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Method for extracting antibody in serum by using aqueous two-phase system

A two-phase, antibody-based technology, applied in the field of phase extraction, achieves the effect of unchanged yield, low equipment requirements, and strong operability

Inactive Publication Date: 2014-10-01
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Traditionally, the method of extracting antibodies from serum is the salting-out method, and the detection rate of antibodies extracted by this method is 48% at the laboratory level. The detection yield is about 40%, which not only affects the immune activity of antibody molecules, but less than half of the yield results in serious waste of antibody molecules in the extraction process, and also makes antibody drugs expensive and cannot be widely used
[0004] The method of using two-phase system to extract antibodies in serum has not been reported in the literature, especially the "sodium acetate" involved in the two-phase extraction technology is only used by people. It can be used as a food flavor agent to promote the absorption of protein, improve the taste of food and increase appetite, or as a preservative to kill enzyme bacteria and improve the storage time of food
And "sodium acetate" is used as a kind of saline solution of the two-phase system to extract the antibody in the serum, there is no literature report

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] 1. Preparation of immune serum.

[0024] Immunization of healthy horses with inactivated mink parvovirus. Blood was collected from the immunized horses after the antibody titer reached the desired value after immunization. The collected blood is placed statically to precipitate the serum, which is the immune serum;

[0025] 2. Prepare a two-phase aqueous system;

[0026] Prepare a stock solution of polyethylene glycol (molecular weight: 3350) with a concentration of 50% (w / w); saline solution with a concentration of 40% (w / w) (the mass ratio of ammonium sulfate to sodium acetate is 3:1) ;Take 4.5ml of polyethylene glycol storage solution, 4.5ml of saline solution, and 1ml of serum, mix well, and set the pH to 7.0;

[0027] 3. Stand still at room temperature to completely separate the aqueous two phases. After layering, place in a centrifuge and centrifuge at 1500r / min for 15 minutes. The two-phase aqueous system after centrifugation is divided into three layers, th...

Embodiment 2

[0031] 1. Preparation of immune serum;

[0032] Immunization of healthy horses with inactivated mink parvovirus. Blood was collected from the immunized horses after the antibody titer reached the desired value after immunization. The collected blood is placed statically to precipitate the serum, which is the immune serum;

[0033] 2. Prepare a two-phase aqueous system;

[0034] Prepare a stock solution of polyethylene glycol (molecular weight: 3350) with a concentration of 50% (w / w); saline solution with a concentration of 40% (w / w) (the mass ratio of ammonium sulfate to sodium acetate is 3:1) ;Take 45ml of polyethylene glycol storage solution, 45ml of saline solution, and 10ml of serum and mix well, and set the pH to 7.0;

[0035] 3. Stand still at room temperature to completely separate the aqueous two phases. After layering, place in a centrifuge and centrifuge at 1500r / min for 15 minutes. The two-phase aqueous system after centrifugation is divided into three laye...

Embodiment 3

[0039] 1. Preparation of immune serum;

[0040] Immunization of healthy horses with inactivated mink parvovirus. Blood was collected from the immunized horses after the antibody titer reached the desired value after immunization. The collected blood is placed statically to precipitate the serum, which is the immune serum;

[0041] 2. Prepare a two-phase aqueous system;

[0042] Prepare a stock solution of polyethylene glycol (molecular weight: 3350) with a concentration of 50% (w / w); saline solution with a concentration of 40% (w / w) (the mass ratio of ammonium sulfate to sodium acetate is 3:1) ;Take 450ml polyethylene glycol storage solution, 450ml saline solution, 100ml serum and mix well, set the pH to 7.0;

[0043] 3. Stand still at room temperature to completely separate the aqueous two phases. After layering, put it in a centrifuge at 1500r / min for 15 minutes; the two-phase aqueous system after centrifugation is divided into three layers, the upper layer is PEG l...

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Abstract

The invention discloses a method for extracting an antibody in serum by using an aqueous two-phase system. The method is a novel method for extracting the antibody in serum and is used for overcoming defects of low yield, high impurity content, complex preparation process, low activity and the like existing in the traditional method for extracting the antibody in serum. Sodium acetate is used as a salt in a brine phase. The yield of antibodies is increased, and the basis is provided for the application of sodium acetate to industrial production in future. Extraction equipment is simple in requirement and can be operated at room temperature; the extracted antibodies are relatively high in purity, and the activities of the antibodies are not influenced; the yield is greatly increased; the extraction time is greatly shortened; the method can be used for expanded production. The yield is not changed after expanded production.

Description

Technical field: [0001] The invention discloses a method for extracting antibodies in serum by a two-phase aqueous system, which relates to the extraction of biologically active macromolecular proteins by a two-phase aqueous system, and is particularly suitable for the extraction of antibodies in immune serum and the treatment of the extracted antibodies. The antibody can be directly used as an antibody drug to treat corresponding diseases, and belongs to the field of bioengineering extraction technology. Background technique: [0002] Antibody, also known as immunoglobulin (Immune globulin), has immune activities such as antibacterial, antiviral, and neutralizing toxins. It can also opsonize, agglutinate and precipitate antigens and activate complement. It can be used for emergency treatment during an outbreak. Therefore, mass production of antibodies has important value and broad market prospects for the treatment of immune diseases. Since such protein macromolecule...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/06C07K1/14
Inventor 雷连成冯新杜崇涛黄盼盼肖锋侯春苗孙长江孙莹莹王彬
Owner JILIN UNIV
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