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High-sensitivity creatine kinase isoenzyme detection kit and preparation method thereof

A detection kit, creatine kinase technology, applied in the fields of immunity, in vitro diagnosis, and medicine, can solve the problem that the analytical sensitivity cannot meet the clinical requirements, and achieve the effect of convenient clinical application, good stability and high sensitivity

Pending Publication Date: 2021-03-23
NINGBO MEDICAL SYSTEM BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

CN111562372A discloses a creatine kinase isoenzyme MB detection kit based on latex-enhanced immune turbidimetry, but it has the problem that the analytical sensitivity cannot meet the clinical requirements

Method used

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  • High-sensitivity creatine kinase isoenzyme detection kit and preparation method thereof
  • High-sensitivity creatine kinase isoenzyme detection kit and preparation method thereof
  • High-sensitivity creatine kinase isoenzyme detection kit and preparation method thereof

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Effect test

Embodiment 1

[0037] Reagent R1 composition and content: 100mM Tris-HCl buffer, 20g / L sodium chloride, 20g / L polyethylene glycol (PEG4000), 2g / L Tween-20, 1g / L sodium azide, 1g / L blocker.

[0038] The preparation process of reagent R2 is as follows: mix 1mg of anti-mouse IgG-Fc antibody with 0.1M sodium periodate at a ratio of 20:1, stir and react at room temperature for 20min; Dialyze overnight; add 0.2M pH9.5 sodium carbonate buffer, adjust the pH to 9.0, immediately add 0.5mL of 280nm latex microspheres with a solid content of 10%, stir at room temperature for 2h; add 0.02mL of newly prepared 4mg / mL NaBH 4 , mix well, react at 4°C for 2h, centrifuge at 15000rpm for 40mim to remove the supernatant, add buffer to resuspend the precipitate and ultrasonically disperse to prepare the latex-anti-mouse IgG-Fc antibody conjugate; take 2.5mg of two kinds of mouse The anti-human CK-MB monoclonal antibody was mixed with the prepared latex-anti-mouse IgG-Fc antibody conjugate respectively, reacted...

Embodiment 2

[0040] Reagent R1 composition and content: 200mM Tris-HCl buffer solution, 1g / L sodium chloride, 15g / L polyethylene glycol (PEG4000), 1g / L Tween-20, 1g / L sodium azide, 1g / L blocker.

[0041] Reagent R2 composition and content: Take 1mg of anti-mouse IgG-Fc antibody and 0.1M sodium periodate in a ratio of 20:1, mix at room temperature for 20min; react the solution with 1mM pH4.4 acetate buffer at 4°C Dialyze overnight; add 0.2M pH9.5 sodium carbonate buffer, adjust the pH to 9.0, immediately add 0.5mL of 330nm latex microspheres with a solid content of 10%, stir at room temperature for 2h; add 0.02mL of newly prepared 4mg / mL NaBH 4 , mix well, react at 4°C for 2h, centrifuge at 15000rpm for 40mim to remove the supernatant, add buffer to resuspend the precipitate and ultrasonically disperse to prepare the latex-anti-mouse IgG-Fc antibody conjugate; take 2.5mg of two kinds of mouse The anti-human CK-MB monoclonal antibody was mixed with the prepared latex-anti-mouse IgG-Fc anti...

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Abstract

The invention provides a high-sensitivity creatine kinase isoenzyme detection kit and a preparation method thereof. The high-sensitivity creatine kinase isoenzyme detection kit comprises a reagent R1and a reagent R2; the preparation method comprises the steps: reacting an anti-mouse IgG-Fc antibody with amino groups on the surfaces of latex microspheres to form a shift alkali, directionally marking the latex microspheres with the anti-mouse IgG-Fc antibody, connecting the anti-mouse IgG-Fc antibody with at least two mouse anti-human CK-MB monoclonal antibodies, and coupling the latex microspheres with the mouse anti-human CK-MB monoclonal antibodies to prepare a latex microsphere reagent; and adding the latex microsphere reagent into a closed buffer solution, carrying out resuspension ultrasonic treatment, closing, adding a latex preservation solution, carrying out resuspension ultrasonic treatment to prepare latex reagents, and mixing at least two latex reagents to prepare a reagentR2. Compared with the prior art, the kit is high in sensitivity, wide in detection range, good in repeatability, high in specificity and good in stability; compared with other coupling methods, the method provided by the invention has the advantages that the CK-MB monoclonal antibody F (ab) end active sites are prevented from being coupled to the microspheres, meanwhile, the CK-MB monoclonal antibody coupling amount is increased, and the reagent detection sensitivity is obviously improved.

Description

technical field [0001] The invention relates to the fields of medicine, immunity and in vitro diagnosis, in particular to a high-sensitivity creatine kinase isoenzyme detection kit and a preparation method thereof. Background technique [0002] Creatine kinase (CK) is an important capacity-regulating enzyme in the myocardium, which mainly catalyzes the transfer of high-energy phosphate bonds from ATP to creatine to generate phosphocreatine. Phosphokinase is a dimeric enzyme, which mainly exists in myocardial tissue, and also has a small amount of distribution in skeletal muscle. Its structure is composed of two subunits: muscle subunit (M) and brain subunit (B). CK-MM, CK-BB, CK-MB and mitochondrial creatine kinase have four isoenzyme forms, among which CK-MM mainly exists in skeletal muscle, and CK-BB mainly exists in brain tissue. Among them, CK-MB mainly exists in myocardial tissue. CK-MB has a molecular weight of 80kD and is composed of two subunits M and B (each subun...

Claims

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Application Information

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IPC IPC(8): G01N33/573G01N33/577G01N33/543
CPCG01N33/573G01N33/577G01N33/54346G01N2333/9123
Inventor 邹继华田燕丹刘献文方亮
Owner NINGBO MEDICAL SYSTEM BIOTECHNOLOGY CO LTD
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