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Method for detection of cancer

A detection method and detection reagent technology, applied in biochemical equipment and methods, microbial determination/inspection, measuring devices, etc.

Inactive Publication Date: 2010-09-15
TORAY IND INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0013] In order to accurately diagnose cancer, laparotomy is required, which poses a big problem in terms of the physical burden of the dog and the cost burden of the breeder.

Method used

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  • Method for detection of cancer
  • Method for detection of cancer
  • Method for detection of cancer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment A-1

[0143] Example A-1: ​​Preparation of novel cancer antigen protein by SEREX method

[0144] (1) Construction of cDNA library

[0145] Total RNA was extracted from testicular tissue of healthy dogs using the acid-guanidium-phenol-chloroform method (Acid guanidium-Phenol-Chloroform method), and polyA was purified using the Oligotex-dT30mRNA purification Kit (manufactured by Takara Shuzo Co., Ltd.) according to the instructions attached to the kit. RNA.

[0146] The obtained mRNA (5 µg) was used to construct a canine testis cDNA phage library. When constructing the cDNA phage library, use the cDNA Synthesis Kit, ZAP-cDNASynthesis Kit, ZAP-cDNA GigapackIII Gold Cloning Kit (manufactured by STRATAGENE Co., Ltd.) to construct the library according to the operating instructions attached to the kit. The size of the constructed cDNA phage library was 1.3×10 6 pfu / ml.

[0147] (2) Use serum to screen cDNA library

[0148] The above-mentioned cDNA phage library derived from canine te...

Embodiment A-2

[0157] Example A-2: Preparation of novel cancer antigen protein

[0158] (1) Preparation of recombinant protein

[0159] Based on the gene of SEQ ID NO: 1 obtained in Example A-1, a recombinant protein was prepared by the following method. PCR was carried out as follows: each reagent and additional buffer were added to make the total amount 50 μl, wherein the carrier for sequence analysis prepared from the phagemid solution obtained in Example A-1 was 1 μl, and the vector containing NdeI and XhoI restriction The two primers for the enzyme-cleaved sequence (as shown in SEQ ID NOs: 11 and 12) were each 0.4 μM, dNTP was 0.2 mM, and PrimeSTARHS polymerase (manufactured by Takara Shuzo Co., Ltd.) was 1.25 U. Using a Thermal Cycler (manufactured by BIO RAD Co., Ltd.), 98°C-10 seconds, 55°C-15 seconds, and 72°C-1 minute constitute one cycle, and this cycle is repeated 30 times. It should be noted that the region encoding the full-length amino acid sequence of SEQ ID NO: 2 can be ob...

Embodiment A-3

[0169] Example A-3: Cancer diagnosis using canine recombinant protein

[0170] (1) Cancer diagnosis in dogs

[0171] Blood was collected from 486 dogs confirmed to have malignant or benign tumors and 6 healthy dogs, and serum was separated. Using the canine recombinant protein and anti-canine IgG antibody prepared in Example A-2, the IgG antibody titer in the serum specifically reacting with the protein was determined according to the ELISA method.

[0172] The prepared protein was immobilized by adding 100 μL / well of a recombinant protein solution diluted to 50 μg / mL with phosphate-buffered saline to a 96-well Immobilizer Amino Plate (manufactured by NUNC), and standing at 4° C. overnight. Blocking was carried out as follows: 50 mM sodium bicarbonate buffer solution (pH 8.3) containing 0.5% BSA (bovine serum albumin (bovine serum albumin), manufactured by Sigma-Aldrich Japan Co., Ltd.) was added at 100 μL / well (hereinafter referred to as blocking solution) , shake at room t...

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Abstract

Disclosed is a method for detecting cancer by employing the expression of a specific polypeptide as an indicator. The polypeptide is isolated as a polypeptide capable of binding to an antibody that occurs in a serum collected from a cancer-bearing living body by the SEREX method using a cDNA library derived from a canine testis and a serum collected from a cancer-bearing dog. The polypeptide can react with an antibody occurring specifically in a serum from a cancer patient. Therefore, the occurrence of cancer in a living body can be detected by measuring the antibody in a sample. The occurrence of cancer in a living body can be also detected by measuring an antigenic protein for the antibody or mRNA encoding the antigenic protein.

Description

technical field [0001] The present invention relates to a novel method for detecting cancer. Background technique [0002] Cancer is the number one cause of death among all diseases, and the current treatment is mainly symptomatic therapy that uses radiotherapy and chemotherapy in combination with surgical therapy. With advances in medical technology, cancer has become a highly treatable disease if detected early. Therefore, there is a need for a cancer detection method that can be easily tested using serum, urine, or the like without physical or economic burden on cancer patients. [0003] As a method for diagnosing cancer using blood or urine, methods for measuring tumor products such as tumor markers have recently become popular. The so-called tumor products refer to tumor-related antigens, enzymes, specific proteins, metabolites, tumor genes, tumor gene products and tumor suppressor genes, etc., carcinoembryonic antigen CEA, glycoprotein CA19-9, CA125, prostate specifi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/574C12Q1/68G01N33/53C12N15/09
CPCG01N2333/723G01N33/57484G01N2333/4727C12N15/1037C12Q1/6886G01N33/68G01N33/57407G01N33/6854G01N2333/47C12Q2600/158
Inventor 冈野文义铃木佳奈
Owner TORAY IND INC
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