Process for preparing high-flux monoclonal antibody

A monoclonal antibody and immunization technology, applied in chemical instruments and methods, measuring devices, cells modified by introducing foreign genetic material, etc. It is difficult to improve the throughput of antibody preparation and reduce the efficiency of mAb production and screening.

Inactive Publication Date: 2006-12-20
SHANGHAI BIOCHIP
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AI Technical Summary

Problems solved by technology

However, the monoclonal antibody obtained by this preparation method can only recognize a specific epitope, and in practice, several antibody-producing hybridomas can be obtained by one fusion, and the manpower and other factors that need to be consumed during screening are very huge.
Due to the huge workload, the high-tech technology of monoclonal antibody preparation has become a manpower-consuming technology, so it has become a limiting factor and a speed-limiting factor in the work.
Another important aspect is that the properties of monoclonal antibodies, especially the specificity of epitope recognition at the recognition site, usually need to be further verified after the antibody is obtained, which greatly reduces the production process of monoclonal antibodies. Screening efficiency
[0009] If multiple immunogens are used to immunize at the same time to increase throughput, one-time fusion, the screening method is limited by the common ELISA screening method
Traditional ELISA screening requires the use of a large number of immunogens to coat plates and a large amount of cell culture supernatant for detection. In practice, immunogens are often very small and precious, and the culture supernatant of each hybridoma It is only 100-200 microliters, and it is difficult to increase the throughput of antibody preparation from the actual experimental operation
[0010] In summary, so far there is no satisfactory method for the efficient and rapid preparation of multiple monoclonal antibodies

Method used

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  • Process for preparing high-flux monoclonal antibody
  • Process for preparing high-flux monoclonal antibody
  • Process for preparing high-flux monoclonal antibody

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preparation example Construction

[0040] In a preferred example, the high-throughput monoclonal antibody preparation method of the present invention comprises the following steps:

[0041] (1) Simultaneously immunize mice with 3-10000 (preferably 4-1000) immunogens (such as proteins and polypeptides). For example, non-human mammals (eg, mice, rats) are immunized with cells, tissues, and cell lysates, purified native or recombinantly expressed proteins, synthetic polypeptides, or mixtures thereof. The way of immunization is routine immunization in this field, and booster immunization can be used if necessary;

[0042] (2) Take the spleen of the mouse to make a cell suspension, mix it with myeloma cells in a conventional ratio (such as 5:1-2:1), prepare hybridoma cells under the action of a fusion agent, and collect the fused hybridoma cells, Cultivate for a period of time (such as 7-8 days), and prepare a suspension;

[0043] (3) Using biochips (including protein chips, polypeptide chips and / or tissue chips) ...

Embodiment 1

[0089] High-throughput preparation of tumor marker monoclonal antibodies

[0090] 1. Mouse immunization and preparation of hybridoma cells

[0091] (1) Select known tumor markers CEA, CA19-9, CA242, CA50, CA724 stone species tumor markers as antigens for preparing monoclonal antibodies;

[0092](2) Intraperitoneally inject a total of 200 micrograms of antigens into the mouse, 40 micrograms each, the antigens have been mixed with Freund’s complete adjuvant in equal volumes, two weeks later for booster immunization, the total dose of antigens is 200 micrograms, the antigens are mixed with Freund’s adjuvant After two weeks, intraperitoneally inject 20 micrograms of the mixed antigen into the mouse, and after one week, inject 20 micrograms of the mixed antigen into the tail vein of the mouse;

[0093] (3) On the third day after the last immunization, take the spleen of the mouse to make a cell suspension, mix it with myeloma cells in a certain proportion, prepare hybridoma cells ...

Embodiment 2

[0115] High-throughput preparation and purification of monoclonal antibodies that recognize gastric cancer tumor markers

[0116] 1. Mouse immunization and preparation of hybridoma cells

[0117] (1) Take human gastric cancer tissue, chop it up with a high-speed tissue crusher, and centrifuge to obtain total protein as an antigen for preparing monoclonal antibodies;

[0118] (2) Inject the antigen into the peritoneal cavity of the mouse for immunization, the immunization method is conventional immunization, and the immunization can be strengthened if necessary;

[0119] (3) Take the spleen of the mouse to make a cell suspension, mix it with myeloma cells in a certain proportion, prepare hybridoma cells under the action of the fusion agent, collect the fused hybridoma cells, culture them for 7-8 days, and prepare a suspension ;

[0120] 2. Preparation of biochip

[0121] (1) Using gastric cancer tissue samples from different patients and corresponding negative controls to pr...

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Abstract

The invention discloses a monoclonal antibody preparing method of high-flux, which comprises the following steps: (a) proceeding immune inoculation for non-human mammal animal through multiple immunogen; (b) compounding splenocyte of immune inoculation animal and bone marrow oncocyte to fuse into cross oncocyte; (c) collecting the cross oncocyte; culturing; preparing cross oncocyte suspension; (d) screening cross oncocyte through biological chip to produce cross oncocyte system of immunogen monoclonal antibody.

Description

technical field [0001] The present invention relates to the field of biology, and more specifically relates to a method for high-throughput preparation of monoclonal antibodies by combined use of protein chips, polypeptide chips and tissue chips. Background technique [0002] At present, in the process of preparing monoclonal antibodies, the traditional method is to immunize mice with a protein or antigen, determine the hybridomas producing antibodies by ELISA, clone and establish strains, and then further identify the purified monoclonal antibodies. If necessary, detect its epitope again. This preparation method can often only obtain a specific antibody. [0003] In actual work, when the fusion rate is high, a large number of antibody-producing hybridomas are often obtained, but they cannot be cloned at the same time. The positive cells can only be screened according to the OD value read by the microplate reader, and the clones are constructed. Monoclonal antibodies of di...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/16C12P21/08C07K16/00G01N33/577
Inventor 张庆华宋凯王升年周佳菁肖华胜奚佳捷彭海林卫玲
Owner SHANGHAI BIOCHIP
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