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Preparation method and use of a non-biological nano-artificial antibody specifically targeting Alzheimer's disease markers

An artificial antibody, non-biological technology, applied in the field of bio-nanomedicine, can solve the problems of long screening period, immunogenicity and variability, and achieve the effects of overcoming long preparation period, shortening screening time and high stability

Active Publication Date: 2021-03-26
BEIJING UNIV OF CHEM TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, natural antibodies are mainly obtained by animal immunization, which has the disadvantages of high cost, low preparation efficiency, long screening cycle, variability, difficulty in storage, and immunogenicity. Monoclonal antibodies also have the problem of large batch performance differences. In practical applications has great limitations

Method used

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  • Preparation method and use of a non-biological nano-artificial antibody specifically targeting Alzheimer's disease markers
  • Preparation method and use of a non-biological nano-artificial antibody specifically targeting Alzheimer's disease markers
  • Preparation method and use of a non-biological nano-artificial antibody specifically targeting Alzheimer's disease markers

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1: Preparation of non-biological nano-artificial antibodies targeting pathological markers of Alzheimer's disease based on three-dimensional structure modification and reconstruction of nanoparticles

[0030] 1. Design and synthesis of nanoparticles

[0031] N-isopropylacrylamide (58-X mol%), charged monomer N-(3-dimethylaminopropyl) methacrylamide (X mol%), N-tert-butylacrylamide ( 40 mol%), the crosslinker N,N'-methylenebisacrylamide (2 mol%) and sodium dodecylsulfonate (10 mg) were dissolved in water to give a total monomer concentration of 130 mM. After adding the initiator, polymerization was carried out at 65° C. for 3 hours with a magnetic stirrer under a nitrogen atmosphere. The polymerized solution was purified by dialysis against excess pure water, and the polymer nanoparticles were obtained after freeze-drying ( figure 1 ).

[0032] 2. Preliminary screening of artificial antibodies

[0033] The selected target protein is β-amyloid protein, but the...

Embodiment 2

[0036] Example 2: Raman imaging of β-amyloid nanoartificial antibodies on brain tissue slices

[0037] First, to synthesize gold nanoparticles with smaller diameter, 0.0255g HAuCl 4 Dissolve in 255g of water, heat to boil and then add 15g of 1% sodium citrate aqueous solution to the solution. After the solution turns wine red, continue to boil for 15min, then cool naturally to room temperature. The gold nanospheres were then concentrated 20 times by centrifugation. The concentrated nano-gold balls are connected to the dye DTNB, and the nano-gold balls connected to the dye are added in the process of synthesizing the nanoparticles, and the nano-gold balls are wrapped into the nanoparticles, and the transmission electron microscope (TEM) and the scanning electron microscope ( SEM) such as image 3 shown. Carry out Raman depth scanning to determine the characteristic peaks ( Figure 4 ).

[0038] Then, the paraffin-encapsulated human brain tissue slices were treated with xyl...

Embodiment 3

[0039] Example 3: Fluorescent imaging of β-amyloid nanoartificial antibodies on brain tissue slices

[0040] Firstly, after dialyzing the synthesized nanoparticles, activate the nanoparticles. 1 mL of nanoparticle solution needs to add 5 mg of EDC and 2.5 mg of NHS, and activate it in a shaker at 4° C. for 12 hours. Then, 10 μL of FITC was added to each mL of nanoparticle suspension, and reacted in a shaker at 4° C. for 24 h, protected from light.

[0041] Then, the paraffin-encapsulated human brain tissue slices were treated with xylene and gradient concentration of ethanol, and the synthesized nanoparticles were added dropwise to the slices, and the slices were incubated in a humid box at room temperature for 60 min, and then washed with PBS buffer. The slices were rinsed with 0.1M NaCl solution and dried with nitrogen gas. Laser confocal imaging was then performed.

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Abstract

The invention discloses a preparation method and application of an abiotic nano artificial antibody specifically targeting an Alzheimer's disease marker. A plurality of functional monomers and a cross-linking agent are polymerized according to different components to form a gel nanoparticle artificial antibody, and the affinity and selectivity of the artificial antibody to an Alzheimer's disease pathological marker are regulated and controlled by changing the ratio of the functional monomers. The biological membrane interference technology is used for testing the capacity of the synthesized nano artificial antibody for effectively capturing the Alzheimer's disease pathological marker; the affinity constant KD value of the nano artificial antibody to the marker can reach 8.26 * 10 <-10 > M;the abiotic nano artificial antibody is equivalent to an antibody, and has excellent selectivity, high-selectivity enrichment of low-abundance Alzheimer's disease pathological markers in a serum sample can be realized by combining magnetic nanoparticles, a monolithic column or a micro-fluidic chip; high-sensitivity detection of the Alzheimer's disease pathological marker can be realized through high-sensitivity Raman spectroscopy, fluorescence quantitative detection, ELISA kits, chemiluminescence kits and other methods.

Description

technical field [0001] The invention belongs to the field of bio-nano medicine, in particular to a preparation method and application of a non-biological nano-artificial antibody specifically targeting Alzheimer's disease markers. Background technique [0002] Alzheimer's disease (AD) is a progressive neurodegenerative disease, an incurable, progressive and fatal degenerative neurological disease. The disease leads to the impairment of the patient's listening, speaking, reading and writing skills, making it impossible for normal people to live, thus aggravating the burden of family life. As the number of elderly people in China reaches its peak, the number of AD patients will also increase sharply. By 2020, the number of AD patients in China will reach 8.93 million, which will bring a heavy burden to the society. Cerebral amyloid angiopathy (CAA), senile plaques, and ganglion tangles are recognized as the three major pathological features of Alzheimer's disease, but the exa...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C08F220/54C08F220/60C08F222/38C08J9/26G01N1/28G01N21/64G01N21/65
CPCC08F220/54C08J9/26C08J2201/0424C08J2333/24G01N1/28G01N21/6458G01N21/65C08F220/60C08F222/385
Inventor 吕永琴王子洁
Owner BEIJING UNIV OF CHEM TECH
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