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Method for separating immunoglobulin IgY from chicken blood

An immunoglobulin and chicken blood technology, applied in the field of isolating immunoglobulin IgY, can solve the problems of wasting natural protein resources and environmental pollution, and achieve the effects of few separation steps, high purity and high separation efficiency

Inactive Publication Date: 2011-01-19
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

On the other hand, my country is a big country for poultry (especially chicken) breeding. Except for a small amount of food, poultry blood is generally discharged as waste, which not only wastes precious natural protein resources, but also causes certain environmental pollution problems.

Method used

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  • Method for separating immunoglobulin IgY from chicken blood

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Experimental program
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Effect test

Embodiment 1

[0023] Take fresh chicken blood, add 0.1M sodium citrate solution for anticoagulation according to the ratio of 9:1 (volume ratio), centrifuge with a centrifuge at 2500 rpm for 20 minutes, remove red blood cells, and obtain plasma. Take 5ml of plasma, add 5ml of 60mM acetic acid-sodium acetate buffer solution (pH4.2) and mix evenly to obtain about 10ml of diluted plasma, add 600μl octanoic acid, stir evenly, let stand at 4°C for 4 hours, and wait until the impurities are completely precipitated. Centrifuge for 30 minutes to obtain 9.6 ml of supernatant. The supernatant was filtered with a 0.45 μm filter membrane, the pH value was adjusted to 7.0, and 1 ml was taken as an injection sample. The chromatographic column (inner diameter 1cm) is filled with 5ml of chromatographic medium with 2-mercapto-1-methylimidazole as the ligand, the equilibration buffer is 20mM Tris-HCl buffer (pH7.0), and the eluent is 20mM acetic acid-sodium acetate buffer solution (pH3.6), the eluted fracti...

Embodiment 2

[0025] Take fresh chicken blood, add 0.1M sodium citrate solution for anticoagulation according to the ratio of 9:1 (volume ratio), centrifuge with a centrifuge at 3000rpm for 10 minutes, remove red blood cells, and obtain plasma. Take 5ml of plasma, add 10ml of 60mM acetic acid-sodium acetate buffer (pH4.2) and mix evenly to obtain about 15ml of diluted plasma, add 450μl octanoic acid, stir evenly, let stand at 8°C for 1 hour, and wait until the impurities are completely precipitated. Centrifuge for 20 minutes to obtain 14.2 ml of supernatant. The supernatant was filtered with a 0.45 μm filter membrane, the pH value was adjusted to 7.5, and 1 ml was taken as an injection sample. The chromatographic column (1cm inner diameter) is filled with 5ml of mixed mode medium with 2-mercapto-1-methylimidazole as ligand, the equilibration buffer is 20mM Tris-HCl buffer (pH7.5), and the eluent is 20mM acetic acid-sodium acetate buffer solution (pH 3.8), the eluted fractions were collecte...

Embodiment 3

[0027] Take fresh chicken blood, add 0.1M sodium citrate solution for anticoagulation according to the ratio of 9:1 (volume ratio), centrifuge with a centrifuge at 2700rpm for 15 minutes, remove red blood cells, and obtain plasma. Take 5ml of plasma, add 15ml of 60mM acetic acid-sodium acetate buffer (pH4.8) and mix evenly to obtain about 20ml of diluted plasma, add 800μl octanoic acid, stir evenly, let stand at 4°C for 1 hour, and wait for the impurities to precipitate completely. After centrifugation for 30 minutes, 19.4 ml of supernatant was obtained. The supernatant was filtered with a 0.45 μm filter membrane, the pH value was adjusted to 8.0, and 1 ml was taken as an injection sample. The chromatographic column (1cm inner diameter) is filled with 5ml of chromatographic medium with 2-mercapto-1-methylimidazole as the ligand, the equilibration buffer is 20mM Tris-HCl buffer (pH8.0), and the eluent is 20mM acetic acid-sodium acetate buffer solution (pH 4.0), the eluted frac...

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Abstract

The invention discloses a method for separating immunoglobulin IgY from chicken blood. The method comprises the following concrete steps: 1) pretreating plasma, selecting fresh chicken blood, centrifuging, and removing erythrocyte to obtain plasma; 2) precipitating octanoic acid, adding the octanoic acid into the plasma to reach a certain concentration, removing foreign protein by precipitating, centrifugally separating, and taking the supernate; 3) carrying out column chromatography: separating the supernate by a chromatographic column filled with mixed adsorbent, and collecting elution peak; and 4) desalting and drying: desalting the collected solution, cooling and drying the desalted collected solution to obtain the high-purity immunoglobulin IgY. The method is characterized in that a new separation process, through which high-purity immunoglobulin IgY can be prepared by separating the chicken blood. The key point of the method is to directly extract the immunoglobulin IgY from the supernate of the octanoic acid, so the method has the characteristics of simple operation steps, high separation efficiency and low cost.

Description

technical field [0001] The invention relates to a method for isolating immunoglobulin IgY from chicken blood. Background technique [0002] Poultry immunoglobulin IgY has biological activity and is widely used. It can be used in serological immunodiagnosis (sandwich method ELISA detection, immunodiffusion, immunoadsorption and complement fixation, etc.) and immunotherapy (viral diseases of chickens and ducks, infants and young livestock rotavirus infection, etc.). Usually IgY is obtained from poultry eggs, mainly after the laying hen is immunized with a specific antigen, the antibody is transferred from the blood to the yolk about 7 to 10 days later, and then IgY is isolated from the yolk. There are many reports on this research, such as Patents CN1752105, CN1935841 and CN101125886. However, the fat content in egg yolk is very high (about 30%). Extracting IgY from egg yolk needs to remove a large amount of lipid substances, and it is difficult to separate IgY on a large sc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/06C07K1/36C07K1/30C07K1/22
Inventor 林东强童红飞姚善泾
Owner ZHEJIANG UNIV
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