 5 mmol / L ATP;

[0089] (2) The composition of Reagent R1 includes: 10 mmol / L Tris-HCL buffer solution, pH 7.5 - 8.0, 0.2 g / L NaN3, purified water, 0.5 g / L disodium EDTA, 20 g / L sucrose, 2.0 g / L NaCl, 3.0 μg / mL CK-MB specific antibody;

[0090] (3) The composition of Reagent R2 includes: 10 mmol / L Tris-HCL buffer solution, pH 7.5 - 8.0, 0.2 g / L NaN3, purified water, 0.5 g / L disodium EDTA, 20 g / L sucrose, 2.0 g / L NaCl, 3.0 μg / mL CK-MM specific antibody; 0.2% latex microspheres.

[0091] Example 4

[0092] In this example, the kits of Examples 1 - 3 and Comparative Examples 1 - 3 were used for experimental evaluation respectively.

[0093] This example provides a preparation method for Reagent R2 as follows:

[0094] (1) Activation. Activation solution: one of 5 - 50 mmol / L MES buffer solution, phosphate buffer solution, glycine buffer solution, pH = 5.5 - 8.0;

[0095] Activation process: The activation solution, latex microspheres, EDC, and NHS are added in sequence, stirred evenly, and activated for 10 - 30 min; among them, the concentration of EDC is 25% - 40% of the volume of the latex microspheres; the concentration of NHS is 25% - 80% of the volume of the latex microspheres;

[0096] (2) Coupling. The activated solution containing latex microspheres is ultrasonicated in a 120 W ultrasonic cleaner for 15 s and repeated twice; add the CK-MM specific antibody to the activated solution, stir evenly, and couple at a constant temperature of 37°C with stirring for 30 min - 200 min;

[0097] (3) Blocking. Blocking solution: one of 5 - 50 mmol / L MES buffer solution, phosphate buffer solution, glycine buffer solution, pH = 5.5 - 8.0; 120 - 320 g / L BSA;

[0098] Procedure: The coupling solution obtained in step (2) is sonicated in a 120W ultrasonic cleaner for 15s and repeated twice; the blocking solution is added and stirred for blocking for 15 - 60 min;

[0099] (4) After blocking, it is sonicated in a 120W ultrasonic cleaner for 15S and repeated twice; centrifuged at 12000 rpm at a temperature of 8 - 15°C; the centrifuged supernatant is discarded, and the other components in reagent R2 are added, then sonicated in a 120W ultrasonic cleaner for 15s and repeated twice; reagent R2 is placed in an incubator at 37°C for aging for 10 - 30h; thus, the preparation of reagent R2 is completed.

[0100] In this example, the kits of Examples 1 - 3 and Comparative Examples 1 - 3 were used to detect dry blood samples, and the steps are as follows:

[0101] (1) Add 300 μL of sample eluent to the dry blood filter paper sample, incubate with shaking at room temperature for 120 min, and use it as a sample for standby;

[0102] (2) The detection instrument used is Hitachi 7180 automatic biochemical analyzer; the instrument software automatically calculates the measurement results and forms a detection report. The measurement results of 20 samples (range: 0.5 ng / mL - 300 ng / mL) are obtained.

[0103] The experimental results are as Figures 1-5 shown, among which, Figure 1 are the comparison data between Example 1 and Comparative Example 1. Figure 1 It shows that when measuring CK-MM samples with different concentrations, the measurement results of Comparative Example 1 are systematically low, and when the sample value is low, zero and negative results will appear.

[0104] Figure 2 is the correlation result graph between Example 1 and Comparative Example 2, Figure 2 indicating that when measuring CK-MM samples with different concentrations, there are obvious differences between the measurement results of Example 1 and those of Comparative Example 2; the measurement results of Comparative Example 2 are systematically low, and when the sample value is low, zero and negative results will appear.

[0105] Figure 3 is the correlation result graph between Example 1 and Comparative Example 3, Figure 3 indicating that when measuring CK-MM samples with different concentrations, there are obvious differences between the measurement results of Example 1 and those of Comparative Example 3; the measurement results of Comparative Example 3 are systematically low, and when the sample value is low, zero and negative results will appear.

[0106] Figure 4 is the correlation result graph between Example 2 and Comparative Example 3, Figure 4It shows that there are obvious differences between the measurement results of Example 2 and those of Comparative Example 3 when measuring CK-MM samples with different concentrations; the measurement results of Comparative Example 3 are systematically low, and zero and negative results will occur when the sample values are low.

[0107] Figure 5 It is the correlation result graph of Example 3 and Comparative Example 3. Figure 5 It shows that there are obvious differences between the measurement results of Example 3 and those of Comparative Example 3 when measuring CK-MM samples with different concentrations; the measurement results of Comparative Example 3 are systematically low, and zero and negative results will occur when the sample values are low.

[0108] In this example, the detection limit, the lowest detection limit, repeatability, and accuracy of the detection kit provided in Example 1 were also tested. The results showed that the detection range of the detection kit provided by the present invention is: 3 - 5000 U / L; the lowest detection limit: 0.5 U / L; repeatability: CV ≤ 10%; accuracy: within the calibration range, the bias between the measured value and the theoretical value of the detection enterprise reference product is within ±5%.

[0109] Control Group 1

[0110] This control group provides a creatine kinase isoenzyme detection kit, and its composition is as follows.

[0111] The composition of the CK-MM kit includes: sample eluent, R1 reagent, and R2 reagent. The components of the sample eluent and R1 reagent are the same as those in Example 1. Only the amount of non-ionic surfactant in the R2 reagent is changed to 0.2 g / L TWEEN-20, 0.2 g / L PEG2000, and 0.2 g / L glycerol. The repeatability detection results of Control Group 1, Example 1, and Comparative Example 3 are shown in Table 1.

[0112] Table 1 Repeatability Detection Results

[0113]

[0114]

[0115] Although the present invention has been described in detail above with general descriptions and specific implementation examples, based on the present invention, some modifications or improvements can be made, which are obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection required by the present invention.

Claims

1. A creatine kinase isoenzyme detection kit, characterized in that, The creatine kinase isoenzyme detection kit includes reagent R1, reagent R2 and sample cleaning solution; Reagent R1 contains 1.0 - 5.0 g / L of non-ionic surfactant and 1 - 5 μg / mL of CK-MB specific antibody; Reagent R2 contains 1.0 - 2.5 g / L of non-ionic surfactant; 3.0 - 8.0 μg / mL of CK-MM specific antibody, 0.02 - 0.3% of latex microspheres; The sample cleaning solution contains 2 - 10 mmol / L of ATP and 0.5 - 1.5 g / L of non-ionic surfactant; The non-ionic surfactant is TWEEN-20.

2. The creatine kinase isoenzyme detection kit according to claim 1, characterized in that, Reagent R1 includes: one of 5 - 50 mmol / L Tris-HCl buffer, HEPES buffer, MOPS buffer, Tricine buffer, MES buffer, glycine buffer; one of 0.1 - 1.0 g / L of Proclin-300, NaN3, thimerosal; 0.5 - 2 g / L of EDTA disodium; one of 5 - 40 g / L of sucrose, trehalose, mannitol; 0.5 - 5.0 g / L of NaCl or KCl; 1.0 - 5.0 g / L of non-ionic surfactant; CK-MB specific antibody; pH is 7.5 - 8.

0.

3. The creatine kinase isoenzyme detection kit according to claim 1, characterized in that, Reagent R2 includes: one of 5 - 50 mmol / L Tris-HCl buffer, HEPES buffer, MOPS buffer, Tricine buffer, MES buffer, glycine buffer; one of 0.1 - 1.0 g / L of Proclin-300, NaN3, thimerosal; 0.5 - 2.0 g / L of EDTA disodium; one of 5 - 40 g / L of sucrose, trehalose, mannitol; 0.5 - 5.0 g / L of NaCl or KCl; 0.5 - 1.0 g / L of TWEEN-20, 0.5 - 1 g / L of PEG2000, 0.5 g / L of glycerol; pH is 7.5 - 8.0; 3.0 - 8.0 μg / mL of CK-MM specific antibody, 0.02 - 0.3% of latex microspheres.

4. The creatine kinase isoenzyme detection kit according to claim 1, characterized in that, The sample cleaning solution includes: one of 5 - 50 mmol / L Tris-HCl buffer, HEPES buffer, MOPS buffer, Tricine buffer, MES buffer, glycine buffer; 0.2 - 1 g / L of EDTA disodium; 0.5 - 5.0 g / L of NaCl or KCl; 0.5 - 1.5 g / L of non-ionic surfactant; 20 - 100 g / L of BSA, 2 - 10 mmol / L of ATP; pH is 7.5 - 8.

0.

5. Use of the creatine kinase isoenzyme detection kit according to any one of claims 1-4 in the preparation of a CK-MM detection reagent, characterized in that, The CK-MM detection reagent has high sensitivity.

6. Use of the creatine kinase isoenzyme detection kit according to any one of claims 1-4 in the preparation of a trace CK-MM detection reagent.

7. The use according to claim 5 or 6, characterized in that, The detection sample in the application is dried blood filter paper, serum or urine.

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