Preparation method of heavy metal zinc artificial antigen and application of DOTA in preparation of heavy metal zinc artificial antigen reagents

An artificial antigen and heavy metal technology, which is applied in the field of heavy metal ion immunochemistry, can solve the problems that affect the preparation efficiency of high-specificity and high-sensitivity antibodies, the characteristics of antigenic determinant antigens are not strong, and the characteristics of characteristic functional groups are not strong, so as to achieve high affinity, Strong specificity and high titer of antiserum

Active Publication Date: 2018-07-10
ZHEJIANG GONGSHANG UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Because these conventional chelating agents are ring-opening and straight-chain structures, the complex of heavy metal ions and chelating agents is relatively simple in spatial structure as the antigen recognition site, and the characteristic functional group features are not strong, resulting in the preparation of antigenic determinants. Antigen characteristics are not strong, which directly affects the production efficiency of highly specific and highly sensitive antibodies

Method used

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  • Preparation method of heavy metal zinc artificial antigen and application of DOTA in preparation of heavy metal zinc artificial antigen reagents
  • Preparation method of heavy metal zinc artificial antigen and application of DOTA in preparation of heavy metal zinc artificial antigen reagents
  • Preparation method of heavy metal zinc artificial antigen and application of DOTA in preparation of heavy metal zinc artificial antigen reagents

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Weigh 7mg DOTA, dissolve it in 2ml 4-hydroxyethylpiperazineethanesulfonic acid (HEPES) solution (0.01M, pH7.4), and prepare DOTA chelating agent solution, this reaction solution is A solution;

[0027] Weigh 111.56mg zinc nitrate and dissolve it in 5ml ultrapure water to prepare 7.5×10 -2 The zinc nitrate solution of M, this reaction liquid is B liquid;

[0028] Take 180 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;

[0029] 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;

[0030] Weigh 20mg of bovine serum albumin (BSA) and dissolve it in 4ml of HEPES, stir magnetically at room temperature and mix well, this reaction solution is solution E;

[0031] Add solution D to solution E dropwise, and react in the dark for 24 hours at room temperature, then add 150-200 μL of d...

Embodiment 2

[0070] Weigh 7mg DOTA, dissolve it in 2ml 4-hydroxyethylpiperazineethanesulfonic acid (HEPES) solution (0.01M, pH7.4), and prepare DOTA chelating agent solution, this reaction solution is A solution;

[0071] Weigh 111.56mg zinc nitrate and dissolve it in 5ml ultrapure water to prepare 7.5×10 -2 The zinc nitrate solution of M, this reaction liquid is B liquid;

[0072] Add 180 μl of liquid B to liquid A drop by drop, and react in the dark for 5 hours at room temperature, and this reaction liquid is liquid C;

[0073] 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;

[0074] Weigh 20mg chicken ovalbumin OVA and dissolve it in 4ml HEPES, stir and mix evenly at room temperature, this reaction solution is E solution;

[0075] Add solution D to solution E dropwise, and react in the dark for 24 hours at room temperature, then add 150-200 μL of diethyl malonate solution (84.6...

Embodiment 3

[0133] The antigens prepared in Example 1, Comparative Examples 1, 2, 3, 4 and 9 were respectively immunized to BALB / C mice, and the initial immunization adopted complete adjuvant emulsified antigens, and the amount was 250 μg / mouse (taking protein as the measurement unit ), followed by booster immunization at intervals of 21 days, a total of 3 booster immunizations, emulsified with incomplete adjuvant for booster immunization, immunization dose was 150 μg / mouse, and final immunization, which was carried out by direct intraperitoneal injection of antigen, Immunometry was 300 μg / mouse, and then blood was collected for titer determination of multiple antisera. The detected antigens were respectively Example 2, Comparative Examples 5, 6, 7, 8 and 10, and the results were as follows:

[0134]

[0135]

[0136]The above data show that the combination ratio and antiserum titer of Example 1 are all higher, and because of the lack of reaction step with diethyl malonate in Compara...

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Abstract

The invention discloses a preparation method of a heavy metal zinc artificial antigen. Specifically, 2-S-(4-aminobenzene)-1, 4, 7, 10tetrazacyclononane-1, 4, 7, 10-tetraacetic acid (p-NH2-Bn-DOTA, called DOTA for short) is adopted as the chelating agent, a zinc ion chelating agent compound is coupled with carrier protein bovine serum albumin BSA or chicken ovalbumin OVA to prepare the artificial antigen. The method is added with a diethyl malonate treatment step based on the original tradition, and improves the coupling efficiency and antiserum titer. The invention also discloses application of DOTA in preparation of heavy metal zinc artificial antigen reagents.

Description

technical field [0001] The invention belongs to the technical field of heavy metal ion immunochemistry, and in particular relates to a preparation method of a heavy metal zinc artificial antigen and the application of DOTA in the preparation of a heavy metal zinc artificial antigen reagent. Background technique [0002] Heavy metal pollution mainly refers to the pollution of lead, cadmium, mercury, nickel, chromium, arsenic, zinc, copper and other pollutants to the environment. 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 cause acute or chronic poisoning to the human body, has carcinogenic, teratogenic and mutagenic effects, and is very harmful to the human body. Due to their different properties, different heavy ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/765C07K14/77C07K1/34C07K1/107
CPCC07K14/765C07K14/77C07K19/00
Inventor 金仁耀郭建军
Owner ZHEJIANG GONGSHANG UNIVERSITY
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