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Anti-human CD26 antibody and application of anti-human CD26 antibody in detection kit

A CD26 and antibody technology, applied in the field of anti-human CD26 antibody and its application in immunohistochemical detection kits, can solve the problem of no curative effect of anticancer drugs, reduce non-specific adsorption, meet large-scale clinical applications, Reduced Effects of Antibody Responses

Active Publication Date: 2019-03-29
ZONHON BIOPHARMA INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Studies have shown that some cancer patients have no CD26 expression in tumor tissue, and such patients have no effect on anticancer drugs targeting CD26

Method used

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  • Anti-human CD26 antibody and application of anti-human CD26 antibody in detection kit
  • Anti-human CD26 antibody and application of anti-human CD26 antibody in detection kit
  • Anti-human CD26 antibody and application of anti-human CD26 antibody in detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1. Preparation of anti-human CD26 hybridoma cell line

[0043] 1. Animal immunization

[0044] BALB / c female mice (purchased from Changzhou Cavens Experimental Animal Co., Ltd.) were immunized with recombinant human CD26 protein (produced by our company) according to the general immunization procedure. For specific immunization conditions, please refer to the "Experimental Guidelines for Antibody Preparation and Use". Indirect ELISA was used to track the serum titer of immunized mice, and the immunized mouse with the highest serum titer was selected for fusion experiment of mouse splenocytes and mouse myeloma cells.

[0045] 2. Cell Fusion

[0046] (1). Preparation of spleen cells

[0047] Take the immunized mice, remove their eyeballs, take blood, put them to death by breaking the cervical spine, soak them in 75% (v / v) alcohol for 10 minutes, take out their spleens in a sterile operating table, place them in a cell mesh, and grind the cells fully , passed t...

Embodiment 2

[0057] Example 2. Determination of the variable region sequence of the anti-human CD26 hybridoma cell line antibody

[0058] 1. Extraction of total RNA from anti-human CD26 hybridoma cells

[0059] Passage the anti-human CD26 hybridoma cell line into a 75T culture flask for culture, digest and centrifuge the cells until the cells reach about 90% confluence, and use the High Pure RNA Isolation Kit (Roche) to perform Total RNA on the anti-human CD26 monoclonal hybridoma cell line extract. Then, using the anti-human CD26 Total RNA as a template, use the RevertAid FirstStrand cDNA Synthesis Kit (Thermo) to reverse-transcribe and amplify the first strand of cDNA. The reaction product is stored at -20°C. For long-term storage, it needs to be stored at -70°C.

[0060] 2. PCR amplification of heavy and light chain variable region genes

[0061] Using the first strand of anti-human CD26 hybridoma cDNA as a template, add 1 μL of cDNA, 5 μL of 10×PCR buffer, 1 μL of upstream and downst...

Embodiment 4

[0065] Example 4. Expression of anti-human CD26 chimeric antibody

[0066] 1. Molecular design and codon optimization of anti-human CD26 chimeric antibody

[0067] The heavy chain variable region sequence of the anti-human CD26 hybridoma cell in Step 3 of Example 3 was directly fused with the human monoclonal antibody IgG1 heavy chain constant region to obtain the heavy chain amino acid sequence of the anti-human CD26 chimeric antibody (SEQ ID No: 9), The light chain sequence of anti-human CD26 hybridoma cells was directly fused with the light chain constant region of human monoclonal antibody IgG1 to obtain the light chain amino acid sequence of anti-human CD26 chimeric antibody (SEQ ID No: 11), and the anti-human CD26 hybridoma cells were respectively OptimumGene TM The heavy chain gene (SEQ ID No: 10) and light chain gene (SEQ ID No: 12) of the anti-human CD26 hybridoma cell of the present invention were obtained after codon optimization software.

[0068] The optimized a...

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Abstract

The invention relates to an anti-human CD26 antibody, and preparation and a commercial application of the anti-human CD26 antibody, and belongs to the field of in vitro diagnosis. A heavy chain variable region of the anti-human CD26 antibody comprises the following complementarity-determining regions: HCDR1 with an amino acid sequence shown as SEQ ID NO: (sequence identifier number) 1, HCDR2 withan amino acid sequence shown as SEQ ID NO: 2, and HCDR3 with an amino acid sequence shown as SEQ ID NO: 3; and a light chain variable region of the anti-human CD26 antibody comprises the following complementarity-determining regions: LCDR1 with an amino acid sequence shown as SEQ ID NO: 4, LCDR2 with an amino acid sequence shown as SEQ ID NO: 5, and LCDR3 with an amino acid sequence shown as SEQ ID NO: 6. Relevant detection performance, such as specificity, of the anti-human CD26 antibody can meet performance requirements of an immunohistochemical kit; and a chimeric humanized antibody constructed by the applicant on a basis of the antibody is more convenient to recombine, express and produce massively, reduces a human anti-mouse antibody response and non-specific adsorption and can meet requirements of large-scale clinical application.

Description

technical field [0001] The invention belongs to the field of in vitro diagnosis, in particular to an anti-human CD26 antibody and its application in an immunohistochemical detection kit. Background technique [0002] CD26 is a ubiquitous multifunctional type II transmembrane protein with multiple biological functions and can also exist in plasma in a dissolved form. CD26 often exists in the form of homodimers, and its monomer contains 766 amino acids with a relative molecular mass of about 110kDa. CD26 amino acid residues are divided into 5 parts from inside to outside: intracellular region (aa1~6), transmembrane region (aa7~28), highly glycosylated region (aa29-323), cysteine-rich region ( aa324~551) and C-terminal catalytic domain (552~766). The C-terminal catalytic domain of CD26 exerts dipeptidyl peptidase IV (Dipeptidylpeptidase4, DPPIV) activity, which can hydrolyze various substrates in the body to play biological roles, and the cysteine-rich region can interact wit...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28C12N15/13C12N15/85C12N5/10C07K16/40G01N33/574G01N33/573
CPCC07K16/2896C07K16/40C07K2317/24C07K2317/33C07K2317/56C12N2800/22G01N33/573G01N33/574
Inventor 马永赵利利王安良
Owner ZONHON BIOPHARMA INST
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