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A improvement method for improving the synthesis of heavy metal copper artificial antigen and the purpose of NOTA in preparing heavy metal copper artificial antigen reagents

A technology of artificial antigen and synthesis method, which is applied in the direction of peptide preparation method, chemical instrument and method, hybrid peptide, etc., can solve the problem of weak characteristic functional groups, weak antigenic determinant antigenic characteristics, affecting high specificity and high Sensitivity, antibody preparation efficiency and other issues, to achieve high affinity and strong specificity

Active Publication Date: 2020-12-22
ZHEJIANG GONGSHANG UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

These conventional chelating agents are due to the ring-opening and straight-chain structures combined with heavy metal ions, the complex of heavy metal ions and chelating agents as the antigen recognition site is relatively simple in spatial structure, and the characteristic functional group characteristics are not strong, resulting in the prepared The antigenic determinant antigenic characteristics are not strong, which directly affects the preparation efficiency of highly specific and highly sensitive antibodies

Method used

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  • A improvement method for improving the synthesis of heavy metal copper artificial antigen and the purpose of NOTA in preparing heavy metal copper artificial antigen reagents
  • A improvement method for improving the synthesis of heavy metal copper artificial antigen and the purpose of NOTA in preparing heavy metal copper artificial antigen reagents
  • A improvement method for improving the synthesis of heavy metal copper artificial antigen and the purpose of NOTA in preparing heavy metal copper artificial antigen reagents

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Weigh 6 mg NOTA, dissolve it in 2 ml 4-hydroxyethylpiperazineethanesulfonic acid (HEPES) solution (0.01 mol / L, pH7.4), and prepare NOTA chelating agent solution, this reaction solution is A solution;

[0028] Weigh 70.33 mg copper nitrate and dissolve it in 5 ml ultrapure water to prepare 7.5X 10-2 The copper nitrate solution of M, this reaction solution is B liquid;

[0029] Take 185 μl of liquid B and add it dropwise to liquid A, and react in the dark for 3 hours at room temperature, and this reaction liquid is liquid C;

[0030] Add 650 μl, 20 mM glutaraldehyde solution dropwise to solution C, and react overnight at room temperature in the dark, this reaction solution is solution D;

[0031] Weigh 20 mg of bovine serum albumin (BSA) A and dissolve it in 3 ml of HEPES, stir and mix evenly at room temperature, this reaction solution is solution E;

[0032] Add solution D to solution E dropwise, and react in the dark for 24 hours at room temperature, then add 200 μl s...

Embodiment 2

[0036]Weigh 7 mg NOTA, dissolve it in 2 ml 4-hydroxyethylpiperazineethanesulfonic acid (HEPES) solution (0.01 mol / L, pH7.4), and prepare NOTA chelating agent solution, this reaction solution is A solution;

[0037] Weigh 70.33 mg copper nitrate and dissolve it in 5 ml ultrapure water to prepare 7.5X 10-2 The copper nitrate solution of M, this reaction solution is B liquid;

[0038] Take 200 μl of liquid B and add it dropwise to liquid A, and react in the dark for 5 hours at room temperature, and this reaction liquid is liquid C;

[0039] Add 700 ul, 20 mM glutaraldehyde solution dropwise to liquid C, and react overnight at room temperature in the dark, this reaction liquid is liquid D;

[0040] Weigh 20 mg chicken ovalbumin OVA and dissolve it in 3 ml HEPES, and stir magnetically at room temperature to mix well. This reaction solution is E solution;

[0041] Add solution D to solution E dropwise, and react in the dark for 24 hours at room temperature, then add 200 ul sodium ...

Embodiment 3

[0045] Weigh 7 mg NOTA, dissolve it in 2 ml 4-hydroxyethylpiperazineethanesulfonic acid (HEPES) solution (0.01 mol / L, pH7.4), and prepare NOTA chelating agent solution, this reaction solution is A solution;

[0046] Weigh 70.33 mg copper nitrate and dissolve it in 5 ml ultrapure water to prepare 7.5X 10-2 The copper nitrate solution of M, this reaction solution is B liquid;

[0047] Take 200 μl of liquid B and add it dropwise to liquid A, and react in the dark for 5 hours at room temperature, and this reaction liquid is liquid C;

[0048] Add 700 μl, 20 mM glutaraldehyde solution dropwise to solution C, and react overnight at room temperature in the dark, this reaction solution is solution D;

[0049] Weigh 20 mg of bovine serum albumin BSA and dissolve it in 3 ml of HEPES, stir magnetically at room temperature and mix well, this reaction solution is solution E;

[0050] Add liquid D dropwise to liquid E, and react in the dark for 24 hours at room temperature to obtain liqui...

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Abstract

The invention provides a synthesis method for preparing a heavy metal copper artificial antigen based on a novel difunctional chelating agent NOTA (2-S-(4-amino-benzene)-1,4,7-triazacyclononane-1,4,7-triacetic acid). According to the preparation method, NOTA is taken as the chelating agent, a copper ion chelating agent compound is coupled with BSA (bovine serum albumin) as carrier protein or chicken OVA (ovalbumin), and the artificial antigen is prepared. According to the method, the step of sodium borohydride reduction is added on the original traditional basis, so that the coupling efficiency is improved. The invention further provides an application of NOTA to preparation of the heavy metal copper artificial antigen.

Description

technical field [0001] The invention relates to a synthesis technology of an artificial antigen targeting heavy metal copper using a novel bifunctional chelating agent NOTA as a linking carrier, which belongs to the technical field of artificial antigen preparation in immunochemical technology and can be used for subsequent high-specificity anti-heavy metal copper antibodies Research on preparation and immunoassay technology, and used for the detection of trace and constant heavy metal copper in environmental samples. Background technique [0002] Heavy metal pollution mainly refers to lead, cadmium, mercury, nickel, chromium, arsenic, zinc, copper and other pollutants. Heavy metals are widely distributed and difficult to degrade. They can enter the human body through the atmosphere, water, and food chain, and interact with proteins and various enzymes in the body to deactivate them and accumulate in certain organs. If they exceed the tolerance limit of the human body , can...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/765C07K14/77C07K1/107
CPCC07K14/765C07K14/77C07K19/00
Inventor 金仁耀郭建军
Owner ZHEJIANG GONGSHANG UNIVERSITY
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