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.
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[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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