Method for production of antibody

A technology for antibody preparation and antibody, applied in the direction of antibodies, chemical instruments and methods, anti-animal/human immunoglobulin, etc., can solve the problems of time-consuming and laborious, high cost of preparing antibodies, relying on professionals, etc., and achieve the goal of improving operation efficiency Effect

Inactive Publication Date: 2010-11-10
ALLIED COLLOIDS LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The above-mentioned methods for preparing monoclonal antibodies are time-consuming and laborious, and need to rely on professionals, and the cost of preparing antibodies is high

Method used

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  • Method for production of antibody
  • Method for production of antibody
  • Method for production of antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0110] In this example, the S100A10 protein was used as an antigen example to illustrate the preparation method of antibody IgG1.

[0111] Extraction of spleen cells (splenocyte)

[0112] After identifying the mouse spleen with naked eyes, take it out and wash it. Two spleens were placed on a mesh, broken with a cell scraper, and cells were collected by centrifugation (1300 rpm×3 minutes, 4° C.). Next, after suspending the cells in 3 mL of ACK lysis buffer, 10 mL of PBS(-) was added. The cells were recovered by centrifugation (1300 rpm×3 minutes, 4°C), and suspended in 6 mL of RPMI1640(-). Pass through a cell strainer to remove insoluble fat etc.

[0113] in vitro immunization

[0114] The concentration of the S100A10 protein antigen was measured using a detection kit (Dc protein assay kit, manufactured by BioRad). At this time, BSA (manufactured by Sigma) was used as a standard. The known concentration of S100A10 protein antigen was dispensed according to the specif...

Embodiment 2

[0134] In this example, the extraction of the anti-S100A10 antibody gene and the steps of expressing the antibody in Escherichia coli will be described.

[0135] DNA extraction, preparation of single-chain antibody scFv plasmid

[0136] Use Isogen (manufactured by Nippongene) from 1.5×10 6 Total RNA was obtained from B cells or hybridomas after immunization of cells. Among them, immune cells obtained by the in vitro immunization step described in Example 1 above can also be used in the step.

[0137] Next, it was confirmed by 1.2% agarose gel (Agarose Gel) electrophoresis. The result obtained is as Figure 1A and Figure 1B shown.

[0138] Using the obtained total RNA as a template, single-stranded cDNA was synthesized using ReverTra Ace (manufactured by TOYOBO) reagent using mouse VH gene-specific primers or mouse VL gene-specific primers. Then, using the prepared cDNA as a template, use the primer-VH forward primer mixture / VH reverse primer mixture or VL forward prime...

Embodiment 3

[0152] The procedure described in Example 1 was repeated. However, in this example, S100A1 protein and S100A10 protein were used as antigens, and the same method as in Example 1 above was used to implement in vitro immunization, and the IgG1 positive rate was calculated. Figure 6 show the results thus obtained. From Figure 6 It can be seen that class switching to IgG1 occurs at a high probability of 60% or higher, although it may vary depending on the antigen.

[0153] In the in vitro (in vitro) immunization process of the present invention, LPS (40 μg / mL), IL-4, IL-5 (10ng / mL), anti-CD38 antibody, anti-CD40 antibody (1 μg / mL) are added once after antigen immunization. mL) and other stimuli, culturing for 4 days in the presence of stimuli can achieve effective class switching.

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Abstract

Disclosed is a method for producing an antibody, which comprises the steps of: immunizing a tissue containing a cell to be immunized in vitro in a liquid culture medium containing an antigen and a stimulant; selecting an immunized cell; and producing the antibody from the selected immunized cell. The method enables to produce an antibody in large quantity within a short period, and therefore can contribute to the widespread use of an immunological test.

Description

technical field [0001] The present invention relates to methods for preparing antibodies. Background technique [0002] As we all know, in terms of disease diagnosis and treatment, clarifying the defense function of the organism, especially its core immune function, is gradually becoming an important issue. For example, among various methods used in diagnosing various diseases other than lifestyle-related diseases such as cancer and diabetes, immunocolloid chromatography, which can visually detect the concentration and amount of markers closely related to diseases, has been proposed The field of reagents for research, reagents for diagnosis, reagents for monitoring various substances, immunological diagnosis, and treatment has been further expanded, but the stability of antibodies in these methods will become more and more important in the future. more important. In addition, the antibody library preparation technology using the phage display system has been developed, and...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P21/08C07K16/00C12N15/09A61K39/395
CPCC07K16/3038C07K2317/622C07K2317/92C07K16/00C07K16/18
Inventor 稻垣贵之吉见达成
Owner ALLIED COLLOIDS LTD
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