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CN-119269794-B - Kit for detecting creatine kinase isozyme and application thereof

CN119269794BCN 119269794 BCN119269794 BCN 119269794BCN-119269794-B

Abstract

The embodiment of the application belongs to the technical field of detection, and relates to a kit for detecting creatine kinase isoenzyme and application thereof, and the kit comprises a first detection reagent and a second detection reagent, wherein the first detection reagent comprises Hepes buffer solution, naCl, PEG-6000, nonylphenol polyoxyethylene ether, BSA, naN 3 , a chemical blocking agent and a biological blocking agent, and the second detection reagent comprises CKBB sensitized latex particles, hepes buffer solution, a blocking agent, tween-20 and NaN 3 . The application effectively improves the detection sensitivity of creatine kinase isozymes.

Inventors

  • XIE LONG
  • QI WENCHUANG
  • JIANG XIWEN
  • LIU QIONG
  • DUAN SHAOQING
  • Lu Huadan

Assignees

  • 广州达安基因股份有限公司

Dates

Publication Date
20260505
Application Date
20230707

Claims (6)

  1. 1. A kit for detecting creatine kinase isozymes, comprising: A first detection reagent and a second detection reagent; The first detection reagent consists of Hepes buffer solution, naCl, PEG-6000, polyoxyethylene nonylphenol ether, BSA, naN 3 , a chemical blocker and a biological blocker, wherein the chemical blocker is sodium dodecyl sulfate, and the biological blocker is goat anti-human IgG; The second detection reagent consists of CKBB sensitized latex particles, hepes buffer, tween-20 and NaN 3 , wherein the CKBB sensitized latex particles are treated by a blocking agent, the CKBB sensitized latex particles comprise carboxyl latex particles with the particle size of 185nm and carboxyl latex particles with the particle size of 359nm, the volume ratio of the carboxyl latex particles with the particle size of 185nm to the carboxyl latex particles with the particle size of 359nm is 10:1, and the blocking agent consists of glycine, HSA and sucrose.
  2. 2. The kit for detecting creatine kinase isoenzyme according to claim 1, wherein the concentration of Hepes buffer solution of the first detection reagent is 10-30 mmol/L, the concentration of NaCl is 10-50 g/L, the concentration of PEG-6000 is 10-30 g/L, the concentration of nonylphenol polyoxyethylene ether is 1-10 g/L, the concentration of chemical blocker is 1-10 g/L, the concentration of BSA is 1-5 g/L, the concentration of NaN 3 is 0.1% and the concentration of biological blocker is 0.02% -0.04%, and/or The concentration of Hepes buffer solution of the second detection reagent is 10-30 mM, the concentration of CKBB 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 NaN 3 is 0.1%.
  3. 3. The kit for detecting creatine kinase isozymes according to claim 1, wherein the second detection reagent is prepared by the steps of: Placing a beaker with a magnetic rotor on a magnetic stirrer, sequentially adding 185nm carboxyl latex particles and 359nm carboxyl latex particles, and performing activation treatment on the 185nm carboxyl latex particles and 359nm carboxyl latex particles; Adding CKBB antibody, reacting for 2-4 hours at room temperature, adding a blocking agent, and reacting for 1-4 hours at room temperature; centrifuging at 12000rmp for 30min, and discarding supernatant to obtain CKBB sensitized latex particles; Resuspension the CKMB priming latex particles, adding Hepes, tween-20, naN 3 , and obtaining the second detection reagent.
  4. 4. The kit for detecting creatine kinase isozymes according to claim 3, wherein the step of activating the 185nm carboxyl latex particles and 359nm carboxyl latex particles comprises: And adding Hepes buffer solution, 5-20 g/L NHS and 5-20 g/L EDC, and performing activation treatment.
  5. 5. The kit for detecting creatine kinase isozymes according to claim 4, wherein the activation time of the activation treatment is 20-40 min.
  6. 6. Use of the kit according to claim 5 for the non-diagnostic detection of creatine kinase isozymes.

Description

Kit for detecting creatine kinase isozyme and application thereof Technical Field The application relates to the technical field of detection, in particular to a kit for detecting creatine kinase isozymes and application thereof. Background Creatine kinase (CREATINEKINASE, CK) is widely present in various tissues and is involved in the regeneration of Adenosine Triphosphate (ATP), maintaining intracellular adenosine triphosphate concentrations at physiological levels. CK is a heterodimer, subunits of which are M type and B type, and are mainly divided into three isozymes of CK-BB, CK-MM and CK-MB, and the mitochondria also contain other CK-Mt isozymes. CK-BB exists mainly in brain, prostate and other organs, CK-MM exists mainly in bones and cardiac muscle, and CK-MB exists mainly in cardiac muscle. The content of CK-MB in serum of normal people is lower than 5% of total activity, and when chest pain occurs after acute myocardial infarction, the content of CK-MB in serum is increased in 4 hours, the peak value is reached in 24 hours, and recovery is achieved in 3-4 days. CK-MB is one of the important markers of myocardial damage. Current methods for detecting CK-MB include agarose gel electrophoresis, enzyme-rate immunosuppression, chemiluminescent immunoassay and latex turbidimetry. Enzyme rate immunosuppression methods are susceptible to multiple factors and have high false positives. It uses a monoclonal antibody against CK-M to inhibit the activity of CK-M subunit in specimen, does not affect the activity of CK-B subunit, and detects the activity of B subunit in residual CK-MB and CK-BB by enzymatic reaction. When brain, prostate, stomach intestine, lung and other tissue cells are damaged, ischemic and necrotic, CK-BB is released into serum, so that the false increase of CK-MB is easily caused, and meanwhile, giant CK released by tumor and immune dysfunction people (an oligomeric mitochondrial CK) can participate in enzymatic reaction, the giant CK cannot be inhibited by anti-CK-M subunit antibodies, the activity is detected by 100 percent, the detection result of CK-MB is multiplied, and the false increase of CK-MB is caused. Agarose gel electrophoresis is a common method for separating and detecting myocardial creatine kinase isozymes, and uses agarose gel as a medium to separate proteins and calculate CKMM, CKMB, CKBB percent. The method needs manual operation, has long detection time and cannot realize automation. The chemiluminescent immunoassay method is based on the principle of a double-antibody sandwich method, a specific mouse monoclonal antibody is coated on a carrier in advance, a sample to be detected and an enzyme-labeled specific monoclonal antibody are added to form an immune complex of the specific antibody-antigen-enzyme-labeled antibody, photons are emitted after excitation, and the mass number of CK-MB in the sample is obtained according to the size of excitation light. The method has strong specificity but high price. A relatively stable and accurate humoral protein homogeneous phase immunity turbidimetry detection method by a latex immunity turbidimetry method. When CKBB is combined with the microsphere of the coupling antibody CKBB, the CKBB is rapidly aggregated, the absorbance of the reaction solution is changed, the reaction solution is in a linear relationship within a certain range, and the concentration of the CKBB can be obtained through conversion. The creatine kinase isoenzyme in the sample is combined with the anti-creatine kinase isoenzyme antibody sensitization latex in the reagent to form an insoluble immune complex, turbidity is formed in a specific buffer environment, the turbidity is in proportional relation with the content of the object to be detected in a detection range, and the content of the creatine kinase isoenzyme in the sample can be obtained by comparing the turbidity with a calibrator operated under the same condition. The existing creatine kinase isoenzyme detection kit has the following problems of (1) poor reagent stability and easiness in precipitation, (2) narrow reagent linear range and poor sensitivity which are generally 3-180ng/mL, (3) easiness in reagent interference by triglyceride, and (4) easiness in reagent interference by rheumatoid factor. In conclusion, the current creatine kinase isoenzyme detection kit has poor detection sensitivity on creatine kinase isoenzyme. Disclosure of Invention The embodiment of the application aims to provide a kit for detecting creatine kinase isozymes and application thereof, and the kit can effectively improve the detection sensitivity of the creatine kinase isozymes. In order to solve the technical problems, the embodiment of the application provides a kit for detecting creatine kinase isoenzyme, which adopts the following technical scheme: a kit for detecting creatine kinase isozymes, comprising: A first detection reagent and a second detection reagent; The first detection reagent comprises Hepes buffer s