Preparation method of heavy metal cadmium artificial antigen and application of DOTA in preparation of heavy metal cadmium artificial antigen reagent

An artificial antigen and heavy metal technology, applied in the field of heavy metal ion immunochemistry, can solve the problems of weak functional group characteristics, affecting the preparation efficiency of high-specificity and high-sensitivity antibodies, and weak antigenic characteristics of antigenic determinants. The effect of strong specificity and high antibody affinity

Active Publication Date: 2018-06-12
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 cadmium artificial antigen and application of DOTA in preparation of heavy metal cadmium artificial antigen reagent
  • Preparation method of heavy metal cadmium artificial antigen and application of DOTA in preparation of heavy metal cadmium artificial antigen reagent
  • Preparation method of heavy metal cadmium artificial antigen and application of DOTA in preparation of heavy metal cadmium artificial antigen reagent

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 88.66mg cadmium nitrate and dissolve it in 5ml ultrapure water to prepare 7.5×10 -2 M cadmium nitrate solution, this reaction solution is B solution;

[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...

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 88.66mg cadmium nitrate and dissolve it in 5ml ultrapure water to prepare 7.5×10 -2 M cadmium nitrate solution, this reaction solution is B solution;

[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...

Embodiment 3

[0133] The antigens prepared in Example 1, Comparative Examples 1, 2, 3, 4 and 9 were immunized to BALB / C mice respectively, and the initial immunization adopted complete adjuvant emulsified antigens, and the amount was 250 μg / mouse (taking protein as the unit of measurement) ), 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 binding ratio and antiserum titer of Example 1 are all higher, because the reaction step with sodium borohydride is lacking in comparat...

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Abstract

The invention discloses a preparation method of heavy metal cadmium artificial antigen, which applies 2-S-(4- aminobenzene)-1, 4, 7, 10 tetraazacyclo cyclononane-1, 4, 7, 10-acetate tetrahydrate(p-NH2-Bn-DOTA, DOTA for short) as cheating agent, and couples cadmium ion chelating agent composite with carrier protein bovine serum albumin BSA or egg albumin OVA; thus the artificial antigen is prepared. The method adds the sodium borohydride processing step on the basis of traditional method, and improves the coupling efficiency and antiserum titre. The invention further discloses an application ofDOTA in preparation of the heavy metal cadmium artificial antigen.

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 heavy metal cadmium artificial antigen and the application of DOTA in the preparation of heavy metal cadmium 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. Therefore, strengthening the detection of heavy m...

Claims

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Application Information

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