Anti-human Tim-3 monoclonal antibody 8E11 and preparation method thereof

A monoclonal antibody and antibody technology, applied in the fields of botanical equipment and methods, biochemical equipment and methods, chemical instruments and methods, etc., can solve problems such as cytotoxic effects, and achieve high binding efficiency, high titer, and specificity. high sex effect

Inactive Publication Date: 2017-05-10
常州格露康生物医药科技有限公司
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  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

Another type of tumor-killing cells is NK cells, which can spontaneously kil

Method used

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  • Anti-human Tim-3 monoclonal antibody 8E11 and preparation method thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Example 1 Preparation and acquisition of anti-human Tim-3 monoclonal antibody 8E11

[0061] 1. Animal immunity

[0062] Using purified recombinant human Tim-3 protein (R&D Systems, 2365-TM) as the immunogen, immunize BALB / c mice aged 6-8 weeks, once every 2 weeks, and immunize 3 times in total, 4 days before fusion , again to boost immunity.

[0063] 2. Preparation of myeloma cells

[0064] Resuscitate the mouse myeloma cells Ag8 10 days before fusion, and culture them in DMEM medium containing 10% fetal bovine serum. After the cells grow vigorously, the shape is good (round, transparent, uniform in size), and the cell viability is greater than 95% (Terpan Blue staining) can be used for fusion. 24-36 hours before fusion, adjust the cell concentration to 3×10 with fresh medium 5 / mL.

[0065] 3. Establishment of hybridoma cell lines

[0066] Before the experiment started, the DMEM basal medium and PEG solution were pre-warmed in a 37°C water bath. Collect 2×10 7 ...

Embodiment 2

[0081] Example 2 Titer evaluation of anti-human Tim-3 monoclonal antibody

[0082] Culture L929-Tim3 cells, transfer to 96-well plate, 1×10 per well 5Cells were suspended in 100 μl of PBS containing 1% FBS. The same amount of 0.2 μg of antibodies was added to each well, including the anti-human Tim-3 monoclonal antibody 8E11 prepared in Example 1, an isotype control, and anti-human Tim-3 antibodies from R&D and eBioscience. Incubate at 4°C in the dark for 20 minutes, then centrifuge to discard the supernatant, resuspend in PBS containing 1% FBS, and detect by flow cytometry.

[0083] The result is as figure 1 As shown, compared with the known antibodies of R&D Company and eBioscience Company, the anti-human Tim-3 monoclonal antibody of the present invention has a higher binding rate at the same concentration.

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Abstract

The invention relates to the technical field of biology, and particularly relates to an anti-human Tim-3 monoclonal antibody 8E11 and a preparation method thereof. The antibody contains a light chain variable region and a heavy chain variable region, wherein the light chain variable region contains an amino acid sequence shown in SEQ ID NO:1, and the heavy chain variable region contains an amino acid sequence shown in SEQ ID NO:2. The monoclonal antibody has high specificity and high potency, thereby providing a brand-new means for Tim-3 detection.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an anti-human Tim-3 monoclonal antibody 8E11 and a preparation method thereof. Background technique [0002] Tim-3 gene and Tim-3 structural protein: the mouse Tim-3 gene is located on chromosome 11, and the full-length transcript is 1015 bases; while the human Tim-3 gene is located on chromosome 5, and the transcript is complete The length is 1116 bases. The Tim family has at least four members, namely Tim-1, Tim-2, Tim-3, and Tim-4. Among them, Tim-1 is the receptor of hepatitis A virus, Tim-2 can be specifically expressed in Th2 cells, Tim-3 protein is the surface marker of activated Th1 cells, and Tim-4, as the ligand molecule of Tim-1, is expressed in Mature DC. [0003] Current data show that the mouse Tim-3 molecule is composed of 281 amino acids and is related to Th1-related responses and diseases; the human Tim-3 molecule is a type I membrane protein composed of 301 amino...

Claims

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Application Information

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IPC IPC(8): C07K16/28C12N15/13G01N33/68G01N33/577
CPCC07K16/2803C07K2317/56
Inventor 夏瑞王信洁
Owner 常州格露康生物医药科技有限公司
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