Cancer detection method

A sequence, amino acid technology, used in the field of cancer detection

Active Publication Date: 2011-08-31
TORAY IND INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, there has never been a report that CAPRIN-1 is expressed at a higher level in breast cancer cells etc. than in normal cells

Method used

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Examples

Experimental program
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Embodiment 1

[0107] Embodiment 1: Obtain new cancer antigen protein by SEREX method

[0108] (1) Construction of cDNA library

[0109] Total RNA was extracted from testicular tissues of healthy dogs by the acid guanidinium-phenol-chloroform method, and polyA RNA was purified using the Oligotex-dT30 mRNA Purification Kit (Takara Shuzo Co., Ltd.) according to the instructions contained therein.

[0110] A canine testis cDNA phage library was synthesized using the mRNA (5 µg) thus obtained. Using cDNA Synthesis Kit, ZAP-cDNA Synthesis Kit, and ZAP-cDNA GigapackIII Gold Cloning Kit (STRATAGENE), a cDNA phage library was constructed according to the respective instructions included in each kit. The size of the cDNA phage library thus constructed was 7.73×10 5 pfu / ml.

[0111] (2) Use serum to screen cDNA library

[0112] Immune screening was performed using the canine testis cDNA phage library constructed above. Specifically, host Escherichia coli (XL1-BlueMRF') was infected with phage on an...

Embodiment 2

[0132] Example 2: Preparation of new canine and human cancer antigen proteins

[0133] (1) Preparation of recombinant protein

[0134] A recombinant protein was prepared in the manner described below based on the gene of SEQ ID NO: 5 obtained in Example 1. By adding reagents (1 μl of the vector prepared from the phagemid solution obtained in Example 1 and subjected to sequence analysis, two types of primers containing NdeI and KpnI restriction sites (each 0.4 μM, according to SEQ ID NO: 37 and 38), 0.2mM dNTP and 1.25U PrimeSTAR HS polymerase (Takara Shuzo Co., Ltd.)) and the accompanying buffer were adjusted to a total volume of 50 μl to prepare a reaction solution, and then using a Thermal Cycler (BIO RAD), the The resultant was subjected to PCR by repeating 30 cycles of reactions of 98°C / 10 seconds and 68°C / 1.5 minutes. The above two primers were used to amplify the region (P47) encoding the full-length amino acid sequence of SEQ ID NO:6. After PCR, the amplified DNA was...

Embodiment 3

[0145] Example 3: Preparation of Anti-CAPRIN-1 Antibody

[0146] (1) Preparation of polyclonal antibodies against CAPRIN-1-derived peptides

[0147] To obtain an antibody that binds to CAPRIN-1, a CAPRIN-1-derived peptide (Arg-Asn-Leu-Glu-Lys-Lys-Lys-Gly-Lys-Leu-Asp-Asp-Tyr-Gln (SEQ ID NO: 43 )). 1 mg of the peptide as an antigen was mixed with an incomplete Freund's adjuvant (IFA) solution equal to the amount of the peptide. The mixture was administered subcutaneously to rabbits 4 times every two weeks. Subsequently, blood was collected, whereby antiserum containing polyclonal antibodies was obtained. Furthermore, the antiserum was purified using protein G carrier (GE Healthcare Bio-Sciences), and a polyclonal antibody against CAPRIN-1-derived peptide was obtained. Next, the reactivity of the obtained polyclonal antibody with the surface of breast cancer cells was checked. Specifically, 10 6 MDA-MB-231V human breast cancer cell line was centrifuged in a 1.5ml microcentr...

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PUM

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Abstract

Disclosed is a cancer detection method comprising measuring the expression of a polypeptide in a sample separated from a living body, wherein the polypeptide has a reactivity to bind, through an antigen-antibody reaction, to an antibody directed against CAPRIN-1 protein comprising an amino acid sequence depicted in any even-numbered sequence selected from SEQ ID NO:2 to SEQ ID NO:30 shown in the Sequence Listing. Also disclosed is a cancer detection reagent comprising CAPRIN-1 protein or a fragment thereof, an antibody directed against CAPRIN-1 protein or the fragment thereof, or a polynucleotide encoding CAPRIN-1 protein or the fragment thereof.

Description

technical field [0001] The present invention relates to a method for detecting cancer using CAPRIN-1 as a tumor marker. Background technique [0002] Cancer is the leading cause of death. Currently, the treatment for cancer is mainly symptomatic therapy in which surgery is the mainstay and radiotherapy and chemotherapy are used in combination. With the advancement of medical technology, cancer is almost a curable disease if it can be detected early. Therefore, there is a need for a cancer detection method that can be easily tested using serum, urine, or the like without requiring much 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 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, g...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/574C12N15/09C12Q1/68G01N33/53
CPCC12Q2600/112G01N33/57415C12Q1/6886G01N33/57407G01N33/6893G01N33/53G01N33/574
Inventor 冈野文义铃木佳奈
Owner TORAY IND INC
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