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Preparation of monoclonal antibody for mouse anti-human type BRAF V600E mutant protein and immunohistochemical application of monoclonal antibody

A monoclonal antibody and mutant protein technology, applied in the field of immunology, can solve the problems of clinical application limitations, time-consuming, high DNA quality requirements, etc.

Inactive Publication Date: 2019-08-30
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

However, molecular detection technology is relatively expensive, time-consuming, requires high DNA quality, and requires professional reagents and equipment. Therefore, not all hospitals can carry out molecular detection, which limits its clinical application.

Method used

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  • Preparation of monoclonal antibody for mouse anti-human type BRAF V600E mutant protein and immunohistochemical application of monoclonal antibody
  • Preparation of monoclonal antibody for mouse anti-human type BRAF V600E mutant protein and immunohistochemical application of monoclonal antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1 Preparation of anti-BRAF V600E monoclonal antibody

[0023] 1. Preparation of immune source: According to the sequence information of BRAF gene (NP-004324), order and synthesize human BRAF V600E polypeptide carrying V600E gene mutation and N-terminal with BSA sequence (to improve the immunogenicity of the polypeptide) for immunization of experimental animals and ELISA to screen positive clones.

[0024] 2. Screening and preparation of monoclonal antibodies

[0025] 1. Animal immunization: The BRAF V600E polypeptide synthesized above was emulsified with complete Freund's adjuvant, and immunized 6-8 week-old BALB / c mice by subcutaneous or intraperitoneal injection. For the second immunization, emulsify with incomplete Freund's adjuvant, and the immunization dose is 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 immuniz...

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 perform 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 dewaxing solution (Leica). 3 times. Antigen retrieval solution 2 (ER2, Leica) was used for antigen retrieval 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 second...

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 monoclonal antibody for mouse anti-human type BRAF V600E mutant protein. The monoclonal antibody is high in specificity, stable in expression and high in potency, can be applied to BRAF V600E mutant protein in malignant tumors, and has a certain medicine-based research value. The invention also relates to application of the monoclonal antibody to preparation of an immunohistochemical detecting tool for detecting the BRAF V600E mutant protein. According to the monoclonal antibody and the application of the monoclonal antibody disclosed by the invention, an immunohistochemical technology based on the BRAF V600E monoclonal antibody is built and is used in auxiliary diagnosis, selection of treatment schemes and prognosis of the malignant tumors, wherein the malignanttumors mainly comprise malignant melanoma, lung adenocarcinoma, colorectal carcinoma, gastrointestinal stromal tumors, papillary thyroid carcinoma, astrocytoma, hairy cell leukemia, lynch syndrome andthe like. Based on simple operation and less charging of the immunohistochemical method, the method can be used as a first-choice detection method of BRAF V600E gene mutation in clinic work.

Description

technical field [0001] The invention relates to the field of immunology, in particular to a mouse monoclonal antibody against human BRAF V600E mutant protein and the immunohistochemical application of the antibody. Background technique [0002] BRAF (V-raf murine sarcoma viral oncogene homolog B1) gene is one of the most important proto-oncogenes in humans. It is located on human chromosome 7p34 and is about 190kb long. It is an important transduction factor in the Ras-Raf-MEK-ERK signal transduction pathway . BRAF has a mutation rate of about 8% in human tumors. More than 40 BRAF mutations have been discovered so far, and more than 80% of them are V600E mutations. This mutation leads to continuous activation of the downstream MEK-ERK signaling pathway, which acts through the serine-threonine protein kinase in the silk protein kinase pathway, which binds receptors and RAS proteins on the cell surface through MEK and ERK to transcription in the nucleus Factors are connected...

Claims

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Application Information

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IPC IPC(8): C07K16/32G01N33/68G01N33/574
CPCC07K16/32G01N33/68G01N33/5743G01N33/57423G01N33/57419G01N33/57446G01N33/57426G01N33/57484G01N2800/52
Inventor 李雪方洁羽李军李沛祥高源远
Owner 南京基诺米医疗科技有限公司
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