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Method for preparing heavy metal holoantigen with high coupling rate and high activity

A technology with high activity and coupling rate, applied in the field of immunochemistry and residue analysis biology, can solve the problems of protein denaturation, low activity, poor immune effect, etc., and achieve high coupling rate, good immunogenicity and high repeatability Effect

Inactive Publication Date: 2013-01-16
NANCHANG UNIV
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AI Technical Summary

Problems solved by technology

[0005] Aiming at the characteristic that protein denaturation is easily caused by rapid and massive combination of heavy metal and protein, the present invention aims to solve the problems of low coupling rate, low activity and poor immune effect of the whole antigen synthesized by the existing method, improve the traditional one-step chelation method, and prepare Heavy metal whole antigen with high coupling rate, high activity and good immunogenicity

Method used

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  • Method for preparing heavy metal holoantigen with high coupling rate and high activity
  • Method for preparing heavy metal holoantigen with high coupling rate and high activity

Examples

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Effect test

Embodiment 1

[0023] Embodiment 1, take the preparation of lead whole antigen as an example:

[0024] 1. Preparation of the solution: Weigh 103.5 mg of lead pellets and dissolve them in 1 mL of concentrated nitric acid, react until the lead is completely dissolved, and prepare a lead nitrate solution with a concentration of 0.5 mol / L. Use 0.1M N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid HBS solution at pH7.4 as the buffer system, and prepare a concentration of 4 × 10 ?3 mol / L bifunctional chelating agent p-NH 2 -Bn-CHX-A"-DTPA solution;

[0025] 2. Preparation of lead ion-chelating agent complex: take 5 ml of the bifunctional chelating agent solution prepared in step 1, add 80 μL of lead nitrate solution prepared in step 1 with shaking, add one drop every 2 s; 8 h, take it out, adjust the pH to 7.0 with 0.1 mol / L NaOH solution, centrifuge at 3000r / min for 10min to remove the precipitate, and obtain solution A;

[0026] 3. Preparation of lead whole antigen: put the above solution A...

Embodiment 2

[0027] Embodiment 2, take the preparation of cadmium whole antigen as an example:

[0028] 1. Preparation of the solution: Weigh 91.7 mg of cadmium chloride and dissolve it in 1 mL of ultrapure water, dissolve completely, and prepare a cadmium chloride solution with a concentration of 0.5 mol / L. Use 0.1M, pH7.4 N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid HBS solution as the buffer system, and prepare a concentration of 10 × 10 ?3 mol / L bifunctional chelating agent P-Nh2-Bn-Chx-A"-DTPA solution;

[0029] 2. Preparation of cadmium ion-chelating agent complex: Take 3 ml of the bifunctional chelating agent solution prepared in step 1, add 240 μL of cadmium chloride solution prepared in step 1 with shaking, add one drop every 2 seconds; React for 12 hours, take it out, adjust the pH to 9.0 with 0.1 mol / L NaOH solution, centrifuge at 3000r / min for 10min to remove the precipitate, and obtain solution A;

[0030] 3. Preparation of cadmium whole antigen: put the above-mentioned ...

Embodiment 3

[0031] Embodiment 3, taking the preparation of copper holoantigen as an example:

[0032] 1. Solution preparation: Weigh 120.8 mg of copper nitrate and dissolve it in 1 mL of ultrapure water, dissolve completely, and prepare a copper nitrate solution with a concentration of 0.5 mol / L. Use 0.1M, pH7.4 N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid HBS solution as a buffer system, and prepare a concentration of 5 × 10 ?3 mol / L bifunctional chelating agent P-Nh2-Bn-Chx-A"-DTPA solution;

[0033] 2. Preparation of copper ion-chelating agent complex: Take 4 ml of the bifunctional chelating agent solution prepared in step 1, add 120 μL of cadmium chloride solution prepared in step 1 by shaking, add one drop every 2 s; React for 20 hours, take it out, adjust the pH to 8.0 with 0.1 mol / L NaOH solution, centrifuge at 3000r / min for 10min to remove the precipitate, and obtain solution A;

[0034] 3. Preparation of copper whole antigen: put the above solution A on a shaker, and drop...

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Abstract

The invention discloses a method for preparing heavy metal holoantigen with high coupling rate and high activity. The method is implemented by improving the synthetic sequence of the heavy metal holoantigen and comprises the following steps of: reacting superfluous heavy metal ion with bifunctional chelator completely, regulating the pH value by using a NaOH solution; performing centrifugal precipitation; removing the superfluous heavy metal ion to obtain a metal ion and chelator compound, so that the metal ion and chelator compound cannot be well reacted with a biomacromolecule; coupling the metal ion and chelator compound with carrier protein, so that the activity of protein in the prepared holoantigen is guaranteed; and thus obtaining the holoantigen with high coupling rate and high activity. Immune tests in mice prove that the prepared holoantigen has high immunogenicity and is simple and practicable, and the preparation method has high repeatability.

Description

technical field [0001] The invention relates to a preparation method of a heavy metal whole antigen with high coupling rate and high activity, and belongs to the field of immunochemistry and residue analysis biotechnology. Background technique [0002] Heavy metal refers to the specific gravity greater than 5 g cm -3 There are about 45 kinds of metal elements in nature. However, since the toxicity of different heavy metals varies greatly, people usually focus on Hg, Cd, Pb, Cr, Cu, Co, Ni, etc. in environmental science. As long as trace amounts of heavy metals in natural water can produce toxic effects, heavy metals are a kind of accumulative substances that are permanently harmful to the environment and cannot be converted into harmless substances through chemical or biological restoration processes. Organisms ingest heavy metals from the environment can be enriched tens of millions of times in higher organisms through the biomagnification of the food chain, and then ente...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/765C07K14/77C07K14/795C07K14/47C07K1/113
Inventor 罗舜菁刘成梅王文飞邹常春万婕钟业俊
Owner NANCHANG UNIV
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