Preparation of hybridoma cell, monoclonal antibody secreted by hybridoma cell and application of monoclonal antibody

A technology of hybridoma cells and cells, applied in the field of bioengineering, monoclonal antibodies and applications, to achieve the effects of improving process efficiency, efficient secretion, and simplifying the screening process

Inactive Publication Date: 2015-12-02
GETEIN BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Commercial mAbs have been successfully prepared abroad, but there is no such mAb that can be successfully used in clinical diagnosis in China

Method used

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  • Preparation of hybridoma cell, monoclonal antibody secreted by hybridoma cell and application of monoclonal antibody
  • Preparation of hybridoma cell, monoclonal antibody secreted by hybridoma cell and application of monoclonal antibody
  • Preparation of hybridoma cell, monoclonal antibody secreted by hybridoma cell and application of monoclonal antibody

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Experimental program
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Effect test

Embodiment 1

[0033] Example 1: Preparation of immune splenocytes

[0034] The purified H-FABP recombinant protein is used as an antigen, and the amino acid sequence of the recombinant protein is shown as SEQ ID NO: 1. The amino acid sequence includes specific antigenic epitopes SEQINNO: 2 and SEQINNO: 3 and non-specific antigenic epitopes SEQINNO: 4, conventional Methods 6-week-old BALB / c mice were immunized, and the first basic immunization was subcutaneously injected with 200 μg of antigen, using Freund’s complete adjuvant; once every 2 weeks, a total of three times, 100 μg / mouse / time, using Freund’s incomplete adjuvant , 2 weeks after the last basic immunization, dock the tail to detect the anti-H-FABP antibody titer in the serum of the mice, and make the serum antibody titer reach 1:10 4 The above mice were used as the source of immunized splenocytes, and 50 μg / ml recombinant FABP antigen was immunized into the peritoneal cavity of the mice, and the spleens of the mice were obtained 3 ...

Embodiment 2

[0035] Embodiment 2: the preparation of hybridoma cell

[0036] (1) Preparation of feeder cells

[0037] The peritoneal macrophages of BALB / c mice were used as feeder cells, and the preparation method was as follows: take BALB / c mice, kill them by pulling the neck, disinfect the body surface with 75% alcohol, and lift the abdomen from the hind abdomen with sterile scissors Skin, exposed peritoneum, wipe the peritoneum with 75% alcohol cotton ball for disinfection. Inject 10ml of DMEM culture solution into the peritoneal cavity with a syringe, rinse repeatedly, recover the washing solution, centrifuge at 1000r / min for 10 minutes, discard the supernatant, and obtain feeder cells. The pellet was resuspended in DMEM medium containing 15% calf serum, and the cell concentration was adjusted to 2×10 5 pieces / ml. Add the above cell suspension into a 96-well plate, 0.1ml per well, set at 37°C, 5% CO 2 overnight in an incubator and used the next day.

[0038] (2) Preparation of imm...

Embodiment 3

[0065] Embodiment 3: Preparation of monoclonal antibody

[0066] The hybridoma cells obtained in (6) above were cultured with serum-free medium, and purified by protein A affinity column chromatography to obtain a small amount of antibody.

[0067] In the present invention, the solution for mass production of monoclonal antibodies is the mouse ascites method, and the steps are as follows:

[0068] In this protocol, the hybridoma cells obtained above were inoculated into the peritoneal cavity of mice, the hybridomas were grown in the peritoneal cavity of mice, and ascites was produced to obtain a large amount of ascites monoclonal antibody.

[0069] The specific method is: BALB / c mice are inoculated with liquid paraffin intraperitoneally, 0.5 ml per mouse, and two weeks later, hybridoma cells diluted with serum-free medium are inoculated intraperitoneally, 1×10 per mouse. 6After a 5-day interval, the production of ascites in the mice was observed every day. When the abdomen of...

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Abstract

The invention disclose preparation of hybridoma cell, a monoclonal antibody secreted by the hybridoma cell and application of the monoclonal antibody and belongs to the field of microbial engineering. During screening of fusion cells prepared with the hybridoma cell, an antigen conjugated with magnetic beads and capable of specifically combining with a nonspecific antibody generated in cell culture is utilized; therefore the inhibitory effect of the nonspecific antibody upon positive reactions during the screening of the fusion cells is eliminated, and preparation efficiency is effectively improved. The invention further discloses H-FABP (Heart fatty acid-binding protein) resistant hybridoma cell strain, a monoclonal antibody produced by the strain, and application of the monoclonal antibody. The strain is capable of stably and effectively secreting high-specific H-FABP resistant monoclonal antibodies; the antibodies are of high potency and are applicable to various branch fields of in vitro diagnosis, especially immunodiagnosis.

Description

technical field [0001] The invention relates to a method for preparing hybridoma cells in the field of bioengineering, a monoclonal antibody and application fields, in particular to a method for preparing hybridoma cells of anti-H-FABP monoclonal antibody, a monoclonal antibody and applications. Background technique [0002] The basic principle of hybridoma cell technology is to maintain the main characteristics of both cells by fusing them. These two kinds of cells are antigen immunized mouse spleen lymphocytes and mouse myeloma cells respectively. The main characteristics of spleen lymphocytes are its antibody secretion function and its ability to grow in selective media, while mouse myeloma cells can divide and proliferate indefinitely under culture conditions, which is the so-called immortality. Under the action of selective medium, only the hybrid cells fused with splenocytes and myeloma cells have the ability to proliferate continuously, forming cells with two charact...

Claims

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Application Information

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IPC IPC(8): C12N15/06C12N5/20C07K16/18G01N33/68G01N33/577
Inventor 苏恩本陈玲杨艳金晶
Owner GETEIN BIOTECH
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