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Preparation method and uses of nanometer artificial antibody targeting brain natriuretic peptide

A brain natriuretic peptide and artificial antibody technology, applied in the field of biomedicine, can solve the problems of lack of selectivity and specificity of low-abundance BNP, large differences in the performance of monoclonal antibody batches, complex composition of plasma samples, etc., and achieve the synthesis process. Simple regeneration process, overcoming the effect of long preparation cycle and shortening screening time

Inactive Publication Date: 2019-12-13
BEIJING UNIV OF CHEM TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Under normal circumstances, the level of BNP in plasma is very low, only 0-100ng / L. In the case of heart failure, the natriuretic peptide system can be stimulated to be activated and release BNP. However, due to the complex composition of plasma samples, it is currently true Detection of low-abundance BNP in samples still lacks selectivity and specificity
[0004] Natural antibodies are widely used in the selective recognition and detection of BNP. 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 preservation, and immunogenicity. There is a problem of large batch performance differences, which has great limitations in practical applications

Method used

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  • Preparation method and uses of nanometer artificial antibody targeting brain natriuretic peptide
  • Preparation method and uses of nanometer artificial antibody targeting brain natriuretic peptide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1: Preparation of artificial antibodies targeting BNP based on the three-dimensional structure modification of nanoparticles and the reconstruction of the recognition region

[0026] 1. Design and synthesis of nanoparticles

[0027] N-isopropylacrylamide (58-X mol%), charged functional monomer (3-acrylamidopropyl) trimethylammonium chloride (X mol%), N-tert-butylacrylamide (35mol %), 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.

[0028] 2. Preliminary screening of artificial antibodies

[0029] BNP was selected as the target object, molecularly imprinted polymer nanoparti...

Embodiment 2

[0033] Example 2: The nano-artificial antibody targeting BNP is combined with the monolithic column stationary phase as a ligand

[0034]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 ...

Embodiment 3

[0036] Example 3: Nano-artificial antibody monolithic column realizes selective enrichment of low-abundance BNP in serum samples

[0037] Serum samples containing BNP at a concentration of 0.001 to 10 μg / L were pumped into the monolithic column of nanoartificial antibodies through a microsyringe pump at a flow rate of 0.1 to 0.5 μL / min for selective enrichment. After enrichment, 100 mM phosphate buffer solution ( pH=7) to elute impurity proteins non-specifically adsorbed on the monolithic column. Then the enriched BNP was eluted, and the enrichment factor and recovery rate were calculated.

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Abstract

The invention discloses a preparation method and uses of a nanometer artificial antibody targeting brain natriuretic peptide. According to the present invention, a plurality of functional monomers anda cross-linking agent are polymerized to form a gel nanoparticle artificial antibody, wherein the affinity and the selectivity of the artificial antibody to BNP are regulated by changing the ratio ofthe functional monomers; by combining with a molecular imprinting technology, the specificity and the selectivity of the nanometer artificial antibody to BNP are further improved by using the full polypeptide or partial polypeptide fragments of the BNP as a template; after screening, the affinity constant KD value of the nanometer artificial antibody to BNP reaches 1.41*10<-11> M, and is equivalent to the affinity constant KD value of antibodies, and the nanometer artificial antibody has good selectivity; the obtained nanometer artificial antibody can achieve the high-selectivity enrichment of low-abundance BNP in serum samples by combing with magnetic nanometer particles, monolithic columns or micro-fluidic chips, and can achieve high-sensitivity detection of BNP through high-sensitivityRaman spectroscopy, fluorescence quantitative detection, ELISA kits, chemiluminescence kits, test paper strips and other methods.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to a preparation method and application of a nano-artificial antibody targeting brain natriuretic peptide (BNP). The non-biological nano-artificial antibody has high affinity and high Selective, can be used to replace natural antibodies and immune antibodies. Background technique [0002] Heart failure is a common clinical cardiovascular disease, the ultimate destination of most cardiovascular diseases, and has a high mortality rate. It is myocardial injury caused by myocardial infarction, cardiomyopathy, hemodynamic overload, inflammation, etc. Changes in myocardial structure and function eventually lead to a complex clinical syndrome of ventricular pumping or filling dysfunction. In recent years, with the progress of society, the improvement of people's living standards, the proportion of elderly population has been increasing, and the number of patients with hear...

Claims

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Application Information

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IPC IPC(8): C08F220/54C08F220/60C08F222/38C08J9/26G01N33/543G01N33/68
CPCC08F220/54C08J9/26C08J2201/0424C08J2333/24G01N33/54326G01N33/68C08F220/60C08F222/385
Inventor 吕永琴王子洁谭天伟
Owner BEIJING UNIV OF CHEM TECH
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