Anti-HER2 recombinant rabbit monoclonal antibody and application thereof

A technology of monoclonal antibody and heavy chain, which is applied in the field of immunochemistry to achieve high sensitivity, accurate cancer and strong positive signal

Active Publication Date: 2022-07-12
苏州百道医疗科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Amplification of HER2 gene inevitably leads to overexpression of its protein product

Method used

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  • Anti-HER2 recombinant rabbit monoclonal antibody and application thereof
  • Anti-HER2 recombinant rabbit monoclonal antibody and application thereof
  • Anti-HER2 recombinant rabbit monoclonal antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] This embodiment is the preparation and screening of anti-HER2 recombinant rabbit monoclonal antibody, and the steps include:

[0036] (1) Antigen preparation

[0037] The specific sequence of the HER2 antigen is shown in SEQ ID NO. 1 below.

[0038] SEQ ID No. 1: PTAENPEYLGLDVPV.

[0039] The above-mentioned polypeptide sequence is selected by analyzing the HER2 molecular sequence according to the structure, antigenicity, hydrophobicity and secondary structure of the HER2 protein molecule on the cell membrane. The polypeptide of the sequence shown in SEQ ID NO. 1 was artificially synthesized, and the synthesized polypeptide was used as an antigen for immunizing rabbits. During immunization, the polypeptide of the sequence shown in SEQ ID NO. 1 was conjugated with KLH or OVA as an immunogen to immunize rabbits.

[0040] (2) Immunity

[0041] The polypeptide sequence of SEQ ID NO.1 (HER2 antigen) was mixed with complete Freund's adjuvant (1:1) and emulsified, and multip...

Embodiment 2

[0067] This example is the immunohistochemical detection of anti-HER2 recombinant rabbit monoclonal antibody as the primary antibody, and the method is as follows:

[0068] (1) Preparation of sample slices: The formalin-fixed paraffin-embedded human breast cancer slices were baked in a 60°C incubator for 1-2 hours, and stored for later use.

[0069] (2) Section dewaxing: paraffin sections were first placed in fresh xylene for dewaxing, and soaked for 2 times for 10 min each time.

[0070] (3) Slice hydration: soak in absolute ethanol, absolute ethanol, 95% ethanol, 85% ethanol, and 70% ethanol for 5 minutes in order for hydration, and then rinse with purified water twice, 3 minutes each time.

[0071] (4) Antigen retrieval: It is recommended to use the high temperature thermal repair method for 3 minutes (if using an automatic repair instrument, you can set the high temperature repair at 98°C for 20 minutes). After the slices are naturally cooled to room temperature, the tissu...

Embodiment 3

[0081]This example is for the determination of the affinity of the 246G0D3 anti-HER2 recombinant rabbit monoclonal antibody, and the commercially available anti-HER2 antibody is used as a control. The determination method is as follows:

[0082] (1) Take out the labeled HER2 polypeptide from 4°C and return to room temperature. Dilute to a concentration of 1 μg / ml, add 100 μL / well to a 96-well microtiter plate and incubate at 4°C overnight, followed by blocking with 2% BSA at 4°C overnight.

[0083] (2) The HER2 recombinant rabbit monoclonal antibody cloned from 246G0D3 and the commercially available anti-HER2 antibody were diluted to an initial concentration of 0.5 μg / mL, and were successively diluted by 2-fold gradients. A total of 7 concentration gradients were set for comparison.

[0084] (3) Add 100 μL / well of anti-HER2 recombinant rabbit monoclonal antibody and commercial anti-HER2 antibody diluted in 7 gradient concentrations to a 96-well microtiter plate with peptides, ...

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Abstract

The invention relates to an anti-HER2 recombinant rabbit monoclonal antibody which comprises a heavy chain variable region and a light chain variable region, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID No.4; the amino acid sequence of the light chain variable region is as shown in SEQ ID No.5. Compared with a commercially available anti-HER2 monoclonal antibody, the anti-HER2 recombinant rabbit monoclonal antibody provided by the invention has higher affinity with HER2 protein, can recognize and detect expression of the HER2 protein on tumor cells or immune cells in a high-specificity and high-sensitivity manner, and has the advantages of high specificity and high sensitivity. The kit can be applied to the detection and screening fields such as immunohistochemistry (IHC), indirect ELISA, Western blotting, antibody chip preparation and flow cytometry, more accurate detection and evaluation results can be obtained, and the detection cost and the interference of background signals are reduced.

Description

technical field [0001] The invention belongs to the technical field of immunochemistry, and in particular relates to an anti-HER2 antibody and applications thereof, in particular to applications in immunohistochemical detection. Background technique [0002] HER2 (also known as HER2 / neu, c-erbB2, ERBB2, or neu) is a transmembrane receptor tyrosine kinase first discovered more than 20 years ago in a rat neuroblastoma cell line (neu). HER2 is a proto-oncogene whose activation causes malignant transformation, increasing the malignant potential of cells (cell proliferation, invasion, etc.). In human cancers, HER2 is activated through gene amplification, a genomic mutation in which a small segment of the 17q12-q21 chromosomal band is multiplied 50-100-fold in cells. HER2 is considered to be the target oncogene driving amplification, although adjacent genes may sometimes co-amplify with HER2. Amplification of the HER2 gene necessarily leads to overexpression of its protein produ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/32C07K16/28C07K16/40C12N15/13G01N33/68G01N33/577G01N33/58
CPCC07K16/32C07K16/2863C07K16/40G01N33/74G01N33/577G01N33/581C07K2317/56C07K2317/92G01N2333/71
Inventor 刘杨宋砚明吴纯
Owner 苏州百道医疗科技有限公司
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