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Preparation method of mouse anti-human SAA monoclonal antibody

A mouse-derived, antigen-based technology, applied in the direction of anti-animal/human immunoglobulin, etc., can solve the problems of large amount of antigen and unsatisfactory genetic stability of hybridoma cells, so as to promote growth, increase cell fusion rate and positive rate , the effect of simple process

Pending Publication Date: 2019-08-09
上海钹乐诗生物技术有限公司
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Problems solved by technology

However, the current general mouse programmed immunization requires a large amount of antigen, and the cell fusion rate, positive rate, and the genetic stability of the obtained hybridoma cells are not ideal

Method used

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  • Preparation method of mouse anti-human SAA monoclonal antibody
  • Preparation method of mouse anti-human SAA monoclonal antibody
  • Preparation method of mouse anti-human SAA monoclonal antibody

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Embodiment Construction

[0014] The technical solution of the present invention will be described in detail below in conjunction with the embodiments.

[0015] 1. Antigen acquisition

[0016] The antigen is human recombinant SAA expressed by the bacterial (E.coli) expression system, the protein size is 122AA, the N' end has a His tag, and the mutation sites are 60: D→N; 71: H→R.

[0017] 2. Animal immunization

[0018] Each mouse immunization only consumes 260 μg of natural SAA protein, and only the first and second immunizations are emulsified with adjuvant, and the subsequent booster immunization and shock immunization are all immunized with water, and the applicant is Desai Diagnostic System (Shanghai) Co., Ltd., the application number is 201710834698.0, and the patent application for the invention name is anti-human SAA monoclonal antibody and its preparation method and application. It is necessary to immunize mice four times. emulsified with complete Freund's adjuvant.

[0019] (1) The first i...

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Abstract

The invention belongs to the technical field of monoclonal antibodies and particularly relates to a preparation method of a mouse anti-human SAA monoclonal antibody. The method comprises the followingsteps of (1) antigen preparation; (2) animal immunization; (3) cell fusion, wherein preparation work including preparation of spleen cells and preparation of a feeding layer is carried out before fusion; (4) screening and cloning of hybridoma cells; (5) expanded production of the monoclonal antibody; (6) determination of the type, subtype and ascites titer of the monoclonal antibody. In step 2, amulti-point injection mode is adopted during first immunization and second immunization, and an emulsified antigen is used and injected into the intradermal cells, foot pads and the abdominal cavityseparately; at the same time, shock immunization is performed once three days before fusion. According to the preparation method, a multi-point combined immunization mode and a shock immunization modeare adopted, the antigen dosage is low, the process is simple, little damage is caused to mice, and the immunization effect is good. The preparation process of the feeding layer is optimized, the growth of the hybridoma cells is promoted, and the cell fusion rate and the positive rate are increased.

Description

technical field [0001] The invention relates to the technical field of monoclonal antibodies, in particular to a preparation method of mouse-derived anti-human SAA monoclonal antibody. Background technique [0002] Human serum amyloid A protein (SAA) is a protein consisting of 104 amino acids, which has a significant relationship with acute-phase response (APR). Typically, serum SAA levels are low, approximately 20-50 mcg / ml in the blood of healthy individuals. However, SAA serum levels rise as much as 1000-fold within 24 hours of APR onset. Therefore, the determination of changes in human serum amyloid A is of great significance for clinical diagnosis of the above diseases, monitoring of disease changes and guiding clinical treatment. [0003] Using human SAA natural protein to immunize mice can make mouse immune cells produce targeted antibodies. Then the splenocytes of the immunized mice were taken out and fused with myeloma cells to obtain hybridoma cells. Through scr...

Claims

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Application Information

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IPC IPC(8): C07K16/18
CPCC07K16/18
Inventor 徐娅佳高健谌敦华陈旭华汪渊
Owner 上海钹乐诗生物技术有限公司
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