CHI3L1 monoclonal antibody as well as preparation method and application thereof

A CHI3L1, monoclonal antibody technology, applied in the fields of botanical equipment and methods, biochemical equipment and methods, applications, etc., can solve the problems of single antigenic site, affecting the application of diagnostic reagents, and few antibodies, and achieve high affinity and specificity. performance, meet the needs of clinical diagnosis, and meet the effect of mass production

Pending Publication Date: 2022-01-21
NINGBO MEDICAL SYSTEM BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] CHI3L1 is a new diagnostic marker discovered in recent years. There are few antibodies available. Monoclonal antibodies prepared from peptides as immunogens have better specificity. However, due to the single antigenic site and limited selectivity, it is difficult to satisfy different methodologies. testing needs
Protein folding is mainly based on the internal force of the primary amino acid sequence and the effect of protein modification. The structure of the recombinant protein is closer to the natural protein, while the similarity between the polypeptide sequence and the natural protein structure will be less, so it will affect the obtained monoclonal antibody and natural protein. The binding force of protein has an impact on detection reagents that require high sensitivity
In addition, the difficulty in controlling batch-to-batch differences in polyclonal antibody production will also affect its application in the development of diagnostic reagents

Method used

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  • CHI3L1 monoclonal antibody as well as preparation method and application thereof
  • CHI3L1 monoclonal antibody as well as preparation method and application thereof
  • CHI3L1 monoclonal antibody as well as preparation method and application thereof

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Experimental program
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Effect test

Embodiment 1

[0041] 1. Antigen preparation

[0042] 1. Gene synthesis and vector construction

[0043]Synthesize the gene sequence P1 corresponding to 31-149aa in the full-length CHI3L1 sequence, add a 6×His tag to the N-terminus, and add restriction sites BamH1 and HindIII at both ends; The cut pet30a plasmid was ligated and transformed into E. coli competent cells. Synthesize the gene sequence P2 corresponding to 248-383aa in the full-length sequence of CHI3L1, add 6×His tags to the C-terminus, and add restriction sites EcoR1 and Xho1 at both ends; The cut pet30a plasmid was ligated and transformed into E. coli competent cells. Five single clones were picked and sent for sequencing.

[0044] 2. Induced expression and purification

[0045] Correctly sequenced clones were inoculated in 500ml LB medium (Kan+) at 0.5%, cultured for 5 hours, added with a final concentration of 0.5mM IPTG, and induced overnight at 25°C.

[0046] Collect the induced solid bacteria, wash twice with PBS at 7...

Embodiment 2

[0073] 1. Preparation of Kit 1

[0074] 1. Preparation of luminescence reagent:

[0075] Take about 0.1 mg of P2-derived monoclonal antibody, add acridinium ester labeling at a molar ratio of 1:5-1:15, mix and react in the dark at room temperature for 2 hours, dialyze and desalt PB in the dark, and the dialysate volume is 20 times larger than the labeled volume. Change the medium every 4 hours, at least 3 times, measure the concentration and dilute to 1-10 μg / mL with PBS-BSA for later use.

[0076] 2. Solid phase reagent preparation:

[0077] Take about 0.1mg of P1-derived monoclonal antibody, add biotin labeling at a molar ratio of 1:5-1:15, mix and react at room temperature for 2 hours, dialyze and desalt PB, the volume of the dialysate is 20 times larger than the labeled volume, and change the medium every 4 hours , change the medium at least 3 times, measure the concentration and dilute to 0.1-1mg / mL with PBS-BSA for later use.

[0078] Take about 0.5mL magnetic beads, ...

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Abstract

The invention provides a CHI3L1 monoclonal antibody as well as a preparation method and application thereof. The preparation method comprises the following steps: S10, immunizing an animal by using an antigen to obtain immune cells; S20, fusing the immune cells and fusion partner cells, and selecting positive fusion cells; and S30, performing subcloning on the positive fusion cells to obtain cloned fusion cells, wherein the cloned fusion cells can secrete a monoclonal antibody, wherein the antigen is a recombinant antigen P1 or a recombinant antigen P2, the recombinant antigen P1 corresponds to a 31-149aa gene sequence in a CHI3L1 sequence, the recombinant antigen P2 corresponds to a 248-383aa gene sequence in the CHI3L1 sequence, and the monoclonal antibody is a monoclonal antibody from P1 or a monoclonal antibody from P2. The invention has the beneficial effects that the recombinant protein is used as the immunogen, the yield is high, the preparation is convenient, the multi-site monoclonal antibody can be obtained, and multiple pairing modes can meet the requirements of different methodology detection.

Description

technical field [0001] The invention relates to the technical field of chemiluminescence immunodiagnosis, in particular to a CHI3L1 monoclonal antibody and its preparation method and application. Background technique [0002] Chitinase 3-like protein 1 (ehifinase 3-like 1, CHI3L1), often called YKL-40, is a monomeric glycosylated protein with 383 amino acids and a molecular weight of 42.6KD, and is a member of the glycosyl hydrolase family one. It can bind chitin, but has no chitinase activity. [0003] CHI3L1 is highly specifically expressed in the liver and can be induced to express in hepatic Kupffer cells to activate hepatic stellate cells, which may be directly involved in the formation and maintenance of liver fibrosis. CHI3L1 liver fibrosis detection can be used to assist in the diagnosis of liver fibrosis and liver cirrhosis caused by viral hepatitis, alcoholic liver cirrhosis and fatty liver. Guidance on anti-fibrotic therapy has certain significance. The expres...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/40C12N15/70C12N15/66C12N15/56G01N33/577G01N33/573G01N33/543G01N21/76
CPCC07K16/40C12N15/70C12N9/2434G01N33/577G01N33/573G01N33/54326G01N21/76C07K2317/92G01N2333/942G01N2800/085
Inventor 邹继华何进军张华杰贾江花瑞亚·富兰克林·沃克曼
Owner NINGBO MEDICAL SYSTEM BIOTECHNOLOGY CO LTD
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