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Preparation method of polystyrene fluorescent microsphere coupled with antibody

A technology of fluorescent microspheres and polystyrene, applied in fluorescence/phosphorescence, material excitation analysis, etc., can solve the problems of strict experimental conditions and complicated fluorescein labeling process, and achieve good dispersion, weakened agglomeration, and specific binding enhanced effect

Inactive Publication Date: 2015-04-22
NANJING UNIV OF POSTS & TELECOMM
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The fluorescein labeling process is complicated and the experimental conditions are strict

Method used

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  • Preparation method of polystyrene fluorescent microsphere coupled with antibody
  • Preparation method of polystyrene fluorescent microsphere coupled with antibody

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Experimental program
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Effect test

Embodiment 1

[0033] Step 1: Use 0.01M MES buffer as the activation buffer, take 100 μL of fluorescent microspheres with carboxyl groups on the surface and add them to a 1.5 mL centrifuge tube, wash with MSE buffer for 3 times, discard the supernatant in a refrigerated centrifuge, and then centrifuge Add MES buffer to the tube, add freshly prepared 0.5g / mL EDC solution and 0.25g / mL sulfo-NHS solution at a volume ratio of 1:2, shake and mix with a vortex mixer, and rotate to activate the carboxyl groups on the surface of the microspheres at room temperature 10 Minutes, and then wash the fluorescent microspheres with MSE buffer 2 times.

[0034] Step 2: Use 0.02M BST buffer as the buffer in the coupling process. After washing, add 10 μg of monoclonal antibody, then add BST buffer, keep the concentration of fluorescent microspheres at 3 mg / ml, and rotate for 1 hour at room temperature, then The supernatant was separated and collected by a refrigerated centrifuge, and the coupling rate was ...

Embodiment 2

[0038]Step 1: Use 0.01M MES buffer as the activation buffer, add 100 μL of fluorescent microspheres with carboxyl groups on the surface into a 1.5 mL centrifuge tube, wash with MSE buffer for 3 times, then use a refrigerated centrifuge to discard the supernatant. After washing 3 times, add MES buffer to the centrifuge tube, add freshly prepared 0.5g / mL EDC solution and 0.25g / mL sulfo-NHS solution at a volume ratio of 1:2, shake and mix with a vortex mixer, and rotate at room temperature Activate the carboxyl groups on the surface of the microspheres for 10 minutes, then wash the microspheres twice with MSE buffer.

[0039] In step 2, use 0.02M BST buffer as the buffer for the coupling process. After washing, add 10 μg of monoclonal antibody, and then add BST buffer, rotate and react at room temperature for 2 hours, then separate and collect the supernatant with a refrigerated centrifuge, and detect that the coupling rate is 85%.

[0040] Step 3, add BST buffer and 5.0 μL glyc...

Embodiment 3

[0043] Step 1: Use 0.01M MES buffer as the activation buffer, add 100 μL of fluorescent microspheres with carboxyl groups on the surface into a 1.5 mL centrifuge tube, wash with MSE buffer for 3 times, then use a refrigerated centrifuge to discard the supernatant. After washing 3 times, add MES buffer to the centrifuge tube, add freshly prepared 0.5g / mL EDC solution and 0.25g / mL sulfo-NHS solution at a volume ratio of 1:2, shake and mix with a vortex mixer, and rotate at room temperature Activate the carboxyl groups on the surface of the microspheres for 10 minutes, then wash the microspheres twice with MSE buffer.

[0044] In step 2, use 0.02M BST buffer as the buffer for the coupling process. After washing, add 10 μg of monoclonal antibody, and then add BST buffer, rotate at room temperature for 2 hours, then separate and collect the supernatant with a refrigerated centrifuge, and detect that the coupling rate is 93%.

[0045] Step 3, add BST buffer and 7.5 μL glycine t...

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Abstract

The invention discloses a preparation method of a polystyrene fluorescent microsphere coupled with an antibody, which comprises the following steps: 1. activating a carboxyl group on the surface of a fluorescent microsphere; 2. coupling an amino group of a monoclonal antibody with the carboxyl group on the surface of the fluorescent microsphere; 3. blocking activated groups not subjected to complete reaction on the surface of the fluorescent microsphere; and 4. preserving the polystyrene fluorescent microsphere coupled with the antibody. The preparation method disclosed by the invention is simple, easy to implement, low in cost and suitable for industrialization.

Description

technical field [0001] The invention relates to the field of fluorescently labeled antibodies, in particular to a method for preparing antibody-coupled polystyrene fluorescent microspheres. Background technique [0002] Polystyrene fluorescent microspheres are uniform microspheres dyed with fluorescent reagents. Fluorescent microspheres refer to solid particles with diameters ranging from nanometers to micrometers, loaded with fluorescent substances, and excited by external energy to fluoresce. Its shape can be any shape, and the typical shape is spherical. The carriers of fluorescent microspheres are mostly organic or inorganic polymer materials. It has a relatively stable morphological structure and luminescent behavior, and is less affected by external conditions such as solvents, heat, electricity, and magnetism than pure fluorescent compounds. As a new type of carrier material, it has been widely used in the field of biomedicine. [0003] Generally, the seed polymeri...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C08J7/12G01N21/64
Inventor 葛·瑞金侯晓纯吴琼杨昕黄维宋怡静
Owner NANJING UNIV OF POSTS & TELECOMM
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