Kit for detecting creatine kinase isoenzyme and application

Abstract

The embodiment of the invention belongs to the technical field of detection, and relates to a kit for detecting creatine kinase isoenzyme and application, the kit comprises a first detection reagent and a second detection reagent; the first detection reagent comprises a Hepes buffer solution, NaCl, PEG-6000 (Polyethylene Glycol), nonylphenol polyoxyethylene ether, BSA (Bovine Serum Albumin), NaN3, a chemical blocking agent and a biological blocking agent; the second detection reagent is prepared from CKMB sensitization latex particles, a Hepes buffer solution, a sealing agent, tween-20 and NaN3. According to the application, the detection sensitivity of the creatine kinase isoenzyme is effectively improved.

Description

Technical Field

[0001] The present application relates to the field of detection technology, and in particular to a kit for detecting creatine kinase isoenzymes and its application. Background Art

[0002] Creatine kinase (CK) is widely present in various tissues and is related to the regeneration of adenosine triphosphate (ATP), maintaining the intracellular adenosine triphosphate concentration at physiological levels.

[0003] CK is a heterodimer, with subunits of M type and B type, mainly divided into three isozymes: CK-BB, CK-MM, and CK-MB. Mitochondria also contain another CK-Mt isozyme. CK-BB is mainly found in organs such as the brain and prostate, CK-MM is mainly found in bones and myocardium, and CK-MB is mainly found in myocardium. The CK-MB content in normal human serum is less than 5% of the total activity. When chest pain occurs after acute myocardial infarction, the CK-MB content in serum increases in 4 hours, reaches a peak value in 24 hours, and recovers in 3-4 days. CK-MB is one of the important markers of myocardial injury.

[0004] Currently, the methods for detecting CK-MB include agarose gel electrophoresis, enzyme rate immunoinhibition method, chemiluminescence immunoassay and latex immunoturbidimetry.

[0005] The enzyme rate immunosuppression method is susceptible to multiple factors and has a high false positive rate. It uses a monoclonal antibody against CK-M to inhibit the activity of the CK-M subunit in the specimen, does not affect the activity of the CK-B subunit, and measures the activity of the B subunit in the remaining CK-MB and CK-BB through an enzymatic reaction. When the brain, prostate, gastrointestinal tract, lung and other tissue cells are damaged, ischemic, or necrotic, CK-BB will be released into the serum, which can easily cause a false increase in CK-MB; at the same time, giant CK (a kind of oligomeric mitochondrial CK) released by people with tumors or immune disorders will also participate in the enzymatic reaction when present. Giant CK will not be inhibited by anti-CK-M subunit antibodies, and its activity will be detected 100%, which will multiply the CK-MB test results and cause a false increase in CK-MB.

[0006] Agarose gel electrophoresis is a common method for separating and detecting myocardial creatine kinase isoenzymes. It uses agarose gel as a medium to separate proteins and calculate the percentage of CKMM, CKMB, and CKBB. This method requires manual operation, takes a long time to detect, and cannot be automated.

[0007] The chemiluminescent immunoassay is based on the principle of double antibody sandwich method. The specific mouse monoclonal antibody is pre-coated on the carrier, and the sample to be tested and the enzyme-labeled specific monoclonal antibody are added to form an immune complex of specific antibody-antigen-enzyme-labeled antibody. After excitation, photons are emitted, and the mass number of CK-MB in the sample is obtained according to the size of the excitation light. This method has strong specificity, but is expensive.

[0008] Latex immunoturbidimetry is a relatively stable and accurate homogeneous immunoturbidimetric method for detecting body fluid proteins. When CKMB combines with the microspheres conjugated with the antibody CKMB, it aggregates rapidly, changing the absorbance of the reaction solution, forming a linear relationship within a certain range, and the concentration of CKMB can be converted.

[0009] The creatine kinase isoenzyme in the sample combines with the anti-creatine kinase isoenzyme antibody-sensitized latex in the reagent to form an insoluble immune complex, which forms turbidity in a specific buffer environment. The turbidity is proportional to the content of the analyte within the detection range. By comparing with the calibrator operated under the same conditions, the content of the creatine kinase isoenzyme in the sample can be calculated.

[0010] The current creatine kinase isoenzyme detection kits have the following problems: (1) The reagents are poorly stable and easily precipitate; (2) The reagents have a narrow linear range and poor sensitivity, generally 3-180 ng / mL; (3) The reagents are easily interfered by triglycerides; (4) The reagents are easily interfered by rheumatoid factors.

[0011] In summary, the current creatine kinase isoenzyme detection kit has poor detection sensitivity for creatine kinase isoenzymes. Summary of the invention

[0012] The purpose of the embodiments of the present application is to provide a kit and application for detecting creatine kinase isoenzymes, so as to effectively improve the detection sensitivity of creatine kinase isoenzymes.

[0013] In order to solve the above technical problems, the present application embodiment provides a kit for detecting creatine kinase isoenzymes, which adopts the following technical solution:

[0014] A kit for detecting creatine kinase isoenzymes, comprising:

[0015] a first detection reagent and a second detection reagent;

[0016] The first detection reagent includes Hepes buffer, NaCl, PEG-6000, nonylphenol polyoxyethylene ether, BSA, NaN3, chemical blocking agent and biological blocking agent;

[0017] The second detection reagent includes CKMB sensitized latex particles, Hepes buffer, blocking agent, Tween-20 and NaN3.

[0018] Furthermore, the blocking agent includes glycine, HSA and sucrose.

[0019] Furthermore, the CKMB sensitized latex particles include carboxyl latex particles with a particle size of 185 nm and carboxyl latex particles with a particle size of 359 nm.

[0020] Furthermore, the volume ratio of the 185 nm carboxyl latex particles to the 359 nm carboxyl latex particles is 10:1.

[0021] Further, the chemical blocking agent includes sodium dodecyl sulfate; and / or

[0022] The biological blocking agent includes goat anti-human IgG.

[0023] Further, by mass fraction, the concentration of the Hepes buffer of the first detection reagent is 10-30 mmol / L, the concentration of the NaCl is 10-50 g / L, the concentration of the PEG-6000 is 10-30 g / L, the concentration of the nonylphenol polyoxyethylene ether is 1-10 g / L, the concentration of the chemical blocker is 1-10 g / L, the concentration of the BSA is 1-5 g / L, the concentration of the NaN3 is 0.1% and the concentration of the biological blocker is 0.02%-0.04%; and / or

[0024] The concentration of the Hepes buffer of the second detection reagent is 10-30 mM, the concentration of the CKMB sensitized latex particles is 2-4 g / L, the concentration of the blocking agent is 1%-5%, the concentration of Tween-20 is 0.1%-1% and the concentration of NaN3 is 0.1%.

[0025] Furthermore, the second detection reagent is prepared by the following steps:

[0026] A beaker with a magnetic rotor is placed on a magnetic stirrer, 185 nm latex particles and 359 nm carboxyl latex particles are added in sequence, and the 185 nm latex particles and the 359 nm carboxyl latex particles are activated;

[0027] Add CKMB antibody and react at room temperature for 2-4 hours; add blocking agent and react at room temperature for 1-4 hours;

[0028] Centrifuge at 12000 rpm for 30 min, discard the supernatant, and obtain CKMB-sensitized latex particles;

[0029] The CKMB sensitized latex particles were resuspended, and Hepes, trehalose, and NaN3 were added to obtain the second detection reagent.

[0030] Further, the step of activating the 185nm latex particles and the 359nm carboxyl latex particles comprises:

[0031] Hepes buffer solution, 5-20 g / L NHS and 5-20 g / L EDC were added for activation treatment.

[0032] Furthermore, the activation time is 20 to 40 minutes.

[0033] In order to solve the above technical problems, the present application embodiment also provides an application of the above kit in detecting creatine kinase isoenzymes for non-diagnostic purposes, using the following technical solution:

[0034] Application of the above kit in detecting creatine kinase isoenzymes for non-diagnostic purposes

[0035] Compared with the prior art, the embodiments of the present application have the following beneficial effects:

[0036] The present application optimizes the type and dosage of blocking agents, and the sensitized latex coated with the creatine kinase isoenzyme antibody has good dispersion stability and is not easy to collide, aggregate and precipitate. The ball ratio and process are optimized to improve the linearity and sensitivity of the reagent, and the linearity can reach 0.5 to 300 ng / mL. Improve the ability of the reagent to resist triglycerides. The first detection reagent of the present application uses a chemical blocker (sodium dodecyl sulfate) and a biological blocker in combination to eliminate interference and cross-reactions caused by rheumatoid factors in the specimen for high-concentration endogenous interference and endogenous interference factors of regular content, reduce false positives, and improve the reagent's ability to resist interference from rheumatoid factors. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] In order to more clearly illustrate the scheme in the present application, a brief introduction is given below to the drawings required for use in the description of the embodiments of the present application. Obviously, the drawings described below are some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0038] Figure 1 It is a flow chart of an embodiment of the use of the kit for detecting creatine kinase isoenzymes according to the present application in detecting creatine kinase isoenzymes for non-diagnostic purposes;

[0039] Figure 2 This is a diagram of experimental results according to one embodiment of a kit for detecting creatine kinase isoenzymes of the present application;

[0040] Figure 3 is a diagram of experimental results of another embodiment of a kit for detecting creatine kinase isoenzymes according to the present application;

[0041] Figure 4 is a diagram of experimental results of another embodiment of a kit for detecting creatine kinase isoenzymes according to the present application;

[0042] Figure 5 is a diagram of experimental results of another embodiment of a kit for detecting creatine kinase isoenzymes according to the present application;

[0043] Figure 6 is a diagram of experimental results of another embodiment of a kit for detecting creatine kinase isoenzymes according to the present application;

[0044] Figure 7 This is a diagram of experimental results of another embodiment of the kit for detecting creatine kinase isoenzymes according to the present application. DETAILED DESCRIPTION

[0045] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by technicians in the technical field of the present application; the terms used in the specification of the application herein are only for the purpose of describing specific embodiments and are not intended to limit the present application; the terms "including" and "having" and any variations thereof in the specification and claims of the present application and the above-mentioned drawings are intended to cover non-exclusive inclusions. The terms "first", "second", etc. in the specification and claims of the present application or the above-mentioned drawings are used to distinguish different objects, not to describe a specific order.

[0046] Reference to "embodiments" herein means that a particular feature, structure, or characteristic described in conjunction with the embodiments may be included in at least one embodiment of the present application. The appearance of the phrase in various locations in the specification does not necessarily refer to the same embodiment, nor is it an independent or alternative embodiment that is mutually exclusive with other embodiments. It is explicitly and implicitly understood by those skilled in the art that the embodiments described herein may be combined with other embodiments.

[0047] The following examples are provided to facilitate a better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from conventional biochemical reagent stores unless otherwise specified.

[0048] In order to enable those skilled in the art to better understand the solution of the present application, the technical solution in the embodiments of the present application will be clearly and completely described below in conjunction with the accompanying drawings.

[0049] The present application provides a kit for detecting creatine kinase isoenzymes

[0050] The kit includes a first detection reagent (reagent R1) and a second detection reagent (reagent R2);

[0051] The first detection reagent includes Hepes buffer, NaCl, PEG-6000, nonylphenol polyoxyethylene ether, BSA, NaN3, chemical blocking agent and biological blocking agent;

[0052] The second detection reagent includes CKMB sensitized latex particles, Hepes buffer, blocking agent, Tween-20 and NaN3.

[0053] In this embodiment, the kit for detecting creatine kinase isoenzymes of the present application adopts latex immunoturbidimetric method. It has good specificity, high sensitivity and good stability. The first detection reagent uses nonylphenol polyoxyethylene ether as a fat-removing agent, which can eliminate the interference of fat-turbid samples, improve the ability of the reagent to resist triglycerides, and avoid triglycerides interfering with the detection of the reagent.

[0054] Wherein, by mass fraction, the concentration of the Hepes buffer of the first detection reagent is 10-30 mmol / L, the concentration of the NaCl is 10-50 g / L, the concentration of the PEG-6000 is 10-30 g / L, the concentration of the nonylphenol polyoxyethylene ether is 1-10 g / L, the concentration of the chemical blocker is 1-10 g / L, the concentration of the BSA is 1-5 g / L, the concentration of the NaN3 is 0.1% and the concentration of the biological blocker is 0.02%-0.04%; and / or

[0055] The concentration of the Hepes buffer of the second detection reagent is 10-30 mM, the concentration of the CKMB sensitized latex particles is 2-4 g / L, the concentration of the blocking agent is 1%-5%, the concentration of Tween-20 is 0.1%-1% and the concentration of NaN3 is 0.1%.

[0056] In this embodiment, the concentration of the Hepes buffer of the first detection reagent is 10-30mmol / L, and optionally includes 10-20mmol / L, 20-30mmol / L, for example, 10mmol / L, 15mmol / L, 20mmol / L, 30mmol / L; the concentration of NaCl is 10-50g / L, and optionally includes 10-30g / L, 30-50g / L, for example, 10g / L, 20g / L, 30g / L, 40g / L, 50g / L; the concentration of PEG-6000 is 10-30g / L, and optionally includes 10-20g / L, 20-30g / L, for example, 10g / L, 15g / L, 20g / L, 30g / L; The concentration of polyoxyethylene phenol ether is 1-10g / L, and optionally includes 1-5g / L, 5-10g / L, for example, 1g / L, 5g / L, 7g / L, 10g / L; the concentration of the chemical blocker is 1-10g / L, and optionally includes 1-5g / L, 5-10g / L, for example, 1g / L, 5g / L, 7g / L, 10g / L; the concentration of the BSA is 1-5g / L, and optionally includes 1-3g / L, 3-5g / L, for example, 1g / L, 3g / L, 5g / L; the concentration of the biological blocker is 0.02%-0.04%, and optionally includes 0.02%-0.03%, 0.03%-0.04%, for example, 0.02%, 0.03%, 0.04%.

[0057] The concentration of the Hepes buffer of the second detection reagent is 10-30mM, optionally including 10-20mM, 20-30mM, for example, 10mM, 15mM, 20mM, 30mM; the concentration of the CKMB sensitized latex particles is 2-4g / L, optionally including 2-3g / L, 3-4g / L, for example, 2g / L, 3g / L, 4g / L; the concentration of the blocking agent is 1%-5%, optionally including 1%-2%, 2%-5%, for example, 1%, 2%, 4%, 5%; the concentration of the Tween-20 is 0.1%-1%, optionally including 0.1%-0.5%, 0.5%-1%, for example, 0.1%, 0.5%, 0.7%, 1.0%.

[0058] Specifically, the components and contents of the first detection reagent include: 10mmol / L Hepes buffer, 25g / L NaCl, 15g / L PEG-6000, 5g / L nonylphenol polyoxyethylene ether, 4g / L chemical blocker, 2g / L BSA, 0.1% NaN3 and 0.03% biological blocker; the components and contents of the second detection reagent include: 0.5g / L CKMB sensitized latex particles, 20mmol / L Hepes buffer, 1% blocking agent, 0.5% Tween-20, and 0.1% NaN3.

[0059] As an option in the present application, the chemical blocking agent includes sodium dodecyl sulfate; and / or

[0060] The biological blocking agent includes goat anti-human IgG.

[0061] In this embodiment, sodium dodecyl sulfate is used as a chemical blocking agent to eliminate interference and cross-reaction caused by rheumatoid factors in the specimen, reduce false positives, and enhance the ability of the reagent to resist interference from rheumatoid factors.

[0062] As an option in the present application, the blocking agent of the second detection reagent includes: 60 g / L of glycine, 30 g / L of HSA (human serum albumin) and 100 g / L of sucrose.

[0063] In this embodiment, the present application uses glycine, BSA and sucrose as blocking agents to better block the sites, promote the dispersion stability of the sensitized latex, and significantly improve the real-time stability, accelerated stability and storage stability at room temperature of the test kit. The sensitized latex coated with the creatine kinase isozyme antibody has good dispersion stability and is not easy to collide, aggregate and precipitate. The test kit of the present application can be stored for 14 months at 2-8°C and can also be stored for 12 months at room temperature, with excellent stability.

[0064] As an option in the present application, the CKMB-sensitized latex particles of the second detection reagent include carboxyl latex particles with a particle size of 185 nm and carboxyl latex particles with a particle size of 359 nm.

[0065] In this embodiment, carboxyl latex particles of 185 nm and 359 nm are mixed, and the particle ratio and process are optimized to improve the linearity and sensitivity of the reagent, and the linearity can reach 0.5-300 ng / mL.

[0066] As an option in the present application, the volume ratio of the 185 nm carboxyl latex particles to the 359 nm carboxyl latex particles is 10:1.

[0067] As an option in this application, the first detection reagent is prepared by the following steps:

[0068] Place the beaker containing the magnetic rotor on a magnetic stirrer, and add pure water, Hepes, NaCl, PEG-6000, nonylphenol polyoxyethylene ether, sodium dodecyl sulfate, BSA, and NaN3 in sequence. After fully mixing, adjust the pH with HCL or NaOH, add the blocking agent, make up the volume, and filter.

[0069] As an option in this application, the second detection reagent is prepared by the following steps:

[0070] A beaker with a magnetic rotor is placed on a magnetic stirrer, 185 nm latex particles and 359 nm carboxyl latex particles are added in sequence, and the 185 nm latex particles and the 359 nm carboxyl latex particles are activated;

[0071] Add CKMB antibody and react at room temperature for 2-4 hours; add blocking agent and react at room temperature for 1-4 hours;

[0072] Centrifuge at 12000 rpm for 30 min, discard the supernatant, and obtain CKMB-sensitized latex particles;

[0073] The CKMB-sensitized latex particles were resuspended, and Hepes, Tween-20, and NaN3 were added to obtain the second detection reagent.

[0074] Wherein, the step of activating the 185nm latex particles and the 359nm carboxyl latex particles comprises:

[0075] Hepes buffer solution, 5-20 g / L NHS and 5-20 g / L EDC were added for activation treatment.

[0076] Furthermore, the activation time is 20 to 40 minutes.

[0077] Specifically, the second detection reagent is prepared by the following steps:

[0078] (1) At room temperature, place a beaker containing a magnetic rotor on a magnetic stirrer, add 185 nm latex particles, 359 nm carboxyl latex particles, Hepes buffer solution, NHS 5-20 g / L and EDC 5-20 g / L solution in sequence, and activate for 20-40 min, preferably 30 min.

[0079] (2) After adding CKMB antibody, react at room temperature for 2 to 4 hours;

[0080] (3) Add blocking agent and react at room temperature for 1-4 hours;

[0081] (4) centrifuging the reaction solution obtained in step (3) at 12000 rpm for 30 min, discarding the supernatant, and obtaining CKMB-sensitized latex particles;

[0082] (5) Resuspend the CKMB sensitized latex particles with an ultrasonic cell crusher, and add 20 mM Hepes, 0.5% Tween-20, and 0.1% NaN3 in sequence to obtain the second detection reagent.

[0083] Based on the existing problems of the current creatine kinase isoenzyme detection kit (latex immunoturbidimetry), this application is mainly based on latex immunoturbidimetry to improve:

[0084] (1) The current reagents have poor stability and are prone to precipitation. The second detection reagent of the present application uses glycine, HSA (human serum albumin) and sucrose as blocking agents, which better block the sites, promote the stability of the dispersion of the sensitized latex, and significantly improve the real-time stability, accelerated stability and storage stability at room temperature of the test kit. The present application optimizes the type and dosage of the blocking agent, and the sensitized latex coated with the creatine kinase isozyme antibody has good dispersion stability and is not easy to collide, aggregate and precipitate.

[0085] (2) The current reagents have a narrow linear range and poor sensitivity, generally 3 to 180 ng / mL. The second detection reagent of the present application is a mixture of 185 nm and 359 nm carboxyl latex particles, and the ratio and process of the two carboxyl latex particles are optimized to improve the linearity and sensitivity of the reagent, and obtain a wider linear range of 3 to 300 ng / mL.

[0086] (3) Current reagents are easily interfered by triglycerides. The first detection reagent of the present application uses nonylphenol polyoxyethylene ether as a fat-removing agent to improve the ability of the reagent to resist triglycerides.

[0087] (4) Current reagents are susceptible to interference from rheumatoid factors. The first detection reagent of the present application uses a chemical blocker (sodium dodecyl sulfate) and a biological blocker (sheep anti-human IgG) in combination to eliminate interference and cross-reactions caused by rheumatoid factors in the specimens, reduce false positives, and enhance the reagent's ability to resist interference from rheumatoid factors.

[0088] The present application also provides the use of the above kit in detecting creatine kinase isoenzymes for non-diagnostic purposes, such as Figure 1 As shown, Figure 1 It is a flow chart of an embodiment of the use of the kit for detecting creatine kinase isoenzymes according to the present application in detecting creatine kinase isoenzymes for non-diagnostic purposes;

[0089] The application includes the following steps:

[0090] S1: uniformly mixing the test sample with the first test reagent and the second test reagent to obtain a mixed solution;

[0091] S2: placing the mixed solution in a special detection instrument to obtain absorbance;

[0092] S3: Calculate the concentration of creatine kinase isoenzyme according to the absorbance.

[0093] In this embodiment, the present application detects creatine kinase isoenzymes by latex immunoturbidimetry, thereby achieving rapid and accurate detection of creatine kinase isoenzymes.

[0094] The above scheme is further described below in conjunction with specific implementation examples, but these embodiments are by no means limiting the present application. The preferred embodiments of the present application are described in detail as follows:

[0095] Example 1

[0096] Preparation of the first detection reagent:

[0097] Place the beaker containing the magnetic rotor on a magnetic stirrer, and add pure water, Hepes, NaCl, PEG-6000, nonylphenol polyoxyethylene ether, sodium dodecyl sulfate, BSA, and NaN3 in sequence. After fully mixing, adjust the pH with HCL or NaOH, add the blocking agent, make up the volume, and filter.

[0098] Preparation of the second detection reagent:

[0099] (1) At room temperature, place a beaker containing a magnetic rotor on a magnetic stirrer, add 185 nm latex particles, 359 nm carboxyl latex particles, Hepes buffer solution, 5-20 g / L NHS and 5-20 g / L EDC solution in sequence, and activate for 30 min.

[0100] (2) After adding CKMB antibody, react at room temperature for 2-4 hours;

[0101] (3) Add blocking agent and react at room temperature for 1-4 hours;

[0102] (4) Centrifuge the reaction solution obtained in step (3) at 12000 rpm for 30 min and discard the supernatant;

[0103] (5) Resuspend the latex precipitate with an ultrasonic cell crusher, and add 20 mM HEPES, 0.5% Tween-20, and 0.1% NaN3 in sequence to obtain the second detection reagent.

[0104] Example 2

[0105] The difference between Example 2 and Example 1 is that the fat reducing agent (nonylphenol polyoxyethylene ether) is not added in Example 2, and the other conditions are the same as those in Example 1.

[0106] Example 3

[0107] The difference between Example 3 and Example 1 is that Example 3 does not contain chemical blocking agent (sodium dodecyl sulfate) and biological blocking agent (sheep anti-human IgG), and the other conditions are the same as those of Example 1.

[0108] Example 4

[0109] The difference between Example 4 and Example 1 is that the blocking agent of the second detection reagent in Example 4 is only human serum albumin (HSA), and the other conditions are the same as those in Example 1.

[0110] Example 5

[0111] The difference between Example 5 and Example 1 is that the carboxyl latex particles of the second detection reagent in Example 5 are carboxyl latex microspheres with different particle sizes, and the other conditions are the same as those in Example 1.

[0112] Example 6

[0113] Example 6 includes 6 groups of 185nm and 359nm carboxyl latex particles with different mixing ratios including Example 1.

[0114] This application also provides the experimental results and data analysis of the above embodiments

[0115] 1. Standard curve of Creatine Kinase Isoenzyme Detection Kit

[0116] 1) Experimental methods

[0117] The creatine kinase isoenzyme standard was diluted into 6 gradients, and the concentrations of the calibrators were 0, 5, 10, 75, 150, and 300 ng / mL, respectively. The standard curve of the creatine kinase isoenzymes was output on the Hitachi 7180 instrument. The X-axis represents the theoretical concentration of the calibrator, and the Y-axis represents the absorbance change value △A at the corresponding concentration level.

[0118] 2) Experimental results are as follows Figures 2 to 5 shown. Figure 2 This is a diagram of experimental results according to one embodiment of a kit for detecting creatine kinase isoenzymes of the present application; Figure 3 is a diagram of experimental results of another embodiment of a kit for detecting creatine kinase isoenzymes according to the present application; Figure 4 is a diagram of experimental results of another embodiment of a kit for detecting creatine kinase isoenzymes according to the present application; Figure 5 It is a graph of experimental results of another embodiment of the kit for detecting creatine kinase isoenzymes according to the present application; specifically, it is a graph of standard curve results of embodiments 1-4 of the kit for detecting creatine kinase isoenzymes according to the present application.

[0119] 3) Result analysis

[0120] Depend on Figures 2 to 5It can be seen that the upper limit of the linear measurement range of the kit of the present application in Examples 1-4 can reach 300 ng / mL.

[0121] 2. Evaluation of anti-triglyceride interference

[0122] 1) Experimental methods

[0123] The prepared serum with low interfering substance concentration and serum with high interfering substance concentration were fully mixed in proportion and divided into test samples with five concentration gradients. These interfering substance concentrations were numbered from 1 to 5 in order from low to high concentrations. The numbered interfering samples were tested for indicators in order on the Hitachi HITACHI7180 fully automatic biochemical analyzer, and the average of the three test results was calculated. The average value of the observed indicator in test tube No. 1 was taken as the true value, and the relative deviation between the average value of the three test results of the other tubes and the true value was calculated and analyzed, and this relative deviation was taken as the interference effect obtained in the experiment.

[0124] 2) The experimental results are shown in the following table:

[0125] Table 1 Results of the anti-triglyceride interference evaluation experiment

[0126]

[0127] It can be seen from the above table that the relative deviations of Examples 1, 3, and 4 in terms of anti-triglyceride interference are all within 10%, but the relative deviations of the normal and abnormal value samples of Example 2 exceed 10%. From the results, it can be seen that using nonylphenol polyoxyethylene ether as the R1 fat-removing agent improves the ability of the reagent to resist triglyceride interference.

[0128] 3. Evaluation of anti-rheumatoid factor interference

[0129] 1) Experimental methods

[0130] The prepared serum with low interfering substance concentration and serum with high interfering substance concentration were fully mixed in proportion and divided into test samples with five concentration gradients. These interfering substance concentrations were numbered from 1 to 5 in order from low to high concentrations. The numbered interfering samples were tested for indicators in order on the Hitachi HITACHI7180 fully automatic biochemical analyzer, and the average of the three test results was calculated. The average value of the observed indicator in test tube No. 1 was taken as the true value, and the relative deviation between the average value of the three test results of the other tubes and the true value was calculated and analyzed, and this relative deviation was taken as the interference effect obtained in the experiment.

[0131] 2) The experimental results are shown in the following table:

[0132] Table 2 Results of the anti-rheumatoid factor interference evaluation experiment

[0133]

[0134] The relative deviations of the anti-rheumatoid factor interference in Example 1, Example 2, and Example 4 were all within 10%, but the relative deviations of the normal value and abnormal value samples in Example 3 exceeded 10%. From the results, it can be seen that the chemical blocker (sodium dodecyl sulfate) and biological blocker (sheep anti-human IgG) added in R1 of the present application, respectively, target high-concentration endogenous interference and regular-content endogenous interference factors, and significantly enhance the ability of the reagent to resist rheumatoid factor interference.

[0135] 4. Stability evaluation

[0136] 1) Real-time stability

[0137] The kits of Examples 1, 2, 3, and 4 and their matching calibrators and quality control products were stored at 2°C to 8°C. When stored at the 0th month, the 2nd month, the 4th month, the 8th month, and the 14th month, the blank absorbance of the reagents and the quality control products were measured and analyzed. The experimental results are shown in the following table:

[0138] Table 3 Stability evaluation test results

[0139]

[0140]

[0141] The present application conducted a 14-month real-time stability research experiment. The experimental results show that the blank absorbance, quality control product 1 and quality control product 2 of Examples 1-3 are stored at 2°C to 8°C for 14 months with relative deviations within 10%, while the sealing agent of Example 4 is HSA, which does not make the reagent disperse and stabilize well. The relative deviations of quality control product 1, quality control product 2 and blank absorbance in the fourth month are all over 10%. The creatine kinase isoenzyme assay kit of the present application has good stability.

[0142] 2) Acceleration stability

[0143] The reagent kits of Examples 1, 2, 3, and 4 were placed in a 37±5°C thermostat, and the blank absorbance of the reagents and the quality control products were measured and analyzed on the 0th, 1st, 3rd, 5th, and 7th days. The experimental results are shown in the following table:

[0144] Table 4 Accelerated stability test results

[0145]

[0146]

[0147]

[0148] The present application conducted accelerated stability experiments on Examples 1-4 on days 0, 1, 3, 5 and 7. The experimental results show that the blank absorbance, control product 1 and control product 2 of Examples 1-3 were all within 10% relative deviation within 7 days of storage at 37±5°C, while the sealing agent of Example 4 was HSA, which did not make the reagents disperse and stabilize well, and the relative deviations of control products 1 and 2 were within 10% within the 7th day, but the blank absorbance of the reagents gradually increased within 7 days, and the CKMB-sensitized latex aggregated. The creatine kinase isoenzyme assay kit of the present application has good stability.

[0149] 3) Stability at room temperature

[0150] The kits of Examples 1, 2, 3, and 4 and their matching calibrators and quality control products were stored at room temperature. When stored for 0 months, 2 months, 4 months, 8 months, and 12 months, the blank absorbance of the reagents and the quality control products were measured and analyzed. The experimental results are shown in the following table:

[0151] Table 5 Stability test results under normal temperature conditions

[0152]

[0153]

[0154] The present application conducted a 12-month stability research experiment under normal temperature conditions. The experimental results show that the blank absorbance, control product 1 and control product 2 of Examples 1-3 are all within 10% relative deviation when stored at 2°C to 8°C for 12 months, while the blocking agent of Example 4 is HSA, which does not allow the reagent to have a good dispersion and stabilization effect. The relative deviation of control product 1 in the fourth month exceeds 10%. The present application uses glycine, human serum albumin (HSA) and sucrose as blocking agents in combination, so that the creatine kinase isoenzyme assay kit has good stability.

[0155] 5. Screening of carboxyl latex particles with different particle sizes

[0156] The 10 different carboxyl latex particles of Example 5 were prepared as the second detection reagent, and were analyzed with the same first detection reagent and its matching calibrator. The experimental results are shown in the following table and Figure 6 As shown, Figure 6 This is a diagram of experimental results of another embodiment of the kit for detecting creatine kinase isoenzymes according to the present application.

[0157] Table 6 Screening results of carboxyl latex particles with different particle sizes

[0158]

[0159] The present application conducted a screening test of 10 carboxyl latex particles with different particle sizes. The experimental results show that the reagent R2 prepared from 141nm, 185nm, 201nm, 302nm and 359nm carboxyl latex particles can specifically bind to and show different affinities respectively. As can be seen from the figure, the smaller particle sizes of 141nm, 185nm and 201nm have a wider measurement range, among which the 185nm carboxyl latex particles have the best linear measurement range; the larger particle sizes of 302nm and 359nm have higher sensitivity, among which the 359nm carboxyl latex particles have the highest sensitivity.

[0160] 6. Exploration of the mixing ratio of 185nm and 359nm carboxyl latex particles

[0161] From the screening of 6 groups of carboxyl latex particles with different particle sizes in Example 6, it can be seen that the linear measurement range of 185nm carboxyl latex particles is good, and the sensitivity of 359nm carboxyl latex particles is high. However, the second detection reagent prepared by 185nm and 359nm carboxyl latex particles alone cannot take into account both the measurement range and sensitivity, so the two need to be mixed to obtain the second detection reagent with the best measurement range and sensitivity. The results are shown in the following table and Figure 7 As shown, Figure 7 This is a diagram of experimental results of another embodiment of the kit for detecting creatine kinase isoenzymes according to the present application.

[0162] Table 7 Exploration of the mixing ratio of 185nm and 359nm carboxyl latex particles (185nm: 359nm)

[0163]

[0164] The present application has conducted 6 kinds of 185nm and 359nm carboxyl latex particle mixing ratio exploration tests. The experimental results show that when the ratio of 185nm:359nm is 1:1, 1:2, and 1:4, as the concentration of the calibration substance increases, the reactivity increases, the correlation coefficient gradually decreases, the reaction graph is convex, the zero-point reactivity △A is all>0.0100, and the linear measurement range is not ideal; when the ratio of 185nm:359nm is 5:1, 10:1, and 15:1, the reactivity increases, and the reaction graph gradually changes from convex to straight line, among which the reactivity of the ratio 15:1 is concave, and the low-value sensitivity is not as good as the ratio 5:1 and 10:1. The linear measurement range and sensitivity of the ratio 5:1 and 10:1 meet the requirements, but the zero-point reactivity △A of the ratio 5:1 is 0.0486>0.0100, so the ratio 10:1 of Example 1 is selected as the mixing ratio of 185nm and 359nm carboxyl latex particles.

[0165] It should be understood that, although the steps in the flowchart of the accompanying drawings are displayed in sequence as indicated by the arrows, these steps are not necessarily executed in sequence in the order indicated by the arrows. Unless otherwise specified herein, there is no strict order restriction on the execution of these steps, and they can be executed in other orders. Moreover, at least a part of the steps in the flowchart of the accompanying drawings may include multiple sub-steps or multiple stages, and these sub-steps or stages are not necessarily executed at the same time, but can be executed at different times, and their execution order is not necessarily sequential, but can be executed in turn or alternately with other steps or at least a part of the sub-steps or stages of other steps.

[0166] Obviously, the embodiments described above are only some embodiments of the present application, rather than all embodiments. The preferred embodiments of the present application are given in the accompanying drawings, but they do not limit the patent scope of the present application. The present application can be implemented in many different forms. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present application more thorough and comprehensive. Although the present application is described in detail with reference to the aforementioned embodiments, for those skilled in the art, it is still possible to modify the technical solutions recorded in the aforementioned specific implementation methods, or to perform equivalent replacement of some of the technical features therein. Any equivalent structure made using the contents of the specification and drawings of this application, directly or indirectly used in other related technical fields, is similarly within the scope of patent protection of this application.

Claims

1. A kit for detecting creatine kinase isoenzymes, characterized in that: include: a first detection reagent and a second detection reagent; The first detection reagent includes Hepes buffer, NaCl, PEG-6000, nonylphenol polyoxyethylene ether, BSA, NaN3, chemical blocking agent and biological blocking agent; The second detection reagent includes CKMB sensitized latex particles, Hepes buffer, blocking agent, Tween-20 and NaN3.

2. The kit for detecting creatine kinase isoenzymes according to claim 1, characterized in that: The blocking agents include glycine, HSA and sucrose.

3. The kit for detecting creatine kinase isoenzymes according to claim 1, characterized in that: The CKMB sensitized latex particles include carboxyl latex particles with a particle size of 185 nm and carboxyl latex particles with a particle size of 359 nm.

4. The kit for detecting creatine kinase isoenzymes according to claim 3, characterized in that: The volume ratio of the 185 nm carboxyl latex particles to the 359 nm carboxyl latex particles is 10:

1.

5. The kit for detecting creatine kinase isoenzymes according to claim 1, characterized in that: The chemical blocking agent comprises sodium lauryl sulfate; and / or The biological blocking agent includes goat anti-human IgG.

6. The kit for detecting creatine kinase isoenzymes according to claim 1, characterized in that: In terms of mass fraction, the concentration of the Hepes buffer of the first detection reagent is 10-30 mmol / L, the concentration of the NaCl is 10-50 g / L, the concentration of the PEG-6000 is 10-30 g / L, the concentration of the nonylphenol polyoxyethylene ether is 1-10 g / L, the concentration of the chemical blocker is 1-10 g / L, the concentration of the BSA is 1-5 g / L, the concentration of the NaN3 is 0.1% and the concentration of the biological blocker is 0.02%-0.04%; and / or The concentration of the Hepes buffer of the second detection reagent is 10-30 mM, the concentration of the CKMB sensitized latex particles is 2-4 g / L, the concentration of the blocking agent is 1%-5%, the concentration of Tween-20 is 0.1%-1% and the concentration of NaN3 is 0.1%.

7. The kit for detecting creatine kinase isoenzymes according to claim 1, characterized in that: The second detection reagent is prepared by the following steps: A beaker with a magnetic rotor is placed on a magnetic stirrer, 185 nm latex particles and 359 nm carboxyl latex particles are added in sequence, and the 185 nm latex particles and the 359 nm carboxyl latex particles are activated; Add CKMB antibody and react at room temperature for 2-4 hours; add blocking agent and react at room temperature for 1-4 hours; Centrifuge at 12000 rpm for 30 min, discard the supernatant, and obtain CKMB-sensitized latex particles; The CKMB-sensitized latex particles were resuspended, and Hepes, Tween-20, and NaN3 were added to obtain the second detection reagent.

8. The kit for detecting creatine kinase isoenzymes according to claim 1, characterized in that: The step of activating the 185nm latex particles and the 359nm carboxyl latex particles comprises: Hepes buffer solution, 5-20 g / L NHS and 5-20 g / L EDC were added for activation treatment.

9. The kit for detecting creatine kinase isoenzymes according to claim 8, characterized in that: The activation time is 20 to 40 minutes.

10. Use of the kit according to claim 9 in detecting creatine kinase isoenzymes for non-diagnostic purposes.

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