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Anti-IDH1 R132H antibody as well as preparation method and application thereof

A technology of IDH1R132H and antibodies, which is applied in the fields of botany equipment and methods, biochemical equipment and methods, antibodies, etc., and can solve the problems such as the emergence of monoclonal antibodies without antibodies

Active Publication Date: 2017-07-18
北京爱仁医疗科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Although the mutation of IDH1 R132H has been found in tumors and leukemia, there is no antibody against this site, especially monoclonal antibody.

Method used

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  • Anti-IDH1 R132H antibody as well as preparation method and application thereof
  • Anti-IDH1 R132H antibody as well as preparation method and application thereof
  • Anti-IDH1 R132H antibody as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] 1. Production of anti-IDH1 R132H antibody

[0059] Antigen preparation

[0060] A short peptide containing IDH1 R132H (synthesized by Shanghai Qiangyao Biotechnology Co., Ltd.) was artificially synthesized and coupled with a carrier to immunize mice.

[0061] Hybridoma preparation

[0062] We used standard in vivo immunization and fusion methods to generate anti-IDH1 R132H antibodies. The brief process of this method is as follows:

[0063] Mice were immunized with IDH1 R132H antigen obtained above, and one type of antigen was mixed and emulsified with Freund's complete adjuvant in an equal volume, and injected into muscles of limbs at multiple points, each with 200 microliters per injection. After the first immunization, booster immunization was carried out with the same dose of any other type plus Freund's incomplete adjuvant. After the fourth booster immunization, blood was collected to detect the titer of reaction with it. When the titer reaches 10 6 In the ab...

Embodiment 2

[0071] Detection of specificity of anti-IDH1 R132H antibody.

[0072] Dot blot verification:

[0073] HEK293FT cells were transfected with an overexpression vector expressing the full length of IDH1 with a Flag tag and an overexpression vector expressing IDH1 R132H. After 72 hours, the cells were lysed, and 30ul of the lysate was spotted on the NC membrane, and air-dried at room temperature. Blocked with PBS at room temperature for 1 hr, added TBST 1:5000 diluted monoclonal antibody, reacted at room temperature for 1 hr, rinsed the membrane with TBST for 10 min×3; added diluted secondary antibody (both 1:4000 dilution), incubated at room temperature for 1 h on a horizontal shaker. Absorb the secondary antibody, rinse the membrane with TBST, 10min×4, mix ECL (A:B=100:1 solution), fully cover the surface of the wet NC membrane, and let it stand for 1min; seal the NC membrane with plastic wrap in the dark room, and put The X-ray film was laid flat on the NC film to develop and r...

Embodiment 3

[0076] Isolation of variable region of light chain gene and heavy chain gene of monoclonal antibody

[0077]Polymerase chain reaction is used to isolate the variable region of the antibody gene, wherein VLF and VLR are downstream primers for gene amplification of the light chain variable region, and VLF and VLR are downstream primers for gene amplification of the heavy chain variable region. The template used was the total RNA of the hybridoma cell line extracted by the Trizol method. After the DNA was digested, it was reverse-transcribed into cDNA using a reverse transcription kit. Conditions: The PCR reaction conditions are: pre-denaturation at 95°C for 5 minutes; then 40 cycles of reaction at 95°C for 15s, 55°C for 30s, and 72°C for 50s. The fragment of interest was recovered, cloned into a T-vector, and then sequenced (Intravel Corporation). The sequencing sequences are compared to determine the nucleotide sequence of the antibody variable region, and then determine the c...

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Abstract

The invention relates to an anti-IDH1 R132H antibody as well as a preparation method and application thereof. The antibody or a functional derivative specifically identifies epitope of IDH1 R132H. The monoclonal antibody has (1) an amino acid sequence of a variable region of a heavy chain of antibody or a conservative variable sequence as shown in one of SEQ IDNO:2, 6, 10; and (2) amino acid sequence of a variable region of a light chain of antibody or a conservative variable sequence as shown in one of SEQ IDNO:4, 8, 12. The invention also relates to coded nucleic acid molecules, a construction vector, an expression system, a corresponding human source antibody, a preparation method, an application and a pharmaceutical composition. The antibody has immune specificity on human type I human isocitrate dehydrogenase (IDHI1)R132H, can specifically identify a mutant site of IDH1 R132H related to a malignant disease and is specifically combined therewith for diagnosing and treating diseases which have pathogenic effects after mutation of IDH1 R132H, for example, diseases such as glioma and leukemia.

Description

technical field [0001] The present invention relates to anti-IDHI R132H antibodies and their use in diagnosis and therapy. Background technique [0002] Isocitrate dehydrogenase (IDH) is a key enzyme in the tricarboxylic acid cycle, which catalyzes the oxidative decarboxylation of isocitrate to generate α-ketoglutarate, and utilizes nicotinamide adenine dinucleotide / nicotinamide adenine Dinucleoside phosphate acts as a cofactor to generate NADH / NADPH (reduced nicotinamide adenine dinucleotide / nicotinamide adenine dinucleoside phosphate), which plays a very important role in the process of energy metabolism, vitamin and amino acid synthesis . IDH is mainly divided into IDH1, IDH2 and IDH3 subtypes. The similarity between human IDH1 and IDH2 reaches 70%, and they are encoded by different genes (IDH1 gene is located on chromosome 2q33; IDH2 gene is located on chromosome 5q26). IDH1 is expressed in the cytoplasm and peroxisomes, while IDH2 is mainly present in the mitochondri...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/40C12N15/13G01N33/573G01N33/574A61K39/395A61P35/00A61P35/02
CPCA61K2039/505C07K16/40C07K2317/24C07K2317/54C07K2317/55C07K2317/622G01N33/573G01N33/57407G01N33/57426G01N2333/904
Inventor 龙梅金卫林高兴春
Owner 北京爱仁医疗科技有限公司
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