Anti ERCC1 monoclonal antibody 2E12 and application

A monoclonal antibody, antibody technology, applied in anti-enzyme immunoglobulin, biochemical equipment and methods, instruments, etc., can solve problems affecting the specificity of immunohistochemical results

Active Publication Date: 2013-11-06
北京中杉金桥生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The core of the IHC experiment is the monoclonal antibody that specifically binds to ERCC1. At present, the ERCC1 monoclonal antibody widely used in the first-line clinical pathology at home and abroad is 8F1, but studies have shown that while 8F1 specifically binds to ERCC1, it also crosses with non-specific proteins. Reaction, which directly affects the specificity of immunohistochemical results

Method used

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  • Anti ERCC1 monoclonal antibody 2E12 and application
  • Anti ERCC1 monoclonal antibody 2E12 and application
  • Anti ERCC1 monoclonal antibody 2E12 and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Embodiment 1, construction of ERCC1 recombinant expression plasmid

[0069] The plasmid BC0089302 (containing ERCC1ORF 894bp) obtained from ATCC was used as a template, and 5'-GAGAGCGATCGCCATGGACCCTGGGAAGGACAAAG-3' (SEQ ID NO: 2) was used as a forward primer (the 5' end of which was introduced into the restriction endonuclease SgfI site GCGATCGC) , 5'-CGAGCCCTTCTTGAAAGTACCCACGCGTGAGA-3' (SEQ ID NO: 3) is the reverse primer (the 5' end introduces the restriction endonuclease MluI site ACGCGT), obtains ERCC1ORF through PCR amplification, and introduces it on both sides respectively Recognition sites for restriction enzymes SgfI and MluI. According to the manufacturer's instructions, using restriction endonucleases SgfI and MluI (ABI company), the PCR product was cloned into the expression vector pCMV6-Entry (derived from origene company), thereby constructing the recombinant plasmid pCMV6-ERCC1 expressing ERCC1, the recombinant The plasmid carries Myc tag and Flag tag do...

Embodiment 2

[0070] Example 2, Expression and Purification of ERCC1 Recombinant Antigen

[0071] Expression of ERCC1 recombinant protein

[0072] Using methods well known to those skilled in the art, the pCMV6-ERCC1 recombinant plasmid obtained in Example 1 was transfected into HEK293T cells. Briefly, at 37 °C in 5% CO 2 In the incubator (Thermo Company), HEK293T cells (derived from ATCC) were cultured in DMEM medium (Thermo Fisher Company) supplemented with newborn bovine serum and gentamycin until confluent, and then passaged at a ratio of 1:3. Continue culturing in a 10cm petri dish (Corning); take 7.5ml DMEM medium (without serum and antibiotics) into a 50ml tube, add 300ul PEI MegaTran1.0 (produced by origene) and mix well; then add 75μg pCMV6-ERCC1 recombinant Put the plasmid DNA into the above mixture, mix well and let it stand for 30 minutes; after standing still, take 515 μl into each culture dish and continue to culture the cells; 24 hours after transfection, add 2M sodium bu...

Embodiment 3

[0078] Example 3, Screening and Preparation of Monoclonal Antibody

[0079] animal immunity

[0080] Purified ERCC1 protein was used as antigen to immunize 6-8 week old BALB / c mice. The way of immunization is subcutaneous or intraperitoneal injection. For the first immunization, the antigen was emulsified with complete Freund's adjuvant, and the immunization dose was 50 μg per mouse. Two weeks later, the antigen was emulsified with incomplete Freund's adjuvant for the second immunization with a dose of 50 μg per mouse. After two immunizations, the mouse tail blood was taken to measure the titer of the serially diluted serum by ELISA method (coated with ERCC1 protein, goat anti-mouse antibody as the secondary antibody). Determine whether to further immunize the mice according to the measurement results, and select the mouse with the highest serum antibody titer for subsequent cell fusion.

[0081] cell fusion

[0082] The myeloma cells used were BALB / c-derived sp2 / 0 ce...

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Abstract

The invention relates to a monoclonal antibody 2E12 capable of specifically bonding excision repair cross complementing action group 1 (ERCC1) protein, generated hybridoma of the monoclonal antibody, a composition of the monoclonal antibody, and a usage method and an application of the monoclonal antibody. The monoclonal antibody 2E12 can overcome the non-specificity problem of ERCC1 monoclonal antibody 8F1, can be used for prognosis of tumors such as non-small cell lung cancer, ovary cancer, breast cancer, renal adenocarcinoma, endometrial carcinoma, and can realize the curative effect prediction for auxiliary chemotherapy with platinum containing chemotherapeutics.

Description

technical field [0001] The present invention relates to a monoclonal antibody that can specifically bind to excision repair cross-complementation-action group 1 (ERCC1) protein, a hybridoma producing the monoclonal antibody, and a combination comprising the monoclonal antibody substances, and methods and uses of said monoclonal antibodies. Background technique [0002] Malignant tumors are one of the diseases that seriously threaten human health, and chemotherapy involving platinum-based drugs is an important treatment. Platinum drugs represented by cisplatin are high-efficiency and broad-spectrum inorganic antineoplastic drugs developed in the 1960s. They belong to cytotoxic drugs that destroy the structure and function of DNA. After long-term research and development, cisplatin (cisp1atin ), Carboplatin, Nedaplatin, Oxaliplatin, Sunpla and Lobaplatin are often used in combination with other antineoplastic drugs for ovarian cancer and lung cancer , gastric cancer, cervica...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/40C12N5/20G01N33/577C12R1/91
Inventor 何为无马东辉袁克湖陈思王宜任琪龚世妍
Owner 北京中杉金桥生物技术有限公司
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