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Method for detecting cancer, and antibody capable of recognizing pancreas-specific ribonuclease 1

A ribonuclease and detection method technology, applied in the field of ribonuclease 1 antibody, can solve the problems of low organ specificity and uncertain diagnosis

Active Publication Date: 2015-02-18
TOSOH CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there are disadvantages such as low organ-specificity of these markers, cases where the markers do not respond due to genetic reasons, etc., and cannot be used as a definitive definitive diagnosis.
However, until now, there has been no report linking the presence or absence of sugar chains bound to sites capable of N-type sugar chain modification in glycoproteins to cancer.

Method used

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  • Method for detecting cancer, and antibody capable of recognizing pancreas-specific ribonuclease 1
  • Method for detecting cancer, and antibody capable of recognizing pancreas-specific ribonuclease 1
  • Method for detecting cancer, and antibody capable of recognizing pancreas-specific ribonuclease 1

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Experimental program
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Effect test

Embodiment 1

[0075] The preparation of embodiment 1 antibody

[0076] Modulation of the immunogen

[0077] In order to obtain the full-length polypeptide comprising human pancreas-specific RNase 1, insert the gene sequence encoding mature human pancreas-specific RNase 1 (SEQ ID NO. 2) into a plasmid vector expressible in insect cells, thereby preparing the expression plasmid. Specifically, a vector encoding a human immunoglobulin kappa chain (kappa chain) was inserted sequentially from the 5' upstream side at the multiple cloning site of pIZ / V5His vector (Life Technologies Japan Ltd.), a plasmid for recombinant protein expression in insect cells. In the gene sequence, gene sequence encoding His tag, gene sequence encoding FLAG tag, gene sequence encoding human pancreas-specific RNase 1 (SEQ ID NO: 1), the 1st to 28th amino acids corresponding to the signal peptide were removed The gene region of 84 nucleic acid residues (SEQ ID NO: 2). For the prepared expression plasmid pIZ-KFH-hRNase ...

Embodiment 2

[0086] Specificity determination of embodiment 2 antibody

[0087] Modulation of recombinant human pancreas-specific RNase 1 using a mammalian expression system

[0088]In order to obtain the full-length polypeptide comprising human pancreas-specific RNase 1 in mammalian cells, the gene sequence encoding human pancreas-specific RNase 1 was inserted into the pcDNA3.1-mycHis vector (Life Technologies Japan Ltd.) ( SEQ ID NO: 2), preparing the expression vector. More specifically, the gene sequence encoding the recombinant protein of the insect cell expression plasmid (pIZ-KFH-hRNase 1) prepared for immunogen preparation was inserted into pcDNA3.1-mycHis by molecular biology techniques vector (Life Technologies Japan Ltd.), thereby preparing pcDNA-KFH-hRNase 1. For the prepared mammalian cells, the recombinant human pancreas-specific RNase 1 was confirmed to be secreted into the culture medium with the expression plasmid; the recombinant human pancreas-specific RNase 1 was deri...

Embodiment 3

[0107] Embodiment 3 utilizes sandwich method ELISA to carry out the determination of human serum subject

[0108] A biological sample was used as a sample to perform measurement by the following sandwich method ELISA.

[0109] Measurement of the site to which the N-type sugar chain is not bound among the sites capable of N-type sugar chain modification of pancreas-specific RNase 1 in a biological sample derived from a pancreatic cancer patient.

[0110] In order to measure the portion of pancreas-specific RNase 1 capable of undergoing N-type sugar chain modification in a human biological sample that does not bind to an N-type sugar chain, prepare: healthy human serum (40 subjects), Pancreatic cancer patient serum (50 subjects, BioTheme Company), pancreatic disease patient serum (non-cancerous, 12 subjects, BioTheme Company), breast cancer patient serum (20 subjects BioTheme Company), benign breast tumor Patient serum (18 subjects), prostate cancer patient serum (24 subjects, ...

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Abstract

Pancreatic cancer can be detected by producing a monoclonal antibody which can bind to pancreas-specific RNase 1 when no sugar chain is bound to a site located in the pancreas-specific RNase 1 and capable of being modified with an N-type sugar chain and of which the binding to the pancreas-specific RNase 1 is inhibited when an N-type sugar chain is bound to the site, also producing a monoclonal antibody which can bind to the pancreas-specific RNase 1 simultaneously with the binding of the aforementioned antibody to the pancreas-specific RNase 1, and determining the ratio of A to B using the antibodies, wherein A represents the amount of the site located in the pancreas-specific RNase 1 and capable of being modified with an N-type sugar chain wherein an N-type sugar chain is bound or unbound to the site; and B represents the amount of the site located in the pancreas-specific RNase 1 and capable of being modified with an N-type sugar chain.

Description

technical field [0001] The present invention relates to a method for detecting cancer and an antibody recognizing pancreas-specific ribonuclease 1. More specifically, it relates to a method for detecting cancer by measuring whether or not a sugar chain is bound to a site of a glycoprotein capable of N-type sugar chain modification. Background technique [0002] As a detection method for diagnosing early-stage cancer, it is preferable to use non-invasive biologically derived samples such as blood and urine, which are relatively easy to collect, as the specimen. Serum markers currently used in the diagnosis of pancreatic cancer include CA19-9, DUPAN-2, and the like. However, there are disadvantages such as low organ-specificity of these markers, non-reaction of the markers due to genetic reasons, etc., and cannot be used as a definitive definitive diagnosis. [0003] Pancreas-specific ribonuclease 1 (hereinafter, ribonuclease 1 is referred to as "RNase 1"), which is one of t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/09A61K39/395C07K16/40G01N27/62G01N33/53C12P21/08
CPCG01N33/57438C07K16/303C07K16/40A61P1/18A61P35/00C07K2317/30C07K2317/34G01N33/57488G01N2333/922G01N2440/38
Inventor 仲田大辅
Owner TOSOH CORP
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