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Preparation method of high-coupling-ratio heavy-metal antigen

A heavy metal, coupling rate technology, applied in the field of immunochemistry and residue analysis biology, can solve the problems of low antigen coupling rate, protein denaturation, and poor immune effect, and achieve good immunogenicity and high method repeatability.

Inactive Publication Date: 2010-12-15
NANCHANG UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] Aiming at the characteristic that heavy metals can easily lead to protein denaturation after a large amount of rapid combination with proteins, the present invention aims to solve the problems of low antigen coupling rate and poor immune effect in existing methods, and prepare heavy metal antigens with high coupling rate and high immunogenicity

Method used

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  • Preparation method of high-coupling-ratio heavy-metal antigen

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] 1. Solution preparation: use 0.1M, pH7.3 N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HBS) solution as a buffer system to prepare bifunctional chelating agent p-NH 2 -Bn-CHX-A”-DTPA10mg / ml, bovine serum albumin (BSA) 2.5mg / ml and Pb(NO 3 ) 2 solution.

[0022] 2. Protein cross-linking reaction: take p-NH 2 -Mix 300 μl of Bn-CHX-A”-DTPA solution with 2ml of BSA solution, slowly add 300 μl of 25% glutaraldehyde dropwise into the mixed solution of bifunctional chelating agent and BSA, and carry out protein cross-linking reaction to obtain protein chelating agent complex BSA- GA-p-NH 2 -Bn-CHX-A"-DTPA and dialyzed against HBS buffer for 2 days.

[0023] 3. Chelation of lead ions: Take out the protein chelating agent complex that has been dialyzed, and add Pb(NO 3 ) 2 Solution, the ratio n (lead) of the amount of protein in the lead ion that adds and protein chelating agent complex: n (protein)=40: 1, control dropping rate is 5min / drops, 30 ℃ of incubators afte...

Embodiment 2

[0027] 1. Solution preparation: use 0.1M, pH7.3 N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HBS) solution as a buffer system to prepare bifunctional chelating agent p-NH 2 -Bn-CHX-A”-DTPA10mg / ml, bovine serum albumin (BSA) 2.5mg / ml and Pb(NO 3 ) 2 solution.

[0028] 2. Protein cross-linking reaction: take p-NH 2 -Mix 300 μl of Bn-CHX-A”-DTPA solution with 2ml of BSA solution, slowly add 300 μl of 25% glutaraldehyde dropwise into the mixed solution of bifunctional chelating agent and BSA, and carry out protein cross-linking reaction to obtain protein chelating agent complex BSA- GA-p-NH 2 -Bn-CHX-A"-DTPA and dialyzed against HBS buffer for 2 days.

[0029] 3. Chelation of lead ions: Take out the protein chelating agent complex that has been dialyzed, and add Pb(NO 3 ) 2 Solution, the ratio n (lead) of the amount of protein in the lead ion that adds and protein chelating agent complex: n (protein)=40: 1, control dropping rate is 5min / drops, 30 ℃ of incubators afte...

Embodiment 3

[0034] 1. Solution preparation: use 0.1M, pH7.3 N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HBS) solution as a buffer system to prepare bifunctional chelating agent p-NH 2 -Bn-CHX-A”-DTPA10mg / ml, bovine serum albumin (BSA) 2.5mg / ml and Pb(NO 3) 2 solution.

[0035] 2. Protein cross-linking reaction: take p-NH 2 -Mix 300 μl of Bn-CHX-A”-DTPA solution with 2ml of BSA solution, slowly add 300 μl of 25% glutaraldehyde dropwise into the mixed solution of bifunctional chelating agent and BSA, and carry out protein cross-linking reaction to obtain protein chelating agent complex BSA- GA-p-NH 2 -Bn-CHX-A"-DTPA and dialyzed against HBS buffer for 2 days.

[0036] 3. Chelation of lead ions: Take out the protein chelating agent complex that has been dialyzed, and add Pb(NO 3 ) 2 Solution, the ratio n (lead) of the amount of protein in the lead ion that adds and protein chelating agent complex: n (protein)=40: 1, control dropping rate is 5min / drops, 30 ℃ of incubators after...

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Abstract

The invention discloses a preparation method of a high-coupling-ratio heavy-metal antigen, comprising the step of chelating heavy metal ions in low consistency for many times with a protein chelant compound to prepare the antigens. The obtained antigen has high coupling ratio, good immunogenicity, and the preparation method has high repeatability and simple and easy operation and can be applied to the synthesis of various heavy-metal whole antigens.

Description

technical field [0001] The invention relates to a preparation method of a high coupling rate heavy metal antigen, which belongs to the field of immunochemistry and residue analysis biotechnology. Background technique [0002] Heavy metals mainly refer to Hg, Cd, Pb, Cr, Zn, Cu, Co, Ni, etc., which are a kind of accumulative and permanently harmful substances to the environment, which cannot be transformed into harmless substances through chemical or biological restoration processes. It exists in the form of combination with soil or sediment for a long time, bioaccumulates, enters the human body through the food chain, damages human nerves, kidneys, hematopoiesis, digestion, endocrine and other systems, and directly endangers human health. Areas polluted by heavy metals must be regularly tested. [0003] The methods currently used to measure heavy metal pollutants mainly include atomic absorption spectrophotometry, fluorescence spectroscopy, polarography, and inductively cou...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/765
Inventor 罗舜菁刘成梅涂宗财严杰琳陈发荣陈婷婷高鹏陈臣
Owner NANCHANG UNIV