Preparation and application of mouse monoclonal antibody against human Siglec-15 protein

A monoclonal antibody, mouse anti-human technology, applied in the biological field, can solve the problems that cannot be used in clinical inspection, and achieve the effect of improving specificity and reliability

Inactive Publication Date: 2019-10-29
广东三九脑科医院 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] There is currently no Siglec-15 antibody that can be used for clinical diagnosis in the world. Some antibodies used in scientific research are polyclonal antibodies, which cannot be used in clinical testing due to specificity and stability. Therefore, it is urgent to develop new ones with excellent performance. The anti-Siglec-15 monoclonal antibody fills the gap in this industry at home and abroad

Method used

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  • Preparation and application of mouse monoclonal antibody against human Siglec-15 protein
  • Preparation and application of mouse monoclonal antibody against human Siglec-15 protein
  • Preparation and application of mouse monoclonal antibody against human Siglec-15 protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1 Preparation of anti-Siglec-15 monoclonal antibody

[0023] 1. Preparation of immune source: According to the sequence information of Siglec-15 gene (NM_213602.2), order and synthesize human Siglec-15 polypeptide with N-terminal BSA sequence, which is used to immunize experimental animals and screen positive clones by ELISA.

[0024] 2. Animal immunization: the above-mentioned purified Siglec-15 polypeptide was emulsified with complete Freund's adjuvant, and immunized 6-8 week-old BALB / c mice by subcutaneous or intraperitoneal injection. The second immunization was carried out, emulsified with incomplete Freund's adjuvant, and the immunization dose was 50 μg per mouse. After the two immunizations, the tail blood was taken to measure the serum titer by ELISA gradient dilution; according to the results, it was determined whether to boost the immunization, and the mouse with the highest antibody titer was selected for cell fusion.

[0025] 2. Cell fusion: the mye...

Embodiment 2

[0029] Example 2 Immunohistochemical experiments using the monoclonal antibody of the present invention as the primary antibody

[0030] 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;

[0031] 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 30min, wash with dewaxing solution (Leica) 3 times. For antigen retrieval, antigen retrieval solution 2 (ER2, Leica) was used and incubated at 100°C for 30 min. The antibody of the present invention was used as the primary antibody, diluted with antibody diluent (Leica) to a final concentration of 1.0 μg / ml, 150 μl. The antibody was incubated at room temperature for 30 min. Use 150 μl of matching secondary an...

Embodiment 3

[0034] Example 3 Specific detection of the monoclonal antibody of the present invention

[0035] 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 monoclonal antibody against a human Siglec-15 protein. The specificity is high, the performance is stable and the titer is high. The invention further relates to application of the monoclonal antibody to preparation of immunohistochemical detection tools for the Siglec-15 protein. According to the mouse monoclonal antibody against the human Siglec-15 protein, an immunohistochemistry method is used for detecting expression of Siglec-15 on the tumor cell surface for selection, the curative effect and the prognosis of therapeutic schedules of malignant tumors. Theinvention further relates to the application of the monoclonal antibody to preparation of antibody drugs for immunotherapy of the malignant tumors. The important application value is achieved. The adopted antibody drugs can be full-length or partial sections (Fab, F(ab)'2 or ScFv) of an Siglec-15 monoclonal antibody or combined use of the Siglec-15 monoclonal antibody and a PD-L1 monoclonal antibody. The malignant tumors mainly include colorectal cancer, endometrial cancer, thyroid cancer, bladder cancer, kidney cancer, lung cancer, liver cancer and the like.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a mouse monoclonal antibody against human Siglec-15 protein, and the immunohistochemistry and therapeutic application of the antibody. Background technique [0002] Sialic acid-binding immunoglobulin-type lectins (Sialic acid-binding immunoglobulin-type lectins, Siglecs) are a class of proteins located on the surface of cells, mainly expressed on the surface of immune cells, and belong to type I lectins. Since it was first reported in 1986, 17 members of the family have been identified. Among them, Siglec-15 was originally found to play an important role in physiological bone reorganization, and is also involved in pathological bone loss caused by estrogen deficiency. The latest research results show that Siglec-15mRNA is only expressed in a small amount in most normal human tissues and various immune cell subsets, but it is highly expressed in macrophages. It has more than 30% hom...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28G01N33/68G01N33/577A61K39/395A61P35/00
CPCC07K16/2803G01N33/68G01N33/577A61P35/00A61K2039/505G01N2800/52
Inventor 李智李海南蔡林波李军李沛祥李雪方洁羽
Owner 广东三九脑科医院
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