Preparation and immunohistochemical use of mouse anti-human IDH1 R132H mutant protein monoclonal antibody

A monoclonal antibody, mutant protein technology, applied in the field of immunology, can solve the problems to be improved, the scarcity of antibodies and so on

Inactive Publication Date: 2018-11-02
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

At present, IDH1 R132H antibodies developed at home and abroad are rare, and their specificity and affinity need to be improved. Therefore, it is urgent to develop new anti-IDH1 R132H monoclonal antibodies with excellent performance, so as to fill the gap in the domestic industry, reduce treatment costs, and reduce the burden on patients.

Method used

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  • Preparation and immunohistochemical use of mouse anti-human IDH1 R132H mutant protein monoclonal antibody
  • Preparation and immunohistochemical use of mouse anti-human IDH1 R132H mutant protein monoclonal antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1 Preparation of anti-IDH1 R132H monoclonal antibody

[0023] 1. Preparation of immune source: According to the sequence information of IDH1 gene (BC012846), order and synthesize human IDH-R132H polypeptide carrying R132H gene mutation and N-terminal with BSA sequence (improving the immunogenicity of the polypeptide), which is used to immunize experimental animals and Positive clones were screened by ELISA.

[0024] 2. Screening and preparation of monoclonal antibodies

[0025] 1. Animal immunization: The IDH1 R132H 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 ...

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, and slice them with a Leica RM2235 tissue slicer with a thickness of 4 μm;

[0032] 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 secon...

Embodiment 3

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

[0036] 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 preparation and immunohistochemical use of a mouse anti-human IDH1 R132H mutant protein monoclonal antibody. The antibody is high in specificity, stable in expression and highin valence, can be used for detecting an IDH1 R132H mutant protein in a malignant tumor and has certain medical fundamental research values. The invention further relates to application of the antibody in the preparation of an immunohistochemical detection tool for detecting the IDH1 R132H mutant protein. An immunohistochemical technique based on the IDH1 R132H mutant protein monoclonal antibody is established, is applied to the treatment of the malignant tumor and mainly comprises the procedures of auxiliary diagnosis of neuroglioma and acute myelogenous leukemia, selection of a treatment scheme and prognosis. The immunohistochemical method is easy in operation and relatively low in charge, can be implemented in most pathology laboratories and can be taken as a primary detection method for IDH1 R132H gene mutation in the clinical work.

Description

technical field [0001] The invention relates to the field of immunology, in particular to a mouse monoclonal antibody against human IDH1 R132H mutant protein and the immunohistochemical application of the antibody. Background technique [0002] Isocitrate dehydrogenase 1 (Isocitrate dehydrogenase 1, IDH1) has a full length of 1245bp and encodes 414 amino acids. It is a member of the NADP-dependent β-decarboxylation dehydrogenase family and is a key rate-limiting enzyme in the tricarboxylic acid cycle. Oxidative decarboxylation of acid to α-ketoglutarate (α-KG) and carbon dioxide (CO 2 ), providing energy and biosynthetic precursors for cell metabolism. IDH1 mutation is a diagnostic and prognostic marker for secondary glioma, among which R132H is the most common, accounting for more than 90%. IDH1-R132 gene mutations mainly occur in diffuse astrocytoma, oligodendroglioma, oligodendroglioma, anaplastic astrocytoma, and anaplastic astrocytoma of World Health Organization ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/40G01N33/574
CPCC07K16/40G01N33/57407G01N33/57426G01N33/57484
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
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