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Signal amplification type immunofluorescence probe as well as preparation method and application thereof

An immunofluorescence and amplification technology, applied in fluorescence/phosphorescence, measuring devices, instruments, etc., can solve the problems of high noise interference, low signal, and difficulty in detection by traditional methods, and achieve high detection sensitivity, simple equipment, and shortened detection the effect of time

Inactive Publication Date: 2012-07-11
吴坚
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, when dealing with very small amounts of substances, this detection method also has problems such as low signal, large noise interference, and difficulty in detection by traditional methods.

Method used

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  • Signal amplification type immunofluorescence probe as well as preparation method and application thereof
  • Signal amplification type immunofluorescence probe as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1 Preparation of Signal Amplified Immunofluorescence Probe

[0044] (1) In a three-necked flask, dissolve 1.08g (10mmol) of p-phenylenediamine and 2.10g (10mmol) of trimesic acid in 80ml of N-methyl-2-pyrrolidone under stirring, and then add Mixture 7.5ml of pyridine and 20mmol of triphenylphosphite, stirring continuously and heating to 80°C, after the mixture dissolves into a homogeneous phase, continue to stir and react for 3h; after stopping the reaction, dissolve the reaction mixture in 500ml of methanol containing hydrochloric acid (the concentration of hydrochloric acid 12mol / L), filtered to obtain the precipitated product; the crude product was recrystallized with N-methyl-2-pyrrolidone / methanol, the final product was washed with hot methanol, and dried in an oven at 100°C for 12h; polycarboxy Macromolecules, named PT macromolecules.

[0045] (2) Dissolve 5 mg of PT macromolecule in step (1) in a solution of 10ml PBS (10mM pH 7-8) and 1ml DMSO; take 30ul...

Embodiment 2

[0047] Example 2 Preparation of dot fluorescence immunoassay detection box

[0048] (1) Coat the antibody at a concentration of 10ug / ml on a nitrocellulose membrane (NC membrane), wash with 10mM PBS (pH 7-8) for 3-5 times, and dry at 37°C for later use.

[0049] (2) Open the small plastic box and divide it into a bottom and a cover. The installation sequence from bottom to top is: bottom, water-absorbing material, nitrocellulose membrane, and cover.

Embodiment 3

[0050] Embodiment 3 dot fluorescence immune penetration test detects AFP (sandwich method)

[0051] (1) First use the AFP standard to prepare a series of standard products with concentrations of 0ppb, 1ppb, 5ppb, 20ppb, 50ppb, 100ppb, 200ppb, and 400ppb.

[0052] (2) Take the detection box prepared in Example 2, drop 40ul of blocking solution (10mM PBS, pH 7-8, containing 0.2% BSA and 0.05% Tween-20) on the NC membrane of the detection box, and seal the NC membrane. Wait for it to seep into the box.

[0053] (3) Add 10ul standard concentration of AFP solution dropwise on the NC membrane, and add dropwise 30ul washing solution (50mM PBS, pH 7-8); after the solution is slightly dry, add dropwise 10ul diluted 1000 times prepared The obtained signal amplification type immunofluorescence probe solution (diluent is 10mM PBS solution containing 1%BSA and 1% sucrose, pH 7.2), and 30ul washing liquid is added dropwise; Detect and read the fluorescence signal (each sample was detected...

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Abstract

The invention discloses a signal amplification type immunofluorescence probe as well as a preparation method and application thereof. The preparation method of the probe includes the steps as follows: carrying out condensation reaction of phenylene diamine and trimesic acid to obtain poly-carboxylic macromolecules under the premise that condensing agent exists; and activating the poly-carboxylic macromolecules, and adding an antibody, polylysine sequentially for reaction; labeling the prepared probe through fluorescence label objects. Or the preparation method of the probe includes the steps as follows: adding the antibody and the polylysine in a biotin solution sequentially for reaction, so as to obtain the corresponding compound; labeling the compound through the fluorescence label objects; and mixing the antibody-biotin compound labeled by fluorescence and a streptavidin solution for reaction, so as to obtain the probe. The probe prepared through adopting the method is labeled by more fluorescence label objects, has a stable structure and is easy to store, can be used for fluorescence immune detection, and has the characteristics of high detection sensitivity, short detection time, low cost and the like.

Description

technical field [0001] The invention relates to the technical field of rapid immunoanalysis, in particular to a signal-amplifying immunofluorescence probe and a preparation method and application thereof. Background technique [0002] Immunoassay technology is one of the most basic and commonly used detection technologies in the fields of medicine, biology, food safety and so on. It is widely used in medical biological research, clinical medicine, environmental testing, and detection of food and microbial contamination. Immunoassay technology is a detection method based on the principle of antibody-antigen reaction for quantitative and qualitative analysis of the test object. It has the advantages of strong specificity, high sensitivity, and simplicity. It is an important research method in modern life sciences and has a wide range of applications in the field of biological analysis and detection Application prospects. [0003] Fluorescence immunoassay refers to a method t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/533G01N21/64
Inventor 吴坚陈春香
Owner 吴坚
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