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Immunohistochemical detection kit for human CD26 and clinical application thereof

A technology of immunohistochemistry and CD26, which is applied in the direction of biological testing, material inspection products, etc., can solve the problems of anticancer drugs without curative effect, achieve the effects of reducing non-specific adsorption, facilitating mass production, and reducing antibody reactions

Active Publication Date: 2019-05-03
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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  • Immunohistochemical detection kit for human CD26 and clinical application thereof
  • Immunohistochemical detection kit for human CD26 and clinical application thereof
  • Immunohistochemical detection kit for human CD26 and clinical application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1. Preparation of anti-human CD26 hybridoma cell line

[0045] 1. Animal immunization

[0046] 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". The serum titer of immunized mice was tracked by indirect ELISA method, and the immunized mouse with the highest serum titer was selected for fusion experiment of mouse splenocytes and mouse myeloma cells.

[0047] 2. Cell Fusion

[0048] (1). Preparation of spleen cells. Immunized mice were plucked from the eyeballs to take blood, put to death by breaking the cervical spine, soaked in 75% (v / v) alcohol for 10 minutes, took out the spleen in a sterile operating table, and placed it in In the cell sieve, fully grind the cells, pass through the si...

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 Heavy chain and light chain genes were used Optimum Gene 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 opt...

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Abstract

The invention, which belongs to the field of in vitro diagnosis, relates to an immunohistochemical detection kit for human CD26 and clinical application thereof. According to the disclosed immunohistochemical detection kit, a novel anti-human-CD26 antibody is employed; the heavy chain variable region contains following complementarity determining regions: HCDR1, HCDR2, and HCDR3 having the amino acid sequences shown by SEQ ID NO:1, 2, and 3 respectively; and light chain variable region sequences contain following complementarity determining regions: LCDR1, LCDR2, LCDR3 having the amino acid sequences shown by SEQ ID NO:4, 5, 6. According to the invention, the performance requirement of the immunohistochemical kit can be met by related detection performances like the specificity of the anti-human-CD26 antibody; the antibody can be a mouse antibody or a recombinantly expressed chimeric human antibody and is convenient to product in a large scale; with the recombinantly expressed chimerichuman antibody, the human anti-mouse antibody response is reduced and the non-specific adsorption is reduced; and thus the need of large-scale clinical application can be met. In the application of the CD26 target drug, the effect of precise treatment is realized by using the immunohistochemical detection kit.

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): G01N33/68
Inventor 马永徐银妹赵利利
Owner ZONHON BIOPHARMA INST
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