Fluorescent microsphere and antibody coupling method

A fluorescent microsphere and coupling technology, applied in the field of biological analysis, can solve the problems of low coupling efficiency and coupling strength of fluorescent microspheres and antibodies, and inappropriate labeling methods, so as to improve the coupling efficiency and improve the coupling efficiency. and coupling strength, the effect of avoiding agglutination

Pending Publication Date: 2020-04-28
BEIJING PEPMAGIC BIOTECH CORPORATION LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] For this reason, the embodiment of the present invention provides a coupling method of fluorescent microspheres and antibodies to solve the problem of low coupling efficiency and coupling strength between fluorescent microspheres and antibodies in the prior art due to inappropriate labeling methods

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  • Fluorescent microsphere and antibody coupling method
  • Fluorescent microsphere and antibody coupling method

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Embodiment 1

[0034] This embodiment is a coupling method of fluorescent microspheres and creatine kinase isoenzyme monoclonal antibody, the coupling method comprising the following steps:

[0035] (a) After washing the fluorescent microspheres, ultrasonically disperse them for 3 minutes, dilute them with MES buffer solution with a concentration of 2% polyvinylpyrrolidone until the solid content of the fluorescent microspheres is 0.8‰, and then add them to the diluted fluorescent microsphere solution in sequence Ethanol solution containing NHS and ethanol solution containing EDC, mix well, react at room temperature for 25min, sonicate, centrifuge at 10000r / min for 10min, collect activated fluorescent microspheres, add 0.2% MES buffer solution of polyvinylpyrrolidone, ultrasonic treatment, the microsphere suspension that obtains the solid content of fluorescent microspheres is 0.08%, wherein, the addition amount of the ethanol solution containing NHS and the ethanol solution containing EDC is...

Embodiment 2

[0040] This embodiment is a coupling method of fluorescent microspheres and creatine kinase isoenzyme monoclonal antibody, the coupling method comprising the following steps:

[0041] (a) After cleaning the fluorescent microspheres, ultrasonically disperse them for 2 minutes, dilute them with MES buffer solution with a concentration of 0.05% polyvinylpyrrolidone until the solid content of the fluorescent microspheres is 1.2‰, and then add Ethanol solution containing NHS and ethanol solution containing EDC, mix well, react at room temperature for 35min, sonicate, centrifuge at 10000r / min for 10min, collect activated fluorescent microspheres, add 0.2% MES buffer solution of polyvinylpyrrolidone, ultrasonic treatment, the microsphere suspension that obtains the solid content of fluorescent microsphere is 0.12%, wherein, the addition amount of the ethanol solution containing NHS and the ethanol solution containing EDC are respectively the same as that of the fluorescent microsphere...

Embodiment 3

[0046] This embodiment is a coupling method of fluorescent microspheres and creatine kinase isoenzyme monoclonal antibody, the coupling method comprising the following steps:

[0047](a) After cleaning the fluorescent microspheres, ultrasonically disperse them for 2.5 minutes, dilute them with MES buffer solution with a concentration of 1% polyvinylpyrrolidone until the solid content of the fluorescent microspheres is 1‰, and then pour them into the diluted fluorescent microsphere solution in turn Add the ethanol solution containing NHS and the ethanol solution containing EDC, mix well, react at room temperature for 30 minutes, sonicate, centrifuge at 10000r / min for 10 minutes, collect the activated fluorescent microspheres, add 0.2 MES buffer solution of % polyvinylpyrrolidone, ultrasonic treatment, the microsphere suspension that obtains the solid content of fluorescent microspheres is 0.1%, wherein, the addition amount of the ethanol solution containing NHS and the ethanol s...

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Abstract

The embodiment of the invention discloses a fluorescent microsphere and antibody coupling method. The method comprises the following steps: (a) activating fluorescent microspheres, and resuspending toobtain a microsphere suspension; (b) adding an antibody solution into the microsphere suspension for reaction, carrying out ultrasonic treatment, and continuing the reaction; (c) after the reaction is finished, adding a confining liquid into a reaction liquid for confining, centrifuging, and collecting precipitates; and (d) putting the precipitates into a glycine solution, and carrying out ultrasonic dispersion to obtain a fluorescent microsphere and antibody coupled compound. In the coupling method, the fluorescent microspheres are activated, and sufficient contact of reactants is ensured through the ultrasonic treatment so that coupling efficiency and coupling strength of the fluorescent microspheres and the antibodies can be significantly improved, microsphere surfaces and antibody protein binding sites can be fully combined, stability of the prepared compound can be effectively improved, and an agglutination phenomenon can be avoided; in addition, sensitivity of fluorescence detection can be improved.

Description

technical field [0001] The embodiment of the present invention relates to the technical field of bioanalysis, and specifically relates to a coupling method of fluorescent microspheres and antibodies. Background technique [0002] When detecting certain antigens or specific proteins, immunolabeling techniques are usually applied. Immunolabeling technology is a technology that marks the substance that is easy to measure and has high sensitivity to specific antigen or antibody protein, and shows the nature and content of the antigen or antibody in the reaction system through the enhanced amplification of these markers. [0003] Currently commonly used labels include colloidal gold, latex, fluorescein, fluorescent microspheres, enzymes, and radionuclides. However, in the existing process of labeling antibodies with fluorescent microspheres, due to inappropriate labeling methods, the coupling efficiency and coupling strength between fluorescent microspheres and antibodies are lo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577G01N33/533G01N21/64
CPCG01N33/577G01N33/533G01N21/6428G01N21/6486
Inventor 管静波赵树民孙如石松传郭波
Owner BEIJING PEPMAGIC BIOTECH CORPORATION LTD
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