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Preparation of rabbit anti-human programmed death-ligand 1 (PD-L1) protein monoclonal antibody and immunohistochemical use thereof

A PD-L1, monoclonal antibody technology, applied in the biological field, can solve problems such as high cost, limitations of supporting instruments and applications, and need to be improved, and achieve the effect of improving specificity and reliability

Inactive Publication Date: 2018-11-23
南京基诺米医疗科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the selection of appropriate PD-1 / PD-L1 inhibitors in the world requires corresponding PD-L1 detection methods. Since the PD-L1 antibodies and techniques used in each detection are different, when using these antibodies for auxiliary diagnosis The cost is high, and at the same time, it is limited by foreign suppliers in terms of supporting instruments and applications
However, domestically developed PD-L1 antibodies are scarce, and their specificity and affinity need to be improved. Therefore, it is urgent to develop new anti-PD-L1 monoclonal antibodies with excellent performance to fill the gap in the domestic industry, reduce treatment costs, and reduce the burden on patients.

Method used

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  • Preparation of rabbit anti-human programmed death-ligand 1 (PD-L1) protein monoclonal antibody and immunohistochemical use thereof
  • Preparation of rabbit anti-human programmed death-ligand 1 (PD-L1) protein monoclonal antibody and immunohistochemical use thereof
  • Preparation of rabbit anti-human programmed death-ligand 1 (PD-L1) protein monoclonal antibody and immunohistochemical use thereof

Examples

Experimental program
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Embodiment 1

[0023] Example 1 Preparation of anti-PD-L1 monoclonal antibody

[0024] 1. Preparation of immune source: According to the sequence information of PD-L1 gene (BC113734), order and synthesize human PD-L1 polypeptide with N-terminal BSA sequence, which is used to immunize experimental animals and screen positive clones by ELISA.

[0025] 2. Blood collection before immunization: select purebred New Zealand white rabbits, and take blood from ear veins as pre-immunization blood samples.

[0026] 3. Screening and preparation of monoclonal antibodies

[0027] 1. Animal immunization: The above synthetic human PD-L1 polypeptide with BSA sequence at the N-terminal was fully mixed with an equal volume of Freund's complete adjuvant to prepare a fully emulsified immune source, and the purebred New Zealand white rabbit was first immunized by subcutaneous injection , the immunization dose was 500μg / only. Two weeks later, the second immunization was carried out, emulsified with incomplete Fr...

Embodiment 2

[0030] Example 2 Immunohistochemical experiments using the monoclonal antibody of the present invention as the primary antibody

[0031] 1. Sampling 24 different types of cancer tissues to make tissue microarrays, using a Leica RM2235 tissue slicer to slice with a thickness of 4 μm;

[0032]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 antibody...

Embodiment 3

[0035] Embodiment 3 Specific detection of monoclonal antibody of the present invention

[0036] No positive band can be seen in the detection of untransfected 293T cells by using the antibody of the present invention.

[0037] 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 rabbit anti-human programmed death-ligand 1 (PD-L1) protein monoclonal antibody which can specifically recognize the PD-L1 protein expressed on the cell surface of a cell, and is stable in performance and high in titers, and has no cross reaction with other proteins in cells and has a certain medical basic research value. The invention also relates to the use of the monoclonal antibody in the preparation of an immunohistochemical detection tool for detecting the PD-L1 protein. The invention adopts an immunohistochemical method to detect the expression of the PD-L1 onthe surfaces of tumor cells, mainly including melanoma, non-small cell lung cancer, bladder cancer, renal cancer, gastric cancer, colorectal cancer, prostate cancer, cervical cancer, breast cancer, toprovide a scientific reference for whether to use PD-1 / PD-L1 inhibitors in immunotherapy and prognosis of tumors and effectively reduce treatment cost, and the detection hasrealizing an important application value.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a rabbit monoclonal antibody against human PD-L1 protein and the immunohistochemical application of the antibody. Background technique [0002] Programmed cell death-ligand 1 (PD-L1), a type I transmembrane protein, has a total length of 290 amino acids and a molecular weight of about 40kDa. In recent years, studies have found that the expression of PD-L1 is upregulated on the surface of various tumor cells, which is related to the immune escape of tumors. The body's immune system usually recognizes tumor cells and responds to them, promoting the proliferation of antigen-specific T cells, thereby killing or eliminating tumor cells. However, the body's immune exclusion against tumors is controlled by immune regulation points, the most important of which is programmed cell death-1 (PD-1). When PD-1 on the surface of T cells recognizes PD-L1 on the surface of tumor cells, it can trans...

Claims

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Application Information

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IPC IPC(8): C07K16/28G01N33/68G01N33/574
CPCC07K16/2827G01N33/57484G01N33/6872G01N2333/70532
Inventor 高源远李军李沛祥李雪方洁羽朱禹宣
Owner 南京基诺米医疗科技有限公司