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Antibody labeling method for time resolved fluorescence microsphere coupling and application thereof

A time-resolved fluorescence and antibody labeling technology, applied in the field of biological analysis, can solve the problems of high equipment cost and maintenance cost, unfavorable carrying and handling, complicated operation process, etc., to achieve large-scale application, simple labeling method and repeatability Good results

Pending Publication Date: 2019-01-11
HANGZHOU LAIHE BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For example, the naked eye interpretation of colloidal gold and latex has a certain degree of human subjectivity; the operation process of fluorescein labeling technology, enzyme labeling technology, and radionuclide technology is relatively complicated and requires high equipment costs and maintenance costs, and is not conducive to carrying and handling.
These conditions often determine the objectivity of testing and many tests cannot be carried out in backward and underdeveloped areas.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1: Labeling rabbit IgG antibodies with time-resolved fluorescent microspheres

[0045]Measure 100 μl of time-resolved fluorescent microspheres (purchased from Bangs Company, particle size 200nm), add 0.2mL PBS buffer (0.01M, pH7.4), shake and mix well, centrifuge at 13000rpm, 15min, 2-8°C, remove the supernatant Reconstitute with 0.3ml of PBS buffer, 100W ultrasound for 10s to disperse, then add 20μg of rabbit IgG, shake and react for 0.5h, add 0.1mg of glutaraldehyde solution after the reaction, and react in the dark for 1h, after the reaction is completed, 13000rpm, 15min, Centrifuge at 2-8°C, remove the supernatant, redissolve with 0.3ml of PBS buffer, 100W ultrasonic for 10s to disperse, then add 0.2ml of 0.5% casein solution, react for 30min, after the reaction, centrifuge at 10000rpm, 15min, 2-8°C Take the supernatant, then redissolve it with 0.3ml PBS buffer, disperse with 100W ultrasound for 10s, and store the fluorescently labeled rabbit IgG at 2-8°C un...

Embodiment 2

[0046] Example 2: Labeling of mouse IgG antibodies with time-resolved fluorescent microspheres

[0047] Measure 100 μl of time-resolved fluorescent microspheres (purchased from Bangs Company, particle size 200nm), add 0.2mL PBS buffer (0.01M, pH7.4), shake and mix well, centrifuge at 13000rpm, 15min, 2-8°C, remove the supernatant Reconstitute with 0.3ml of PBS buffer, disperse with 100W ultrasound for 10s, then add 20μg of mouse IgG, shake and react for 0.5h, add 0.1mg of glutaraldehyde solution after the reaction, and react in the dark for 1h. After the reaction, 13000rpm, 15min, Centrifuge at 2-8°C, remove the supernatant, redissolve with 0.3ml of PBS buffer, 100W ultrasonic for 10s to disperse, then add 0.2ml of 0.5% casein solution, react for 30min, after the reaction, centrifuge at 10000rpm, 15min, 2-8°C Take the supernatant, then redissolve it with 0.3ml PBS buffer, disperse with 100W ultrasound for 10s, and store the fluorescently labeled rabbit IgG at 2-8°C until use. ...

Embodiment 3

[0048] Example 3: Labeling of human IgG antibodies with time-resolved fluorescent microspheres

[0049] Measure 100 μl of time-resolved fluorescent microspheres (purchased from Bangs Company, particle size 200nm), add 0.2mL PBS buffer (0.01M, pH7.4), shake and mix well, centrifuge at 13000rpm, 15min, 2-8°C, remove the supernatant Reconstitute with 0.3ml of PBS buffer, 100W ultrasound for 10s to disperse, then add 20μg of human IgG, shake and react for 0.5h, add 0.1mg glutaraldehyde solution after the reaction, and react in the dark for 1h. After the reaction, 13000rpm, 15min, Centrifuge at 2-8°C, remove the supernatant, redissolve with 0.3ml of PBS buffer, 100W ultrasonic for 10s to disperse, then add 0.2ml of 0.5% casein solution, react for 30min, after the reaction, centrifuge at 10000rpm, 15min, 2-8°C Take the supernatant, then redissolve it with 0.3ml PBS buffer, disperse with 100W ultrasound for 10s, and store the fluorescently labeled rabbit IgG at 2-8°C until use.

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Abstract

The invention discloses an antibody labeling method for time resolved fluorescence microsphere coupling and application thereof. The labeling method disclosed by the invention comprises the followingsteps: (1) sufficiently mixing time resolved fluorescence microspheres with a buffer solution, and then performing low-temperature high-speed centrifugation; (2) redissolving precipitate after centrifugation, performing ultrasonic dispersion uniformly, adding a to-be-labeled antibody to a redissolving solution for oscillating reaction; (3) then adding a coupling agent into the solution in step (2)for coupling reaction, and performing low-temperature high-seed centrifugation; (4) redissolving the precipitate after centrifugation, then adding a confining liquid for closed reaction, and then performing low-temperature high-speed centrifugation; (5) redissolving the precipitate after centrifugation, and performing ultrasonic dispersion uniformly, so as to obtain the time resolved fluorescencemicrosphere labeled antibody. The labeling method disclosed by the invention is simple in structure, good in repeatability, and convenient for large-scale application; and the fluorescent antibody labeled by the method can be applied to various different fields of immune diagnosis, and provides a more convenient and rapid detection means for in vitro diagnosis.

Description

technical field [0001] The invention belongs to the technical field of biological analysis, in particular to a time-resolved fluorescent microsphere-coupled antibody labeling method and its application. Background technique [0002] When detecting certain antigens or specific proteins, immunolabeling techniques are usually applied. Immunolabeling technology is a technology that labels substances that are both easy to measure and highly sensitive to specific antigens or antibody proteins, and displays the nature and content of antigens or antibodies in the reaction system through the enhanced amplification of these markers. [0003] Currently commonly used labels include colloidal gold, latex, fluorescein, enzymes, and radionuclides. However, in practical applications, these immunolabeling techniques have different defects. For example, in colloidal gold and latex labeling technology, there are shortcomings of low sensitivity and narrow linear range, which cannot meet the m...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/543G01N33/533
CPCG01N33/533G01N33/54313
Inventor 汪惠泽欧阳云叶肖俊葛秀龙
Owner HANGZHOU LAIHE BIOTECH CO LTD
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