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Preparation method and use of a nano-artificial antibody targeting cardiac troponin I

A cardiac troponin and artificial antibody technology, applied in the field of biomedicine, can solve the problems of large performance differences of monoclonal antibody batches, lack of selectivity and specificity, complex components of serum samples, etc., to overcome the long preparation cycle, high Effects of affinity, synthesis and simplicity of regeneration

Active Publication Date: 2021-06-08
BEIJING UNIV OF CHEM TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the complex composition of serum samples, the detection of low-abundance cTnI in real samples still lacks selectivity and specificity.
[0004] Natural antibodies are widely used in the selective recognition and detection of troponin. However, natural antibodies are mainly obtained by animal immunization, which has disadvantages such as high cost, low preparation efficiency, long screening cycle, variability, difficulty in preservation, and immunogenicity. Antibodies also have the problem of large batch-to-batch performance differences, which has great limitations in practical applications

Method used

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  • Preparation method and use of a nano-artificial antibody targeting cardiac troponin I
  • Preparation method and use of a nano-artificial antibody targeting cardiac troponin I
  • Preparation method and use of a nano-artificial antibody targeting cardiac troponin I

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1: Preparation of Cardiac Troponin I Artificial Antibody Based on Nanoparticle Three-Dimensional Structure Modification and Reconstruction Recognition Region

[0032] 1. Design and synthesis of nanoparticles

[0033] N-isopropylacrylamide (58-X mol%), charged monomer (3-acrylamidopropyl) trimethylammonium chloride (X mol%), N-tert-butylacrylamide ( 35 mol%), the crosslinker N,N'-methylenebisacrylamide (7 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 an excess of pure water, and polymer nanoparticles were obtained after freeze-drying.

[0034] 2. Preliminary screening of artificial antibodies

[0035] Troponin I was selected as the target, molecularly imprinted polymer nanoparticles were used as biomimet...

Embodiment 2

[0042] Example 2: Troponin I Nano-artificial Antibody as Ligand Combined with Monolithic Column Stationary Phase

[0043] First, a silane coupling agent needs to be bonded to the inner wall of the capillary column. Rinse the inner wall of the capillary column with acetone and pure water successively, pump through the capillary column with a sodium hydroxide solution with a concentration of 1 mol / L, so that it is fully and evenly filled with the capillary column, and then place it in an oven at 120°C . Then take out the capillary column that has completed the reaction, and rinse the sodium hydroxide in the capillary column with pure water until the pH of the solution pumped out of the capillary column is neutral. Then continue to wash with acetone and blow dry the capillary column with nitrogen, and put the capillary column in an oven at 120° C. without sealing to fully dry it. Dissolve 0.1 g of silane coupling agent 3-methacryloxypropyltrimethoxysilane in 0.9 g of anhydrous ...

Embodiment 3

[0045] Example 3: Nano-artificial antibody monolithic column realizes selective enrichment of low-abundance troponin in serum samples

[0046] Serum samples containing troponin I at a concentration of 0.001-10 μg / L were pumped into the monolithic column of nanoartificial antibodies through a microsyringe pump at a flow rate of 0.1-0.5 μL / min for selective enrichment, and after enrichment, 100 mM phosphoric acid was used to Buffer solution (pH=7) elutes impurity proteins non-specifically adsorbed on the monolithic column. Then the enriched troponin I was eluted, and the enrichment factor and recovery rate were calculated. Finally, the enrichment factor was 500, and the recovery rate was 98%.

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Abstract

The invention discloses a preparation method and application of a nano-artificial antibody targeting cardiac troponin I. A variety of functional monomers and cross-linking agents are polymerized to form a gel nanoparticle artificial antibody. By changing the ratio of functional monomers To regulate the affinity and selectivity of artificial antibodies to troponin I. Combined with molecular imprinting technology, the specificity and selectivity of nano-artificial antibodies to troponin I are further improved by using the whole protein or polypeptide fragment of troponin I as a template. Affinity constant K of nanoartificial antibodies to troponin I after screening D The value reaches 6.26×10 ‑11 M, comparable to antibodies, and has good selectivity. The obtained nano-artificial antibody combined with magnetic nanoparticles, monolithic column or microfluidic chip can achieve high selective enrichment of low-abundance cardiac troponin I in serum samples, and can be detected by high-sensitivity Raman spectroscopy and fluorescence quantification , ELISA kits, chemiluminescent kits, test strips and other methods to achieve high-sensitivity detection of troponin I.

Description

technical field [0001] The invention belongs to the field of biomedical technology, and in particular relates to a preparation method and application of a nano-artificial antibody targeting cardiac troponin I. The non-biological nano-artificial antibody has high affinity and high selectivity similar to natural antibodies and immune antibodies , can be used to replace natural antibodies and immune antibodies. Background technique [0002] Acute myocardial infarction is myocardial necrosis caused by acute and persistent coronary ischemia and hypoxia. Clinically, there are often severe and persistent substernal pains, which cannot be completely relieved by rest and nitrates, accompanied by increased serum myocardial enzyme activity and progressive ECG changes, which may be complicated by arrhythmia, shock or heart failure, often life-threatening. China has shown a clear upward trend in recent years, with at least 500,000 new cases and at least 2 million existing cases each yea...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C08F220/54C08F220/60C08F222/38C08J9/06G01N33/543G01N33/68
CPCC08F220/54C08J9/06C08J2201/0424C08J2333/24G01N33/54326G01N33/68C08F220/60C08F222/385
Inventor 吕永琴罗静宜付晓鹏
Owner BEIJING UNIV OF CHEM TECH
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