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Methods for preparing monoclonal antibody and hybridoma cell strain thereof by multiple antigens in immune high-flux manner

A hybridoma cell and monoclonal antibody technology, applied in the biological field, can solve the problem of insufficient types of immunization and screening

Inactive Publication Date: 2013-08-21
INST OF RADIATION MEDICINE ACAD OF MILITARY MEDICAL SCI OF THE PLA
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  • Abstract
  • Description
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  • Application Information

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Problems solved by technology

[0005] In the previous study, mice were immunized with four known proteins and monoclonal antibodies were screened by ELISA and protein chips, but the types of immunization and screening were not enough

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  • Methods for preparing monoclonal antibody and hybridoma cell strain thereof by multiple antigens in immune high-flux manner
  • Methods for preparing monoclonal antibody and hybridoma cell strain thereof by multiple antigens in immune high-flux manner
  • Methods for preparing monoclonal antibody and hybridoma cell strain thereof by multiple antigens in immune high-flux manner

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Embodiment 1

[0093] Example 1. High-throughput preparation of hybridoma cells secreting specific monoclonal antibodies by immunization with various antigens

[0094] 1. Acquisition of multiple immune antigens and detection proteins

[0095] 1. Selection of multiple target proteins

[0096] The following eight proteins were selected as target proteins: tumor protein p53 (TP53, NP_000537.3), c-myc (NP_002458.2), S100 calcium-binding protein A8 (S100A8, NP_002955.2), S100 calcium-binding protein A9 (S100A9, NP_002956.1), histone acetylase pCAF (NP_003875.3), calcitonin PCT-1 (NP_001732.1), calcitonin truncated PCT-2 (upper fragment of NP_001732.1, PCT-2 protein ), protein tyrosine phosphatase PTEN (PTEN, NP_000305.3).

[0097] The nucleotide sequences of the genes encoding the above-mentioned various proteins are shown above.

[0098] The amino acid sequence similarity of any two target proteins is listed in Table 1. PCT-1 and PCT2 have a high homology (64%) because they have an identical ...

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Abstract

The invention discloses methods for preparing a monoclonal antibody and a hybridoma cell strain thereof by multiple antigens in an immune high-flux manner. The method for preparing a hybridoma cell strain for secreting a specific monoclonal antibody by multiple antigens in the immune high-flux manner comprises the following steps of: 1) mixing n antigen proteins serving as immune antigens for immunizing a mice, thereby obtaining an immunized mice; 2) combining in-vitro spleen cells and SP2 / 0 myeloma cell of the immunized mice to obtain combined cells; 3) performing primary enzyme-linked immuno sorbent assay (ELISA) selection on the combined cells, and selecting hybridoma cells generating fos-like immunoreactivity; and 4) performing secondary ELISA selection on the hybridoma cells generating the fos-like immunoreactivity, and selecting the hybridoma cells aiming at single-antigen fos-like immunoreactivity. A high-flux technical method for obtaining multiple protein monoclonal antibodies by once cell combination is constructed; the time and the labor cost are saved for the preparation of the monoclonal antibodies; and contributions is made to improvement of the production efficiency of the monoclonal antibodies.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for high-throughput preparation of monoclonal antibodies and hybridoma cell lines by immunizing multiple antigens. Background technique [0002] With the development of proteomics research, thousands of proteins need further functional verification with affinity reagents such as monoclonal antibodies, such as protein qualitative, quantitative analysis, protein separation and purification, functional verification, and protein interaction. Function verification and identification of protein post-translational modifications, etc. [0003] Monoclonal antibody technology is the basic method to produce highly specific antibodies with protein recognition function. Therefore, monoclonal antibody has become an indispensable tool in protein research. However, the traditional monoclonal antibody preparation is time-consuming and inefficient. Many articles have reported attempts to inc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/06C12N5/20C07K16/18C07K16/32
Inventor 刘莹刘静左威郝露张莉莉甄蓓
Owner INST OF RADIATION MEDICINE ACAD OF MILITARY MEDICAL SCI OF THE PLA
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