Kit for detecting creatine kinase isozyme and application

Abstract

This application belongs to the field of detection technology and relates to a kit for detecting creatine kinase isoenzymes and its application, including a first detection reagent and a second detection reagent; the first detection reagent includes Hepes buffer, NaCl, PEG-6000, nonylphenol polyoxyethylene ether, BSA, NaN3, a chemical blocking agent, and a biological blocking agent; the second detection reagent includes CKMB sensitized latex particles, Hepes buffer, a blocking agent, Tween-20, and NaN3. This application effectively improves the detection sensitivity of creatine kinase isoenzymes.

Description

Technical Field

[0001] This application relates to the field of detection technology, and in particular to kits and applications for detecting creatine kinase isoenzymes. Background Technology

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

[0003] CK is a heterodimer with M and B subunits, mainly composed of three isoenzymes: CK-BB, CK-MM, and CK-MB. Mitochondria also contain another isoenzyme, CK-Mt. CK-BB is primarily 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. In normal individuals, serum CK-MB levels are less than 5% of total activity. During acute myocardial infarction followed by chest pain, serum CK-MB levels rise after 4 hours, peak at 24 hours, and recover within 3-4 days. CK-MB is an important biomarker for myocardial injury.

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

[0005] Enzyme rate immunosuppression assays are susceptible to multiple factors and have a high false-positive rate. They use a monoclonal antibody targeting CK-M to inhibit the activity of the CK-M subunit in the sample, without affecting the activity of the CK-B subunit. The remaining CK-MB and the activity of the B subunit in CK-BB are detected through an enzymatic reaction. When cells in tissues such as the brain, prostate, gastrointestinal tract, and lungs are damaged, ischemic, or necrotic, CK-BB is released into the serum, easily causing a false increase in CK-MB. Simultaneously, the presence of macro-CK (an oligomeric mitochondrial CK) released by tumor patients and individuals with immune disorders also participates in the enzymatic reaction. Macro-CK is not inhibited by anti-CK-M subunit antibodies, and its activity is 100% detectable, which can exponentially amplify the CK-MB detection result, leading to a false increase in CK-MB.

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

[0007] Chemiluminescent immunoassay is based on the double-antibody sandwich method. Specific mouse monoclonal antibodies are pre-coated onto a carrier, and the sample to be tested and enzyme-labeled specific monoclonal antibodies are added, forming an immune complex of specific antibody-antigen-enzyme-labeled antibody. Upon excitation, the complex emits photons, and the mass number of CK-MB in the sample is determined based on the magnitude of the excitation light. This method is highly specific but expensive.

[0008] Latex immunoturbidimetry is a relatively stable and accurate homogeneous immunoturbidimetric method for detecting body fluid proteins. When CKMB binds to CKMB-conjugated antibody microspheres, they rapidly aggregate, altering the absorbance of the reaction solution. This alteration exhibits a linear relationship within a certain range, allowing the calculation of the CKMB concentration.

[0009] Creatine kinase isoenzymes in the sample bind to the sensitized latex containing anti-creatine kinase isoenzyme antibodies in the reagent, forming an insoluble immune complex. This complex generates turbidity in a specific buffer environment. Within the detection range, the turbidity is proportional to the analyte content. By comparing this turbidity with that of a calibrator operated under the same conditions, the content of creatine kinase isoenzymes in the sample can be determined.

[0010] The current creatine kinase isoenzyme detection kits have the following problems: (1) poor reagent stability and easy precipitation; (2) narrow linear range and poor sensitivity, generally 3-180 ng / mL; (3) the reagent is easily affected by triglycerides; (4) the reagent is easily affected by rheumatoid factor.

[0011] In summary, current creatine kinase isoenzyme detection kits have poor sensitivity for detecting creatine kinase isoenzymes. Summary of the Invention

[0012] The purpose of this application is to provide a kit for detecting creatine kinase isoenzymes and its application, which effectively improves the detection sensitivity of creatine kinase isoenzymes.

[0013] To address the aforementioned technical problems, this application provides a kit for detecting creatine kinase isoenzymes, employing the following technical solution:

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

[0015] First detection reagent and second detection reagent;

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

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

[0018] Furthermore, the sealing 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 185nm carboxyl latex particles to the 359nm carboxyl latex particles is 10:1.

[0021] Furthermore, 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 in 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 blocking agent 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 blocking agent is 0.02%–0.04%; and / or

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

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

[0026] Place a beaker containing a magnetic rotor on a magnetic stirrer, and add 185nm latex particles and 359nm carboxyl latex particles in sequence to activate the 185nm latex particles and 359nm carboxyl latex particles.

[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] Furthermore, the step of activating the 185nm latex particles and the 359nm carboxyl latex particles includes:

[0031] Activation treatment was performed by adding Hepes buffer solution, 5-20 g / L NHS and 5-20 g / L EDC.

[0032] Furthermore, the activation time is 20–40 min.

[0033] To address the aforementioned technical problems, this application also provides an application of the above-mentioned kit in the detection of creatine kinase isoenzymes for non-diagnostic purposes, employing the following technical solution:

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

[0035] Compared with the prior art, the embodiments of this application have the following main advantages:

[0036] This application optimizes the type and dosage of the blocking agent, resulting in a sensitized latex coated with creatine kinase isoenzyme antibodies with good dispersion stability and less susceptibility to collision, aggregation, and precipitation. The sphere ratio and manufacturing process have been optimized to improve the linearity and sensitivity of the reagent, achieving a linearity of 0.5–300 ng / mL. The reagent's ability to resist triglycerides has also been enhanced. The first detection reagent in this application uses a combination of a chemical blocking agent (sodium dodecyl sulfate) and a biological blocking agent to target high-concentration and conventional levels of endogenous interference, eliminating interference and cross-reactions caused by rheumatoid factor and other factors in the sample, reducing false positives, and improving the reagent's ability to resist rheumatoid factor interference. Attached Figure Description

[0037] To more clearly illustrate the solutions in this application, the accompanying drawings used in the description of the embodiments of this application will be briefly introduced below. Obviously, the accompanying drawings described below are some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0038] Figure 1 This is a flowchart of an embodiment of the application of the kit for detecting creatine kinase isoenzymes according to this application in the detection of creatine kinase isoenzymes for non-diagnostic purposes;

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

[0040] Figure 3 This is a graph showing experimental results from another embodiment of the kit for detecting creatine kinase isoenzymes according to this application;

[0041] Figure 4 This is a graph showing experimental results from another embodiment of the kit for detecting creatine kinase isoenzymes according to this application;

[0042] Figure 5 This is a graph showing experimental results from another embodiment of the kit for detecting creatine kinase isoenzymes according to this application;

[0043] Figure 6 This is a graph showing experimental results from another embodiment of the kit for detecting creatine kinase isoenzymes according to this application;

[0044] Figure 7 This is a graph showing experimental results from another embodiment of the kit for detecting creatine kinase isoenzymes according to this application. Detailed Implementation

[0045] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains; the terminology used herein in the specification of the application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application; the terms "comprising" and "having," and any variations thereof, in the specification, claims, and foregoing drawings of this application, are intended to cover non-exclusive inclusion. The terms "first," "second," etc., in the specification, claims, or foregoing drawings of this application are used to distinguish different objects, not to describe a particular order.

[0046] In this document, the term "embodiment" means that a particular feature, structure, or characteristic described in connection with an embodiment may be included in at least one embodiment of this application. The appearance of this phrase in various places throughout the specification does not necessarily refer to the same embodiment, nor is it a separate or alternative embodiment mutually exclusive with other embodiments. It will be explicitly and implicitly understood by those skilled in the art that the embodiments described herein can be combined with other embodiments.

[0047] The following embodiments are provided to facilitate a better understanding of this application, but do not limit the scope of this application. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments were purchased from conventional biochemical reagent stores.

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

[0049] This 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 agents, and biological blocking agents;

[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 this application employs a latex immunoturbidimetric method. This method offers good specificity, high sensitivity, and good stability. The first detection reagent uses nonylphenol polyoxyethylene ether as a lipid-reducing agent, which eliminates interference from lipid-turbid samples, improves the reagent's resistance to triglycerides, and prevents triglycerides from interfering with the reagent's detection.

[0054] Wherein, by mass fraction, the concentration of the Hepes buffer in 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 blocking agent 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 blocking agent is 0.02%–0.04%; and / or

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

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

[0057] The concentration of the Hepes buffer in the second detection reagent is 10–30 mM, optionally including 10–20 mM, 20–30 mM, for example, 10 mM, 15 mM, 20 mM, 30 mM; the concentration of the CKMB sensitized latex particles is 2–4 g / L, optionally including 2–3 g / L, 3–4 g / L, for example, 2 g / L, 3 g / L, 4 g / 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 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: 10 mmol / L Hepes buffer, 25 g / L NaCl, 15 g / L PEG-6000, 5 g / L nonylphenol polyoxyethylene ether, 4 g / L chemical blocking agent, 2 g / L BSA, 0.1% NaN3, and 0.03% biological blocking agent; the components and contents of the second detection reagent include: 0.5 g / L CKMB sensitized latex particles, 20 mmol / L Hepes buffer, 1% blocking agent, 0.5% Tween-20, and 0.1% NaN3.

[0059] Optionally, the chemical blocking agent may include sodium dodecyl sulfate; and / or

[0060] The biological blocking agent includes sheep 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 factor and other factors in the sample, reduce false positives, and enhance the reagent's ability to resist interference from rheumatoid factor.

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

[0063] In this embodiment, glycine, BSA, and sucrose are used as blocking agents to better block the sites, promoting stable dispersion of the sensitized latex and significantly improving the real-time stability, accelerated stability, and room-temperature storage stability of the kit. This results in good dispersion stability of the sensitized latex coated with creatine kinase isoenzyme antibody, making it less prone to collision, aggregation, and precipitation. The kit can be stored at 2-8°C for 14 months and at room temperature for 12 months, demonstrating excellent stability.

[0064] As an option in this 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, 185nm and 359nm carboxyl latex particles were mixed, and the ball ratio and process were optimized to improve the linearity and sensitivity of the reagent, with linearity reaching 0.5–300 ng / mL.

[0066] Optionally, the volume ratio of the 185nm carboxyl latex particles to the 359nm carboxyl latex particles is 10:1.

[0067] Optionally, the first detection reagent may be 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 mixing thoroughly, adjust the pH with HCl or NaOH, add the blocking agent, bring the volume to a final volume, and filter.

[0069] Optionally, the second detection reagent may be prepared by the following steps:

[0070] Place a beaker containing a magnetic rotor on a magnetic stirrer, and add 185nm latex particles and 359nm carboxyl latex particles in sequence to activate the 185nm latex particles and 359nm carboxyl latex particles.

[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] The step of activating the 185nm latex particles and the 359nm carboxyl latex particles includes:

[0075] Activation treatment was performed by adding Hepes buffer solution, 5-20 g / L NHS and 5-20 g / L EDC.

[0076] Furthermore, the activation time is 20–40 min.

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

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

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

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

[0081] (4) Centrifuge the reaction solution obtained in step (3) at 12000 rpm for 30 min, discard the supernatant, and obtain CKMB sensitized latex particles;

[0082] (5) The CKMB sensitized latex particles were resuspended in an ultrasonic cell disruptor, and 20 mM Hepes, 0.5% Tween-20 and 0.1% NaN3 were added in sequence to obtain the second detection reagent.

[0083] Based on the existing problems of current creatine kinase isoenzyme detection kits (latex immunoturbidimetric assay), this application mainly improves upon the latex immunoturbidimetric assay:

[0084] (1) Current reagents suffer from poor stability and are prone to precipitation. The second detection reagent in this application uses glycine, HSA (human serum albumin), and sucrose as blocking agents, which better blocks the sites, promotes stable dispersion of the sensitized latex, and significantly improves the real-time stability, accelerated stability, and room-temperature storage stability of the kit. This application optimizes the type and amount of blocking agent, resulting in good dispersion stability of the sensitized latex coated with creatine kinase isoenzyme antibody, making it less prone to collision, aggregation, and precipitation.

[0085] (2) Current reagents have a narrow linear range and poor sensitivity, generally ranging from 3 to 180 ng / mL. The second detection reagent of this application is made by mixing 185 nm and 359 nm carboxyl latex particles, and the ratio of the two carboxyl latex particles and the process are optimized to improve the linearity and sensitivity of the reagent, resulting in a wider linear range of 3 to 300 ng / mL.

[0086] (3) Current reagents are susceptible to interference from triglycerides. The first detection reagent in this application uses nonylphenol polyoxyethylene ether as a degreasing agent to improve the reagent's ability to resist triglycerides.

[0087] (4) Current reagents are susceptible to interference from rheumatoid factor. The first detection reagent of this application uses a combination of chemical blocking agent (sodium dodecyl sulfate) and biological blocking agent (goat anti-human IgG) to target high-concentration endogenous interference and conventional endogenous interference factors, eliminate interference and cross-reaction caused by rheumatoid factor in the sample, reduce false positives, and improve the reagent's ability to resist rheumatoid factor interference.

[0088] This application also provides the application of the above-mentioned kit in the detection of creatine kinase isoenzymes for non-diagnostic purposes, such as... Figure 1 As shown, Figure 1 This is a flowchart of an embodiment of the application of the kit for detecting creatine kinase isoenzymes according to this application in the detection of creatine kinase isoenzymes for non-diagnostic purposes;

[0089] The application includes the following steps:

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

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

[0092] S3: Calculate the concentration of creatine kinase isoenzyme based on the absorbance.

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

[0094] The above solution will be further described below with reference to specific embodiments, but these embodiments are by no means intended to limit this application. The preferred embodiments of this application are described in detail below:

[0095] Example 1

[0096] Preparation of the first test 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 mixing thoroughly, adjust the pH with HCl or NaOH, add the blocking agent, bring the volume to a final volume, and filter.

[0098] Preparation of the second test reagent:

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

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

[0101] (3) Add a 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) The latex precipitate was resuspended in an ultrasonic cell disruptor, and 20 mM HEPES, Tween-20 0.5% and NaN 30.1% were added in sequence to obtain the second detection reagent.

[0104] Example 2

[0105] The difference between Example 2 and Example 1 is that no degreasing agent (nonylphenol polyoxyethylene ether) is added in Example 2, while all other conditions are the same as in Example 1.

[0106] Example 3

[0107] The difference between Example 3 and Example 1 is that Example 3 does not contain a chemical blocker (sodium dodecyl sulfate) and a biological blocker (goat anti-human IgG), while all other conditions are the same as those in Example 1.

[0108] Example 4

[0109] The difference between Example 4 and Example 1 is that the blocking agent of the second test reagent in Example 4 is only human serum albumin (HSA), while 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 test reagent in Example 5 are carboxyl latex microspheres of different particle sizes, while the other conditions are the same as those in Example 1.

[0112] Example 6

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

[0114] This application also provides 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 to six gradients, with calibrator concentrations of 0, 5, 10, 75, 150, and 300 ng / mL. The standard curve of the creatine kinase isoenzyme 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 ΔA at the corresponding concentration level.

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

[0119] 3) Results Analysis

[0120] Depend on Figures 2 to 5It is known that the upper limit of the linear measurement range of the kits of this 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 samples with low and high concentrations of interfering substances were thoroughly mixed in a specific ratio and divided into five test samples with varying concentrations. These interfering substance concentrations were numbered 1 to 5 in ascending order of concentration. The numbered samples were then analyzed sequentially using a Hitachi 7180 fully automated biochemical analyzer, and the average of three test results was calculated. The average value observed in tube 1 was taken as the true value. The relative deviation between the average value and the true value of the three test results in each of the other tubes was calculated and analyzed. This relative deviation was used as the experimentally determined interference effect.

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

[0125] Table 1. Experimental results evaluating anti-triglyceride interference.

[0126]

[0127] As can be seen from the table above, the relative deviations of the anti-triglyceride interference in Examples 1, 3, and 4 are all within 10%, but the relative deviations of the normal and abnormal value samples in Example 2 exceed 10%. The results show that using nonylphenol polyoxyethylene ether as R1 lipase enhances the reagent's ability to resist triglyceride interference.

[0128] 3. Evaluation of resistance to rheumatoid factor interference

[0129] 1) Experimental methods

[0130] The prepared serum samples with low and high concentrations of interfering substances were thoroughly mixed in a specific ratio and divided into five test samples with varying concentrations. These interfering substance concentrations were numbered 1 to 5 in ascending order of concentration. The numbered samples were then analyzed sequentially using a Hitachi 7180 fully automated biochemical analyzer, and the average of three test results was calculated. The average value observed in tube 1 was taken as the true value. The relative deviation between the average value and the true value of the three test results in each of the other tubes was calculated and analyzed. This relative deviation was used as the experimentally determined interference effect.

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

[0132] Table 2. Experimental results of anti-rheumatoid factor interference evaluation

[0133]

[0134] The relative deviations of the anti-rheumatoid factor interference in Examples 1, 2, and 4 were all within 10%, but the relative deviations of the normal and abnormal value samples in Example 3 exceeded 10%. The results show that the chemical blocker (sodium dodecyl sulfate) and biological blocker (sheep anti-human IgG) added in R1 of this application significantly enhanced the reagent's ability to resist rheumatoid factor interference, targeting high concentrations of endogenous interference and conventional levels of endogenous interference factors, respectively.

[0135] 4. Stability Evaluation

[0136] 1) Real-time stability

[0137] The reagent kits and their accompanying calibrators and quality control samples from Examples 1, 2, 3, and 4 were stored at 2℃–8℃. The blank absorbance of the reagents and the quality control samples were measured and analyzed at 0, 2, 4, 8, and 14 months of storage. The experimental results are shown in the table below:

[0138] Table 3. Experimental Results for Stability Evaluation

[0139]

[0140]

[0141] This application underwent a 14-month real-time stability study. The results showed that, after 14 months of storage at 2℃~8℃, the relative deviations of the blank absorbance, quality control 1, and quality control 2 in Examples 1-3 were all within 10%. However, in Example 4, the blocking agent was HSA, which did not effectively disperse and stabilize the reagents; the relative deviations of quality control 1, quality control 2, and the blank absorbance all exceeded 10% in the fourth month. Therefore, the creatine kinase isoenzyme assay kit of this application exhibits good stability.

[0142] 2) Accelerate stability

[0143] The reagent kits from Examples 1, 2, 3, and 4 were placed in a 37±5℃ incubator. The blank absorbance and quality control samples were measured and analyzed on days 0, 1, 3, 5, and 7. The experimental results are shown in the table below:

[0144] Table 4. Results of accelerated stability experiments

[0145]

[0146]

[0147]

[0148] This application conducted accelerated stability tests on Examples 1-4 at days 0, 1, 3, 5, and 7. The results showed that the blank absorbance and relative deviations of control 1 and control 2 of Examples 1-3 stored at 37±5℃ for 7 days were all within 10%. However, in Example 4, the blocking agent was HSA, which did not effectively disperse and stabilize the reagents. The relative deviations of control 1 and control 2 were within 10% on day 7, but the blank absorbance gradually increased over 7 days, indicating aggregation of the CKMB-sensitized latex. Therefore, the creatine kinase isoenzyme assay kit of this application exhibits good stability.

[0149] 3) Stability under normal temperature conditions

[0150] The reagent kits and their accompanying calibrators and quality control samples from Examples 1, 2, 3, and 4 were stored at room temperature. The blank absorbance of the reagents and the quality control samples were measured and analyzed at 0, 2, 4, 8, and 12 months of storage. The experimental results are shown in the table below:

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

[0152]

[0153]

[0154] This application conducted a 12-month stability study under room temperature conditions. The experimental results showed that the relative deviations of blank absorbance, quality control 1, and quality control 2 in Examples 1-3 stored at 2℃~8℃ for 12 months were all within 10%. However, in Example 4, the blocking agent was HSA, which did not provide good dispersion and stability of the reagents. The relative deviation of quality control 1 exceeded 10% in the fourth month. This application uses a combination of glycine, human serum albumin (HSA), and sucrose as blocking agents to give the creatine kinase isoenzyme assay kit good stability.

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

[0156] The 10 different carboxyl latex particles of the same particle size from Example 5 were used to prepare a second detection reagent, which was then analyzed in conjunction with the same first detection reagent and its matching calibrators. The experimental results are shown in the table below. Figure 6 As shown, Figure 6 This is a graph showing experimental results from another embodiment of the kit for detecting creatine kinase isoenzymes according to this application.

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

[0158]

[0159] This application conducted screening tests on carboxyl latex particles of 10 different sizes. The experimental results showed that reagent R2 prepared from 141nm, 185nm, 201nm, 302nm, and 359nm carboxyl latex particles specifically bound to the latex, exhibiting different affinities. As shown in the figure, the smaller particle sizes of 141nm, 185nm, and 201nm had wider measurement ranges, with 185nm carboxyl latex particles showing the best linear measurement range. The larger particle sizes of 302nm and 359nm exhibited higher sensitivity, with 359nm carboxyl latex particles showing the highest sensitivity.

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

[0161] Screening of six groups of carboxylated latex particles with different particle sizes in Exercise 6 revealed that 185nm carboxylated latex particles exhibited good linearity and 359nm carboxylated latex particles showed high sensitivity. However, the second detection reagent prepared solely from 185nm and 359nm carboxylated latex particles could not simultaneously achieve both measurement range and sensitivity. Therefore, a mixture of the two was necessary to obtain the second detection reagent with the optimal measurement range and sensitivity. The results are shown in the table below. Figure 7 As shown, Figure 7 This is a graph showing experimental results from another embodiment of the kit for detecting creatine kinase isoenzymes according to this application.

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

[0163]

[0164] This application conducted exploratory experiments on the mixing ratios of 185nm and 359nm carboxylated latex particles in six different proportions. The experimental results showed that when the 185nm:359nm ratio was 1:1, 1:2, and 1:4, the reactivity increased with increasing calibrator concentration, the correlation coefficient gradually decreased, and the reaction graph became convex. The zero-point reactivity ΔA was >0.0100, and the linear measurement range did not reach the ideal level. When the 185nm:359nm ratio was 5:1, 10:1, and 15:1, the reactivity increased, and the reaction graph gradually changed from convex to linear. The 15:1 ratio showed a concave reactivity, and the low-value sensitivity was not as good as the 5:1 and 10:1 ratios. The linear measurement range and sensitivity of the 5:1 and 10:1 ratios met the requirements; however, the zero-point reactivity ΔA of the 5:1 ratio was 0.0486 >0.0100. Therefore, the 10:1 ratio from Example 1 was selected as the mixing ratio for 185nm and 359nm carboxylated latex particles.

[0165] It should be understood that although the steps in the flowcharts of the accompanying figures are shown sequentially as indicated by the arrows, these steps are not necessarily executed in the order indicated by the arrows. Unless explicitly stated herein, there is no strict order restriction on the execution of these steps, and they can be executed in other orders. Moreover, at least some steps in the flowcharts of the accompanying figures may include multiple sub-steps or multiple stages. These sub-steps or stages are not necessarily completed at the same time, but can be executed at different times, and their execution order is not necessarily sequential, but can be performed alternately or in turn with other steps or at least some of the sub-steps or stages of other steps.

[0166] Obviously, the embodiments described above are only some embodiments of this application, not all embodiments. The accompanying drawings show preferred embodiments of this application, but do not limit the patent scope of this application. This application can be implemented in many different forms; rather, the purpose of providing these embodiments is to provide a more thorough and comprehensive understanding of the disclosure of this application. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing specific embodiments, or make equivalent substitutions for some of the technical features. Any equivalent structures made using the content of this application's specification and drawings, directly or indirectly applied to other related technical fields, are similarly within the scope of patent protection of this application.

Claims

1. A kit for detecting creatine kinase isoenzymes, characterized in that, include: First detection reagent and second detection reagent; The first detection reagent consists of Hepes buffer, NaCl, PEG-6000, nonylphenol polyoxyethylene ether, BSA, NaN3, a chemical blocking agent, and a biological blocking agent; the chemical blocking agent is sodium dodecyl sulfate, and the biological blocking agent is goat anti-human IgG; The second detection reagent consists of CKMB sensitized latex particles, Hepes buffer, Tween-20, and NaN3; wherein the CKMB sensitized latex particles are treated with a blocking agent, and the CKMB sensitized latex particles include carboxylated latex particles with a particle size of 185 nm and carboxylated latex particles with a particle size of 359 nm, with a volume ratio of 10:1 for the 185 nm carboxylated latex particles and the 359 nm carboxylated latex particles; the blocking agent consists of glycine, HSA, and sucrose.

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

3. The kit for detecting creatine kinase isoenzymes according to claim 1, characterized in that, The second detection reagent is prepared by the following steps: Place a beaker containing a magnetic rotor on a magnetic stirrer, and add 185nm carboxylate particles and 359nm carboxylate particles in sequence to activate the 185nm and 359nm carboxylate particles. 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.

4. The kit for detecting creatine kinase isoenzymes according to claim 3, characterized in that, The step of activating the 185nm carboxyl latex particles and the 359nm carboxyl latex particles includes: Activation treatment was performed by adding Hepes buffer solution, 5-20 g / L NHS and 5-20 g / L EDC.

5. The kit for detecting creatine kinase isoenzymes according to claim 4, characterized in that, The activation time for the activation treatment is 20-40 minutes.

6. The use of the kit as described in claim 5 for the detection of creatine kinase isoenzymes for non-diagnostic purposes.

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