IgM purification method

A purification method, the technology of ammonium sulfate method, applied in chemical instruments and methods, peptide preparation methods, organic chemistry, etc., can solve problems such as difficulties, and achieve the effects of small cross-reaction, product stability, and good reproducibility

Active Publication Date: 2021-02-26
GUANGDONG FAPON BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Although IgM is widely used, it is sti

Method used

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  • IgM purification method
  • IgM purification method

Examples

Experimental program
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Embodiment 1

[0051] Example 1 IgM rough extraction

[0052] 1) Ammonium sulfate precipitation treatment: Dilute the centrifuged serum with PBS (pH=7.0-8.0), add saturated ammonium sulfate to a final concentration of 40%-60%, let it stand at 4 degrees for more than 30 minutes, and discard the supernatant by centrifugation ;

[0053] 2) Reconstitute the collected precipitate with a buffer solution containing more than 150 mM salt, pH = 7.0-9.0; after reconstitution, perform dialysis treatment with purified water at a ratio of 1:1000 or more for 3-5 days;

[0054] 3) Collect the precipitate after dialysis, redissolve it with a buffer solution containing more than 150mM salt, pH = 7.0-9.0, adjust the pH = 3.0-5.0 after redissolution, add n-octanoic acid at a ratio of 1ml: at least 5μl, and keep at 20°C After reacting at ~35°C for 10min~2h, centrifuge to discard the precipitate; adjust the pH of the collected supernatant to 7.0~9.0 and add saturated ammonium sulfate to a final concentration of...

Embodiment 2

[0055] Example 2 IgM rough extraction

[0056] Same as Example 1, the difference is that step 1) is replaced by: use polyethylene glycol precipitation treatment: after centrifugation and clarification of the antibody supernatant secreted by hybridoma cells cultured in serum-free medium, use an equal amount of PBS, pH=7.0 ~8.0 Dilute, add PEG6000 gradually under stirring to make the final concentration 5%, let stand for 30min, discard the supernatant after centrifugation.

Embodiment 3

[0057] Embodiment 3 IgM refined extraction

[0058] The crude extract obtained in Example 1 is further operated:

[0059] (1) Hydrophobic chromatography:

[0060] The supernatant is passed through the hydrophobic medium of GE Company, and the hydrophobic ligand used in the present invention is phenyl.

[0061] Buffer A: 20mM PB+1000mM NaCl, pH=8.0;

[0062] Load the sample after equilibrating the column with Buffer A, and collect the breakthrough sample when the UV absorbance value starts to rise.

[0063] (2) CHT column

[0064] The above breakthrough samples were purified by Bio-Rad's CHT II medium

[0065] Buffer A: 15mM PB+1000mM NaCl, pH=8.0;

[0066] Buffer B: 300mM PB, pH=8.0;

[0067] Load the sample after equilibrating the column with Buffer A, elute with a linear gradient of 0% to 100% Buffer B, and collect the eluted samples.

[0068] (3) G column purification

[0069] The eluted sample was purified by GE's Protein G column

[0070] Buffer A: 30mM PB+1000mM...

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Abstract

The invention relates to the technical field of antibodies, and particularly relates to an IgM purification method. The IgM purification method comprises the following steps of: a, providing a samplecontaining IgM; b, performing at least one time of protein precipitation treatment and at least one time of protein concentration treatment to obtain a protein precipitate; c, redissolving the proteinprecipitate, and removing impure protein by using an octanoic acid-ammonium sulfate method to obtain a crude extract containing the IgM; d, performing hydrophobic chromatography treatment, and collecting a penetrating substance; e, performing hydroxyl phosphate limestone chromatography, and collecting an eluate; and f, performing purification treatment by using affinity chromatography resin capturing IgG, and collecting a penetrating substance. The purity of the IgM in a composition obtained by purification according to the method is 95% or above, the product is stable, precipitates are not easy to appear after cryopreservation, and the antibody activity is good.

Description

technical field [0001] The present invention relates to the technical field of antibodies, in particular to an IgM purification method. Background technique [0002] IgM (immunoglobulin M) is composed of 5 monomers connected by a J chain and disulfide bonds to form a pentamer. The molecular weight is the largest among the 5 types of immunoglobulins, 970kD, and it is called macroglobulin (macroglobulin). This structure makes immunoglobulin M have more antigen-binding valence, and can bind to several target cells at the same time. Therefore, immunoglobulin M plays the role of the main antibody in the body's anti-infection immunity. [0003] IgM is found in the blood and lymph and is usually the first antibody produced in response to an infection, enabling other immune system cells to destroy foreign objects. Although IgM has a wide range of uses, it is still difficult to obtain high-purity IgM. [0004] In view of this, the present invention is proposed. Contents of the i...

Claims

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Application Information

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IPC IPC(8): C07K16/00C07K1/36C07K1/30C07K1/22C07K1/20C07K1/16
CPCC07K16/00C07K1/36C07K1/30C07K1/22C07K1/20C07K1/16
Inventor 柏艳辉成谦孙华坤唐佳
Owner GUANGDONG FAPON BIOTECH CO LTD
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