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Preparation method of hematogenous human IgM antibody purification medium

A technology for antibody purification and medium, which is applied in the field of preparation of blood-derived human IgM antibody purification medium, which can solve the problems of complexity, low yield, cross-reaction, etc., and achieve the effect of satisfying antigen supply, small structural impact, and easy production and scale-up

Pending Publication Date: 2022-03-11
南京京达生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The existing method is relatively complicated, with many steps, and the purity of the human IgM antibody obtained by separation and purification is not high. If it is used as the final product or immunogen, it is easy to cause cross-reaction to the experiment, and it will damage the structure of the human IgM antibody to a certain extent. In addition, the yield of this method is not high, which is unfavorable for large-scale production;

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] A method for preparing a blood-derived human IgM antibody purification medium. A secondary antibody AT2910 that can specifically bind to the human IgM antibody μ chain is coupled to an agarose medium through CNBr coupling to obtain an IgM antibody purification medium.

[0031] The preparation method of the substance comprises the following steps:

[0032] S1. Use coupling buffer (0.1M NaHCO 3 +0.3M NaCl, pH=7.4) to dialyze the AT2910 protein to replace the buffer, dialyze twice according to the volume ratio of 1:40, A 280 Measuring protein concentration;

[0033] S2. Agarose CNBr freeze-dried powder (GE Health Care): AT2910 = 1ml: 4mg (V / W) ratio Weigh the corresponding quality of agarose freeze-dried powder, 1g freeze-dried powder corresponds to 3.5ml finished agarose gel column volume;

[0034] S3. Resuspend the agarose freeze-dried powder in 20mL of 1mM HCl corresponding to each g of the freeze-dried powder, and place it at 4°C for activation for 2 hours;

[0035...

Embodiment 2

[0042] AT2910 is a monoclonal antibody obtained through hybridoma cell fusion technology, which can specifically bind to human IgM-μ chain. The nucleotide sequence of the CDR region of the light and heavy chains is obtained by cell line sequencing. The preparation process of AT2910 is as follows:

[0043] 1. Ascites treatment: 500mL AT2910 ascites was filtered at 25°C or room temperature with slow filter paper with a pore size of 10 microns to remove solid content such as grease, precipitated impurities, tissue fragments, etc.;

[0044] 2. Column balance: Use a Protein G chromatography column with a column volume of 100mL, and equilibrate 5 times the column volume with an equilibration buffer (0.02mM Tris+0.2M NaCl, pH=7.4). After the column is well balanced, the protein UV detector will display The value A280 is adjusted to 0 as the baseline;

[0045] 3. Sample loading: Dilute the ascites with 1000mL volume of equilibration buffer (0.02mM Tris+0.2M NaCl, pH=7.4), and use the ...

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Abstract

The invention discloses a preparation method of a blood-derived human IgM antibody purification medium, which comprises the following steps: coupling a secondary antibody AT2910 (anti-mu chain) capable of being specifically combined with a human IgM antibody with an agarose medium through CNBr coupling to obtain the IgM antibody purification medium; the method disclosed by the invention is simple, easy to produce and amplify, and convenient for producing human IgM in serum on a large scale; the method has small influence on the structure of the human IgM antibody, and the separated and purified human IgM antibody has high purity and strong specificity, and can be used as a better immunogen; the method can be used for large-scale preparation of goat anti-human IgM serum or rabbit anti-human IgM serum, and the antigen supply is met.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a method for preparing a blood-derived human IgM antibody purification medium. Background technique [0002] Existing methods mostly use PEG precipitation or ammonium sulfate (SAS) precipitation, in addition to Gel filtration chromatography (Gel filtration chromatography), ion-exchange column chromatography (Ion-exchange column chromatography, IEC); [0003] The existing method is relatively complicated, with many steps, and the purity of the human IgM antibody obtained by separation and purification is not high. If it is used as the final product or immunogen, it will easily cause cross-reaction to the experiment, and the structure of the human IgM antibody will be damaged to a certain extent. In addition, the method yield is not high, which is unfavorable for large-scale production; [0004] The method of the invention is simple, easy to produce and scale up, has little influence ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/06C07K16/42C07K1/22
CPCC07K16/065C07K16/4283Y02A50/30
Inventor 李永刚李伟吴德风
Owner 南京京达生物技术有限公司