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Anti-C-peptide monoclonal antibody and use thereof in detection of C-peptide content

A monoclonal antibody and application technology, applied in the preparation of reagents or drugs for detecting or diagnosing C-peptide, the field of monoclonal antibodies, can solve the problems of inappropriate immunization antigen or screening antigen, poor stability of C-peptide, easy degradation, etc., to avoid C-peptide is unstable, facilitates purification, increases solubility and stability

Active Publication Date: 2013-05-01
BEIJING LEADMAN BIOCHEM
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] C-peptide is composed of 31 amino acid residues, and the molecule is small. The C-peptide obtained by peptide synthesis is very unstable and easy to degrade. It is not suitable as an immune antigen or a screening antigen.

Method used

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  • Anti-C-peptide monoclonal antibody and use thereof in detection of C-peptide content
  • Anti-C-peptide monoclonal antibody and use thereof in detection of C-peptide content
  • Anti-C-peptide monoclonal antibody and use thereof in detection of C-peptide content

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Example 1 Preparation of C-peptide-GST and C-peptide-Trx fusion proteins

[0019] 1) Experimental materials

[0020] Expression vector: pGEX-4T-1 was purchased from GE healthcare, pET32 was purchased from invitrogen; expression host bacteria: Escherichia coli BL21(DE3), purchased from Beijing Tiangen Biotechnology Co., Ltd.; yeast powder and peptone: both products of Oxide Company;

[0021] 2) Experimental method

[0022] The C-peptide gene sequence (the nucleotide sequence is shown in SEQ ID No.1; the encoded amino acid sequence is shown in SEQ ID No.2) has a BamHI site at the 5' end and an XhoI site at the 3' end , inserted into the pMD-18-T vector, the target gene was double-digested with BamHI and XhoI and then inserted into the pGEX-4T-1 and pET32a vectors treated in the same way to construct the GST-C peptide expression vector pGEX- C peptide and Trx-C peptide expression vector pET32a-C peptide, transfer the plasmid into Escherichia coli BL21 (DE3) competent ce...

Embodiment 2

[0023] Example 2 Preparation of anti-C peptide paired monoclonal antibodies

[0024] 1) Experimental materials

[0025] SP 2 / 0 was purchased from the Cell Center of Shanghai Institute of Biochemistry; Balb / c mice were purchased from the Experimental Animal Center of the Chinese Academy of Medical Sciences; fetal bovine serum: the product of Hangzhou Sijiqing Company; DMEM, HAT and HT are all products of Invitrogen Company; 50% PEG1450 The solution is the product of Sigma Company; other drugs are of analytical grade.

[0026] 2) Experimental method

[0027] 2.1 Immunization of mice

[0028] Select 6-8 female Balb / c mice aged 6-8 weeks to immunize with C-peptide-GST and C-peptide-Trx fusion proteins expressed in Escherichia coli respectively. Immunizations were performed 3 times before cell fusion. First immunization: Take 50 μg / mouse of antigen emulsified with an equal volume of Freund’s complete adjuvant and inject it subcutaneously on the back of the mouse, and inject 0.2...

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Abstract

The invention discloses an anti-C-peptide monoclonal antibody and a use thereof in detection of C-peptide content. According to the invention, C-peptide-GST and C-peptide-Trx are used as immunogens, two hybridoma cell strains are obtained by cell fusion technique-based screening, the microbial preservation numbers of the two hybridoma cell strains are respectively CGMCC NO. 6804 and CGMCC NO.6805, two monoclonal antibodies secreted by the two hybridoma cell strains can mutually matched, the content of C-peptide in serum is detected by double-antibody sandwich ELISA method, and the detection result has high accuracy and sensitivity.

Description

technical field [0001] The present invention relates to monoclonal antibodies, especially to paired anti-C-peptide monoclonal antibodies and hybridoma cell lines secreting the anti-C-peptide monoclonal antibodies. The present invention further relates to their use in the preparation of reagents or drugs for detecting or diagnosing C-peptide The invention belongs to the detection or diagnosis field of C peptide. Background technique [0002] C-peptide (C-Peptide), also known as connecting peptide, is a product secreted by pancreatic β-cells. It has a common precursor with insulin—proinsulin. After enzymatic cleavage, one molecule of proinsulin is split into one molecule of insulin and one molecule of C-peptide. Determination of C-peptide can know the function of pancreatic islet cells, which is of great significance to the diagnosis and treatment of diabetes. C-peptide has no function of insulin, and insulin secreted by pancreatic β-cells has an equimolecular relationship wi...

Claims

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Application Information

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IPC IPC(8): C12N5/20C07K16/18G01N33/68G01N33/577C12R1/91
Inventor 不公告发明人
Owner BEIJING LEADMAN BIOCHEM
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