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Mouse anti-human TIM3 (T cell immunoglobulin domain and mucin domain protein 3) monoclonal antibody and immunohistochemical application thereof

A monoclonal antibody, immunohistochemical technology, applied in the field of immunology, to achieve the effect of improving specificity and reliability

Inactive Publication Date: 2018-11-13
镇江爱必梦生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

TIM3 plays an important role in different levels of anti-tumor immunity and cancer development, but unfortunately there is no registered TIM3 antibody that can be used for clinical in vitro diagnostics (In Vitro Diagnostics, IVD) at home and abroad.

Method used

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  • Mouse anti-human TIM3 (T cell immunoglobulin domain and mucin domain protein 3) monoclonal antibody and immunohistochemical application thereof
  • Mouse anti-human TIM3 (T cell immunoglobulin domain and mucin domain protein 3) monoclonal antibody and immunohistochemical application thereof
  • Mouse anti-human TIM3 (T cell immunoglobulin domain and mucin domain protein 3) monoclonal antibody and immunohistochemical application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Embodiment 1 Preparation of anti-TIM3 monoclonal antibody

[0025] 1. Construction of TIM3 recombinant expression plasmid

[0026] According to the TIM3 gene (Uniprot-Q8TDQ0) reported by uniprot, the complete sequence of the TIM3 gene was obtained by ordering gene synthesis, and restriction sites NheI and XhoI were introduced at both ends of the sequence, and then cloned into the expression vectors pDB-His-GST and pDB respectively -His-MBP plasmid, to establish T3-pDB-GST and T3-pDB-MBP recombinant expression plasmids, which are used to immunize experimental animals and screen positive clones by ELISA respectively.

[0027] 2. Expression and purification of TIM3 recombinant protein

[0028] 1. Transformation of BL21 DE3 Star Competent Cells

[0029] Use the T3-pDB-GST and T3-pDB-MBP plasmids constructed above to transform into BL21 DE3 Star competent cells respectively, pre-culture to obtain 250ml bacterial liquid, and further add the pre-culture liquid to 6L LB conta...

Embodiment 2

[0042] Example 2 Immunohistochemical experiments using the monoclonal antibody of the present invention as the primary antibody

[0043] 1. Sampling 24 different types of cancer tissues to make tissue microarrays, and slice them with a Leica RM2235 tissue slicer with a thickness of 4 μm;

[0044] 2. Use the Leica BondMax immunohistochemical automatic staining machine to carry out immunohistochemical staining test on the antibody of the present invention, using the dewaxing and hydration conditions that come with the machine. The specific steps are: incubate at 60°C for 30 minutes, and wash with Leica's dewaxing solution for 3 all over. For antigen retrieval, Leica's Antigen Retrieval Solution 2 (ER2) was used and incubated at 100°C for 20 min. The antibody of the present invention was used as the primary antibody, which was diluted with Leica antibody diluent to a final concentration of 0.2 μg / ml. 150μl antibody was incubated at room temperature for 30min. Use 150 μl of Lei...

Embodiment 3

[0047] Embodiment 3 Specific detection of monoclonal antibody of the present invention

[0048] No positive band can be seen in the detection of non-transfected CHO-K1 cells by using the antibody of the present invention.

[0049] The antibody of the present invention was used to detect the 96-well plate (Her-2) pre-coated with irrelevant antigens by ELISA, and the result was negative.

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Abstract

The invention discloses a mouse anti-human TIM3 (T cell immunoglobulin domain and mucin domain protein 3) monoclonal antibody. The mouse anti-human TIM3 monoclonal antibody is stable in performance, high in potency and capable of particularly recognizing expression of TIM3 on surfaces of immune cells and tumor cells. The invention further relates to application of the mouse anti-human TIM3 monoclonal antibody in preparation of an immunohistochemical TIM3 detecting tool. An immunohistochemical method is used to detect the TIM3 for the first time, and scientific references are provided for clinical diagnosis and treatment of non-small cell lung cancer, renal cell carcinoma, hepatocellular carcinoma, melanoma, colon cancer, esophageal cancer and other tumors as well as septicopyemia, systemiclupus erythematosus or related infections and autoimmune diseases. The monoclonal antibody generated from different hybridomas is discovered for the first time can recognize different states of TIM3of a same antigen, by the aid of the discovery, the TIM3 monoclonal antibodies can be screened purposefully, and basic medical research value is realized.

Description

technical field [0001] The invention relates to the field of immunology, in particular to a mouse monoclonal antibody against human TIM3 protein and the immunohistochemical application of the antibody. Background technique [0002] T cell immunoglobulin domain and mucindomain protein 3 (T cell immunoglobulin domain and mucindomain protein 3, TIM3) is an immune checkpoint molecule discovered in the study of asthma susceptibility genes in 2001. It is a member of the Tim family. Protein structure includes immunoglobulin-like (IgV) region, mucin (mucin) region, transmembrane region and intracellular region. TIM3 is constitutively or inducibly expressed in Th1, Tcl, Treg, NK, dendritic cells, mast cells, mononuclear macrophages and lymphatic endothelial cells, and these cells are the main components of the tumor microenvironment, so this The role of molecules in tumor immunity has received much attention. On the one hand, TIM3 on Th1 is activated by its ligand galectin-9 (Gal-9...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28G01N33/68G01N33/574
CPCC07K16/2803G01N33/57488G01N33/6893
Inventor 李军李雪方洁羽高源远威尔逊王李沛祥
Owner 镇江爱必梦生物科技有限公司
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