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Preparation method of heavy metal zinc artificial antigen and application of NOTA in preparation of heavy metal zinc 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, weak antigenic determinant antigen characteristics, affecting the preparation efficiency of high specificity and high sensitivity antibodies, etc. The effect of strong specificity and high antiserum titer

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 zinc artificial antigen and application of NOTA in preparation of heavy metal zinc artificial antigen reagent
  • Preparation method of heavy metal zinc artificial antigen and application of NOTA in preparation of heavy metal zinc artificial antigen reagent
  • Preparation method of heavy metal zinc artificial antigen and application of NOTA in preparation of heavy metal zinc artificial antigen reagent

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Weigh 7mg NOTA and dissolve it in 2ml 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES) solution (0.01M, pH7.4) to prepare a NOTA 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 M zinc nitrate solution, this reaction solution is B solution;

[0028] Pipette 180μl of liquid B into liquid A drop by drop, and react for 3h in the dark at room temperature. This reaction liquid is liquid C;

[0029] Add 700μl of 20mM glutaraldehyde solution dropwise to solution C, and react overnight in the dark at room temperature. This reaction solution is solution D;

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

[0031] Add liquid D to liquid E drop by drop, and react for 24 hours in the dark at room temperature, then add 150-200 μL sodium borohydride solution (20 mg dissolved in...

Embodiment 2

[0070] Weigh 7mg NOTA, and dissolve it in 2ml 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES) solution (0.01M, pH7.4) to 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 M zinc nitrate solution, this reaction solution is B solution;

[0072] Pipet 180μl of liquid B and add it to liquid A dropwise, and react for 5h in the dark at room temperature. This reaction liquid is liquid C;

[0073] Add 700μl of 20mM glutaraldehyde solution dropwise to solution C, and react overnight in the dark at room temperature. This reaction solution is solution D;

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

[0075] Add liquid D to liquid E drop by drop, and react for 24 hours in the dark at room temperature, then add 150-200 μL sodium borohydride solution (20 mg...

Embodiment 3

[0133] The antigens prepared in Example 1, Comparative Examples 1, 2, 3, 4, and 9 were respectively immunized against BALB / C mice. The first immunization was performed with a complete adjuvant to emulsify the antigen, and the measurement was 250 μg / mouse (with protein as the measurement unit). ), every 21 days after the booster immunization, a total of 3 boosters, the booster immunization is emulsified with an incomplete adjuvant, the immunological weight is 150 μg / mouse, and the final immunization is performed. The final immunization is carried out by direct intraperitoneal injection of the antigen. The immunoassay was 300 μg / mouse, and the blood was drawn into the titer of polyantiserum. The detected antigens were Example 2, Comparative Examples 5, 6, 7, 8 and 10 respectively. The results are as follows:

[0134]

[0135] The above data shows that the binding ratio and antiserum titer of Example 1 are both higher. Due to the lack of the reaction step with sodium borohydride in C...

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Abstract

The invention discloses a preparation method of heavy metal zinc artificial antigen, which applies 2-S-(4- aminobenzene)-1, 4, 7, 10 tetraazacyclo cyclononane-1, 4, 7, 10-acetate tetrahydrate(p-NH2-Bn-NOTA, NOTA for short) as cheating agent, and couples mercury 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 in the past, and improves the coupling efficiency and antiserum titre. The invention further discloses an application of NOTA in preparation of the heavy metal zinc artificial antigen.

Description

Technical field [0001] The invention belongs to the technical field of heavy metal ion immunochemistry, and specifically relates to a preparation method of heavy metal zinc artificial antigen and the application of NOTA in the preparation of 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 make them inactive and accumulate in certain organs. If they exceed the limit that the human body can tolerate , Can cause acute or chronic poisoning of human body, have carcinogenic, teratogenic and mutagenic effects, and have great harm to human body. Therefore, strengthening the detection of heavy metal residu...

Claims

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Application Information

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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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