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Glycosylation site blocking/protein immobilization magnetic nanoparticles

A technology of magnetic nanoparticles and glycosylation sites, used in instruments, measuring devices, scientific instruments, etc., can solve the problems of complex preparation process of enzyme-labeled antibodies, false positive reactions of cross-reaction, and high requirements for storage conditions, etc. Low cost of raw materials, reduced transportation and storage costs, convenient and simple separation process

Pending Publication Date: 2022-05-06
TIANJIN MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, they all have certain limitations. The preparation process of enzyme-labeled antibodies in ELISA is complicated and time-consuming, and the storage conditions are high and inconvenient to transport; the radioactive substances in radioimmunoassay have high requirements on the experimental environment, and there will be crossover. Reactions and false positive reactions; electrochemical instruments are expensive, bulky, and the number of detection chips is too large, which still needs to be improved; in fluorescence immunoassays, low fluorescence intensity, detection difficulties caused by strong background, and fluorescence quenching caused by biological binding, etc.

Method used

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  • Glycosylation site blocking/protein immobilization magnetic nanoparticles
  • Glycosylation site blocking/protein immobilization magnetic nanoparticles
  • Glycosylation site blocking/protein immobilization magnetic nanoparticles

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0120] Example 1 Preparation and Characterization of Glycosylation Site Blocking / Protein Immobilization Magnetic Nanoparticles

[0121] Glycosylation site blocking / protein immobilized magnetic nanoparticles were prepared as antibody capture materials. Firstly, ferroferric oxide magnetic nanoparticles were coated with tetraethoxysilane self-polymerization, and 3-aminopropyltriethyl Oxysilane connects amino groups on the surface, then grafts glutaraldehyde to introduce aldehyde groups, then immobilizes the antigen human epidermal growth factor receptor-2 protein and passivates the active site, and finally uses the masking agent pentafluorophenylboronic acid to deactivate the sugar Kylation sites are blocked. Perform transmission electron microscopy and infrared characterization of the prepared antibody capture material. The specific steps are as follows:

[0122] Step 1: Disperse 80.10-88.50% (both mass fraction) of ferroferromagnetic nanoparticles in 9.10-17.60% absolute ethan...

Embodiment 2

[0131] Example 2 Effects of Different Masking Agents on Glycosylation Site Blocking / Protein Immobilization Magnetic Nanoparticles

[0132] The preparation method is the same as in Example 1, only the type of masking agent is changed, and the masking agent is as follows: 4-ethylphenylboronic acid, 4-methylbenzeneboronic acid, 4-fluorophenylboronic acid, 4-trifluoromethylphenylboronic acid, 4- Hydroxyphenylboronic acid, 2,3-difluorophenylboronic acid, 3,5-difluorophenylboronic acid, 3,4,5-trifluorophenylboronic acid, 2,3,5,6-tetrafluorophenylboronic acid, pentafluorophenylboronic acid Dissolve the same mass of masking agent in phosphate buffered saline solution (pH=7.4, 20mM), respectively add to ferroferric oxide magnetic nanoparticles with active site passivation, shake at room temperature for 2-5h to carry out the glycosylation site For blocking, wash 3 times with phosphate buffered saline (pH=7.4, 20 mM). Take 0.05mg and add artificial secondary antibody, shake at room temp...

Embodiment 3

[0134] Example 3 Stability detection of glycosylation site blocking / protein immobilization magnetic nanoparticles

[0135] The stability of the glycosylation site blocking / protein immobilized magnetic nanoparticles was investigated, and the effect of its use was tested every ten days during the two months of storage at 4°C. The specific operation steps are as follows:

[0136] Preparation of the glycosylation site blocking / protein immobilization magnetic nanoparticles described in Example 1 and the fluorescent signal amplifier with graphene oxide as the carrier mentioned in the publication number CN113512417A patent application.

[0137] Take 0.05mg glycosylation site blocking / protein immobilization magnetic nanoparticles and add 0.1mL 10ng mL -1 Herceptin solution, incubated at 37°C for 1 h, washed three times with phosphate buffered saline (pH=7.4, 20 mM), added 250 μL of a fluorescent signal amplifier with graphene oxide as the carrier, shaken at room temperature for 30 min...

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Abstract

The invention discloses a glycosylation site blocking / protein immobilization magnetic nanoparticle which is prepared by the following steps: by taking a ferroferric oxide magnetic sphere as a carrier, coating the surface of the carrier with a layer of silicon dioxide, further modifying amino and aldehyde groups, then connecting an antigen to the aldehyde groups, and sealing active sites by using bovine serum albumin, so as to obtain the glycosylation site blocking / protein immobilization magnetic nanoparticle. And finally, adding a glycosylation site screening agent of the antigen for blocking. The magnetic nanoparticles can realize specific capture or separation of an antibody. According to the present invention, the magnetic nanoparticles and the fluorescence signal amplifier using the graphene oxide as the carrier are combined, such that the antibody detection sensitivity is high, the detection range is wide, and compared with the enzyme-linked immunosorbent assay technology, the advantages of short time consumption, simple preparation, low price, simple storage and transportation conditions, good stability and the like are provided.

Description

technical field [0001] The invention relates to the field of biological detection, and relates to glycosylation site blocking / protein immobilization magnetic nanoparticles. Background technique [0002] In the development of many diseases, such as tumors, inflammation and nervous system diseases, the human immune system can resist the disease by producing corresponding antibodies. Therefore, specific antibodies can be used as an important indicator of disease progression, and antibody detection plays a vital role in the diagnosis of diseases, especially immune and infectious diseases. In addition, antibody drugs are one of the fastest growing biopharmaceuticals in recent years. Quantitative detection of antibody-based drugs is of great significance for clinical evaluation of drug delivery efficiency, tracking the toxicity of drug therapy, and judging the therapeutic effect of diseases. Since human body fluids are rich in various antibodies, and the type and quantity of ant...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/543G01N33/533G01N33/532
CPCG01N33/6854G01N33/54326G01N33/54346G01N33/533G01N33/532
Inventor 王涎桦董林毅白晨晨
Owner TIANJIN MEDICAL UNIV
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