Creatine kinase isoenzyme detection reagent
By using template separator and polystyrene latex microspheres with conjugated antibodies in the creatine kinase isozyme detection reagent, the problem of CK-BB and CK-MM interference was solved, and a high-accurate creatine kinase isozyme detection was achieved.
Patent Information
- Application Number
- CN202510631360.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-16
- Publication Date
- 2025-08-05
AI Technical Summary
When the existing magnetic microparticle chemiluminescence method detects creatine kinase isoenzymes, the interference between CK-BB and CK-MM leads to errors in the detection result, affecting the detection accuracy.
Reagent A containing template separator and polystyrene latex microspheres coupled to the antibody are used to reduce the impact on CK-MB by blocking the active sites of CK-BB or CK-MM, and the sample absorbance value is measured by combining magnetic microspheres with CK-MM to improve detection accuracy.
Effectively eliminate interference from CK-BB and CK-MM, improve the detection accuracy of creatine kinase isozyme detection reagents, and reduce the phenomenon of pseudo-enhanced.
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of biological monitoring, and particularly relates to a creatine kinase isoenzyme detection reagent. Background Art
[0002] Creatine kinase (CK) is a dimeric enzyme that has four different forms: mitochondrial isoenzyme, cytoplasmic isoenzyme CK-MM (muscle type), creatine kinase brain isoenzyme CK-BB (brain type), and creatine kinase isoenzyme (CK-MB), among which creatine kinase isoenzyme is mainly located in the myocardium.
[0003] The level of CK-MB in normal human serum is less than 5% of total activity. When chest pain strikes after an acute myocardial infarction, serum CK-MB levels rise within 4 hours, peak within 24 hours, and recover within 3-4 days. CK-MB is one of the key markers of myocardial injury.
[0004] The enzyme rate immunosuppression assay 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, without affecting the activity of the CK-B subunit. The activity of the remaining CK-MB and the B subunit in CK-BB is measured through an enzymatic reaction. When tissue cells in the brain, prostate, gastrointestinal tract, lungs, and other areas are damaged, ischemic, or necrotic, CK-BB is released into the serum, easily causing a false increase in CK-MB. At the same time, the presence of macro-CK (a type of oligomeric mitochondrial CK) released by tumors and people with immune disorders also participates in the enzymatic reaction. Macro-CK is not inhibited by anti-CK-M subunit antibodies, resulting in 100% detection of its activity, which exponentially amplifies the CK-MB test result and falsely increases CK-MB.
[0005] Magnetic microparticle chemiluminescence is a new development in enzyme-labeled immunoassay technology. This technique incorporates magnetic microspheres into the solid phase of the immunoassay, significantly increasing the reaction surface area, increasing the amount of antigen or antibody adsorbed, accelerating the reaction rate, and improving detection sensitivity. Compared to colloidal gold and ELISA methods, it offers high accuracy, good specificity, high precision, a wide linear range, and good reagent stability. Compared to plate-based chemiluminescence, it offers advantages such as good reproducibility, high sensitivity, rapid detection, and reduced detection time. Currently, magnetic microparticle chemiluminescence immunoassay technology has become the most widely used analytical method in the field of immunodiagnosis worldwide and a key development direction for immunoreagents. However, problems with binding to interfering antibodies to CK-BB, CK-MM, or giant CK can lead to errors in the final test results. Summary of the Invention
[0006] In view of this, the object of the present invention is to provide a creatine kinase isoenzyme detection reagent, which can effectively eliminate the influence of CK-BB or CK-MM and has high accuracy.
[0007] In order to achieve the above object, the present invention provides the following technical solutions:
[0008] The present invention provides a creatine kinase isoenzyme detection reagent, comprising reagent A and reagent B;
[0009] The reagent A includes: Hepes buffer 30-60mM, mouse IgG 0.2-0.5g / L, TritonX-100 1-5g / L, stabilizer 0.4-0.8g / L, template separation agent 1-2g / L;
[0010] The reagent B comprises 30-60 mM Hepes buffer, 1-3 g / L blocking agent, 1-5 g / L stabilizer, and 1-3 g / L polystyrene latex microspheres coupled with antibodies.
[0011] Preferably, the preparation method of the template separating agent is:
[0012] The template molecule is mixed evenly with cyclodextrin and methacrylic acid, and then a cross-linking agent and an initiator are added to carry out polymerization. After the polymerization is completed, the first particles are obtained by crushing.
[0013] The first particles are eluted by refluxing a methanol-acetic acid mixture and then washed to obtain a template separating agent;
[0014] The template molecule is CK-BB or CK-MM.
[0015] The present invention adopts the above technical solution, prepares a molecular template of CK-BB or CK-MM to eliminate the interference of the two on the detection of CK-MB, and selectively coats CK-BB or CK-MM to prevent its active site from contacting with the CK-MB antigen, thereby improving the detection accuracy of CK-MB.
[0016] Preferably, the mass ratio of the template molecule, cyclodextrin, methacrylic acid, crosslinking agent and initiator is 1:(0.1-0.2):4:10:(0.1-0.5).
[0017] Preferably, the cross-linking agent is ethylene glycol dimethacrylate, trimethylolpropane trimethacrylate, or divinylbenzene.
[0018] Preferably, the initiator is azobisisobutyronitrile or DMPA.
[0019] Preferably, the stabilizer is bovine serum albumin, trehalose, or chitosan.
[0020] Preferably, the blocking agent is HSA, glycine, or sucrose.
[0021] Preferably, the preparation method of the antibody-coupled polystyrene latex microspheres is:
[0022] The carboxyl polystyrene latex microspheres were activated and then resuspended in MOPS buffer, CK-MB antibody was added, incubated at 25°C overnight and then washed with PBS; then blocking solution was added, shaken at room temperature and then washed with PBS to obtain antibody-coupled polystyrene latex microspheres.
[0023] Preferably, the mass ratio of the carboxyl polystyrene latex microspheres to the CK-MB antibody is 5:1.
[0024] Preferably, the molar ratio of the antibody-coupled magnetic beads to the fluorescent dye is 1:8.
[0025] Contains at least the following beneficial technical effects:
[0026] The present invention adds a template separation agent to reagent A, which can effectively block the active sites of CK-BB or CK-MM, and reduces the influence on CK-MB by selecting the reagent, while facilitating the binding of CK-BB or CK-MM to the template. Polystyrene latex microspheres coupled with magnetic antibodies added to reagent B bind to CK-MM, and the content of the analyte is determined according to the change in the sample absorbance value. The creatine kinase isoenzyme detection reagent has high accuracy. DETAILED DESCRIPTION
[0027] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.
[0028] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between any intermediate value within a stated value or stated range and any other stated value or intermediate value within the stated range is also encompassed by the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.
[0029] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.
[0030] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the present invention. The present description and examples are intended to be illustrative only.
[0031] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.
[0032] Unless otherwise specified, the "room temperature" and "normal temperature" mentioned in the present invention are all calculated as 25±2°C.
[0033] Unless otherwise specified, all raw materials and instruments used in the following examples of the present invention are commercially available.
[0034] Preparation of template separating agent:
[0035] CK-MM, cyclodextrin, and methacrylic acid are mixed and dissolved in chloroform, and then trimethylolpropane trimethacrylate and azobisisobutyronitrile are added to carry out polymerization. After the polymerization is completed, the mixture is crushed to obtain first particles with a particle size of 50-100 nanometers; wherein the mass ratio of the template molecule, cyclodextrin, chloroform, methacrylic acid, trimethylolpropane trimethacrylate, and azobisisobutyronitrile is 1:0.1:4:20:10:0.3;
[0036] The first particles were eluted by refluxing with a mixture of methanol and acetic acid (volume ratio 1:9) for 12 h and then washed with distilled water to obtain a template separating agent.
[0037] Preparation of polystyrene latex microspheres coupled to antibodies:
[0038] Carboxyl polystyrene latex microspheres with a particle size of 280 nm were placed in a pH 7.5 buffer solution, an activator was added, and the mixture was reacted at 25°C for 30 minutes. After centrifugation, the excess activator in the supernatant was removed, and the mixture was resuspended in a buffer solution to obtain activated carboxyl polystyrene latex microspheres.
[0039] Then resuspend in MOPS buffer, add CK-MB antibody (20% of the mass of microspheres), incubate at 25°C overnight and wash with PBS; then add blocking solution, shake at room temperature and wash with PBS to obtain antibody-coupled polystyrene latex microspheres.
[0040] Example 1
[0041] This embodiment provides a creatine kinase isoenzyme detection reagent comprising reagent A and reagent B;
[0042] The reagent A includes: Hepes buffer 50mM, mouse IgG 0.4g / L, TritonX-100 2g / L, stabilizer (bovine serum albumin) 0.6g / L, template separation agent 1.5g / L;
[0043] The reagent B comprises 50 mM Hepes buffer, 2 g / L blocking agent (HSA), 2 g / L stabilizer (bovine serum albumin), and 1.5 g / L polystyrene latex microspheres coupled with antibodies.
[0044] Example 2
[0045] This embodiment provides a creatine kinase isoenzyme detection reagent comprising reagent A and reagent B;
[0046] The reagent A includes: Hepes buffer 30mM, mouse IgG 0.2g / L, TritonX-100 1g / L, stabilizer (trehalose) 0.4g / L, template separation agent 1g / L;
[0047] The reagent B comprises 30 mM Hepes buffer, 1 g / L blocking agent (glycine), 1 g / L stabilizer (trehalose), and 1 g / L polystyrene latex microspheres coupled with antibodies.
[0048] Example 3
[0049] This embodiment provides a creatine kinase isoenzyme detection reagent comprising reagent A and reagent B;
[0050] The reagent A includes: Hepes buffer 60mM, mouse IgG 00.5g / L, TritonX-1005g / L, stabilizer (chitosan) 0.8g / L, template separation agent 2g / L;
[0051] The reagent B comprises 60 mM Hepes buffer, 3 g / L blocking agent (sucrose), 5 g / L stabilizer (chitosan), and 3 g / L polystyrene latex microspheres coupled with antibodies.
[0052] Comparative Example 1
[0053] This comparative example is the same as the example, except that cyclodextrin is not added when preparing the template separating agent.
[0054] Comparative Example 2
[0055] This comparative example is the same as the embodiment, except that no template separating agent is added.
[0056] Comparative Example 3
[0057] This comparative example is the same as the embodiment, except that the template separation agent is 0.1 g / L.
[0058] Comparative Example 4
[0059] This comparative example is the same as the embodiment, except that Span 20 is replaced.
[0060] Experimental example
[0061] CK-MB was combined with polystyrene latex microspheres to form a turbid solution, which was then diluted to 0, 5, 10, 75, 150, and 300 ng / mL according to the concentration to establish a standard curve.
[0062] Preparation of experimental samples: The concentrations of CK-MB in each experimental sample were 0, 5, 10, 75, 150, and 300 ng / mL, respectively; each sample also contained an equal amount of CK-MM.
[0063] The experimental samples were tested using the creatine kinase isoenzyme detection reagents in Examples 1-3 and Comparative Examples 1-4, and the relative deviations were statistically analyzed, as shown in Table 1.
[0064] Table 1
[0065] Relative deviation Example 1 3.9％ Example 2 5.8％ Example 3 5.1％ Comparative Example 1 10.6％ Comparative Example 2 26.3％ Comparative Example 3 14.8％ Comparative Example 4 7.4％
[0066] As shown in Table 1, the creatine kinase isoenzyme detection reagent prepared in the present invention has high specificity for CK-MM, can eliminate the error of CK-MM, and has a detection limit of 5-300 ng / mL.
[0067] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.
Claims
1. A creatine kinase isoenzyme detection reagent, characterized in that, Including reagent A and reagent B; The reagent A includes: Hepes buffer 30-60mM, mouse IgG 0.2-0.5g / L, TritonX-100 1-5g / L, stabilizer 0.4-0.8g / L, template separation agent 1-2g / L; The reagent B comprises 30-60 mM Hepes buffer, 1-3 g / L blocking agent, 1-5 g / L stabilizer, and 1-3 g / L polystyrene latex microspheres coupled with antibodies.
2. The creatine kinase isoenzyme detection reagent according to claim 1, wherein The preparation method of the template separating agent is: The template molecule is mixed evenly with cyclodextrin and methacrylic acid, and then a cross-linking agent and an initiator are added to carry out polymerization. After the polymerization is completed, the first particles are obtained by crushing. The first particles are eluted by refluxing a methanol-acetic acid mixture and then washed to obtain a template separating agent; The template molecule is CK-BB or CK-MM.
3. The creatine kinase isoenzyme detection reagent according to claim 2, wherein The mass ratio of the template molecule, cyclodextrin, methacrylic acid, crosslinking agent and initiator is 1:(0.1-0.2):4:10:(0.1-0.5).
4. The creatine kinase isoenzyme detection reagent according to claim 3, wherein The crosslinking agent is ethylene glycol dimethacrylate, trimethylolpropane trimethacrylate, and divinylbenzene.
5. The creatine kinase isoenzyme detection reagent according to claim 3, wherein The initiator is azobisisobutyronitrile or DMPA.
6. The creatine kinase isoenzyme detection reagent according to claim 3, characterized in that The stabilizers are bovine serum albumin, trehalose and chitosan.
7. The creatine kinase isoenzyme detection reagent according to claim 3, characterized in that The blocking agent is HSA, glycine and sucrose.
8. The creatine kinase isoenzyme detection reagent according to claim 3, characterized in that The preparation method of the antibody-coupled polystyrene latex microspheres is as follows: The carboxyl polystyrene latex microspheres were activated and then resuspended in MOPS buffer, CK-MB antibody was added, incubated at 25°C overnight and then washed with PBS; then blocking solution was added, shaken at room temperature and then washed with PBS to obtain antibody-coupled polystyrene latex microspheres.
9. The creatine kinase isoenzyme detection reagent according to claim 8, characterized in that The mass ratio of the carboxyl polystyrene latex microspheres to the CK-MB antibody is 5:1.