Method for purifying antibody by denatured antigen affinity purification

A technology for denaturing antigens and antibodies, applied in chemical instruments and methods, peptide preparation methods, organic chemistry, etc., can solve problems such as the destruction of antibody activity, and achieve the effect of simplifying operation steps

Inactive Publication Date: 2015-08-19
INST OF BOTANY CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, since most inclusion bodies are difficult to refold in aqueous solution, and antibody activity is easily damaged by denat

Method used

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  • Method for purifying antibody by denatured antigen affinity purification
  • Method for purifying antibody by denatured antigen affinity purification
  • Method for purifying antibody by denatured antigen affinity purification

Examples

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Embodiment

[0025] In order to better understand the present invention, this example uses the method of denatured antigen affinity purification antibody to obtain DOF purified antibody from rabbit polyclonal antibody, and uses the obtained antibody to detect the expression of endogenous DOF ​​protein of tomato fruit and the expression of purified antibody and endogenous The immune binding ability of the source DOF protein verifies that the denatured antigen purification antibody system is feasible in the process.

[0026] The experimental methods used in the following examples are conventional methods unless otherwise specified.

[0027] The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

[0028] 1 material

[0029] 1.1 Main experimental equipment and consumables

[0030] 1) Solid phase resin: AminoLink Coupling Resin, 50% Slurry (Thermo company)

[0031] 1) Chromatography column: specification 5ml (Millipore Co...

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Abstract

The invention relates to a novel method for purifying an antibody by denatured antigen affinity purification. In the antigen chromatographic column preparation process, the antigen is coupled with a solid-phase carrier in a urea-containing medium; and as the urea concentration gradually decreases, the antigen connected with the solid phase is utilized to implement in-situ renaturation, thereby ensuring that the subsequent antibody affinity purification process is completed in the water solution medium. The denatured antigen protein (such as prokaryotically-expressed inclusion body protein) can be directly coupled to the solid-phase carrier to purify the antibody, thereby simplifying the operation steps (the soluble antigen protein preparation process). The method has the advantages of high operability and low consumption. The obtained antibody has very high purity and specificity, and is suitable for laboratory preparation and industrial production.

Description

technical field [0001] The present invention is a technical scheme of antibody affinity purification with denatured antigen coupling solid-phase carrier reaction as the main innovation point, especially suitable for polyclonal antibody purification using insoluble protein prepared by prokaryotic expression as antigen, and belongs to antibody affinity purification technology field. Background technique [0002] There are many methods for antibody purification. In the early days, people used the salting out method, and later developed into the method of ion exchange chromatography. At present, the more commonly used method is to use Protein A (type A Staphylococcus aureus cell wall protein), G (type G gold Staphylococcus aureus cell wall protein), A / G or the antigen itself coupled to the activated matrix for antibody affinity purification. In comparison, antibodies obtained by antigen affinity purification have good affinity, strong specificity, and high purity, and are bette...

Claims

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Application Information

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IPC IPC(8): C07K1/22
Inventor 田世平秦国政王豫颖
Owner INST OF BOTANY CHINESE ACAD OF SCI
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