Pancreatic Cancer Detection Methods

A serial number and antibody technology, applied in chemical instruments and methods, biochemical equipment and methods, and microbial determination/examination, etc., can solve the problems of no report on the diagnosis of pancreatic cancer, no report on the expression of CAPRIN-1, etc.

Active Publication Date: 2016-08-17
TORAY IND INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there has been no report that CAPRIN-1 is expressed in pancreatic cancer at all, nor has it been reported that the expression of CAPRIN-1 in the serum of pancreatic cancer patients, the polypeptides that induce antibodies or antigen-antibody reactions in vivo against CAPRIN-1, can induce pancreatic cancer diagnosis possible

Method used

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  • Pancreatic Cancer Detection Methods
  • Pancreatic Cancer Detection Methods
  • Pancreatic Cancer Detection Methods

Examples

Experimental program
Comparison scheme
Effect test

Embodiment

[0092] [Example] Obtaining pancreatic cancer antigen protein by SEREX method

[0093] (1) Preparation of cDNA library

[0094] Total RNA was extracted from testicular tissues of normal dogs by the Acid guanidium-Phenol-Chloroform method (Acid guanidium-Phenol-Chloroform method), and using the Oligotex-dT30 mRNA purification kit (manufactured by Takara Shuzo Co., Ltd.), the polymer was purified according to the instructions attached to the kit. A RNA.

[0095] The thus obtained mRNA (5 µg) was used to synthesize a dog testis cDNA phage library. The cDNA phage library was prepared using cDNA Synthesis Kit, ZAP-cDNA Synthesis Kit, and ZAP-cDNA GigapackIII Gold Cloning Kit (manufactured by STRATAGENE), and the library was prepared according to the instructions attached to the kit. The size of the cDNA phage library produced was 7.73×10 5 pfu / ml.

[0096] (2) Screening of cDNA library by serum

[0097] The dog testis cDNA phage library prepared above was used for immune screen...

Embodiment 2

[0107] [Example 2]: the preparation of dog and human CAPRIN-1 protein

[0108] (1) Preparation of recombinant protein

[0109] Based on the gene of SEQ ID NO: 5 obtained in Example 1, a recombinant protein was prepared by the following method. PCR was carried out as follows: 1 μl of vectors for sequence analysis prepared from the phage solution obtained in Example 1, 0.4 μM and 0.2 mM of each of the two primers (recorded in SEQ ID Nos. 37 and 38) containing NdeI and KpnI restriction enzyme cutting sequences dNTP, 1.25U PrimeSTAR HS Polymerase (manufactured by Takara Shuzo Co., Ltd.), add each reagent and the accompanying buffer solution to make the total amount 50 μl, and use a ThermalCycler (manufactured by BIO RAD Co., Ltd.) to cycle at 98°C / 10 seconds and 68°C / 1.5 minute cycle repeated 30 times. In addition, the above-mentioned two types of primers are primers for amplifying the region encoding the entire length of the amino acid sequence of SEQ ID NO: 6 (dog CAPRIN-1)....

Embodiment 3

[0120] [Example 3]: Preparation of antibodies against CAPRIN-1

[0121] (1) Preparation of polyclonal antibodies against peptides derived from CAPRIN-1

[0122] To obtain an antibody that binds CAPRIN-1, a peptide derived from CAPRIN-1 (Arg-Asn-Leu-Glu-Lys-Lys-Lys-Gly-Lys-Leu-Asp-Asp-Tyr-Gln (SEQ ID NO: 43) ). 1 mg of this peptide was mixed with an equal volume of incomplete Freund's adjuvant (IFA) solution as an antigen, and administered once every two weeks, subcutaneously in rabbits four times. Blood is then collected to obtain antiserum containing polyclonal antibodies. This antiserum was further purified using a protein G carrier (manufactured by GE Healthcare Science) to obtain a polyclonal antibody against a CAPRIN-1-derived peptide. Next, the reactivity of the obtained polyclonal antibody to CAPRIN-1 on the surface of cancer cells was investigated using breast cancer cells. Specifically, centrifuge 10 6 To the human breast cancer cell line MDA-MB-231V, a PBS solut...

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Abstract

The present invention relates to a method for detecting pancreatic cancer by using a new tumor marker. Specifically, the present invention provides a method for detecting pancreatic cancer, which includes determining the antibody that is directed against the CAPRIN‑1 protein through the antigen in a sample isolated from a subject. The antibody response has the presence or amount of bound reactive polypeptides and provides a reagent or kit for detecting pancreatic cancer comprising CAPRIN-1 protein or fragments thereof, antibodies directed thereto, or polynucleotides encoding them.

Description

technical field [0001] The invention relates to a method for detecting pancreatic cancer using CAPRIN-1 as a tumor marker. Background technique [0002] Pancreatic cancer, one of the refractory cancers, is reported to have more than 10,000 patients in my country, and its incidence is increasing year by year, and it is expected to increase in the future. In the case of pancreatic cancer, even if surgery can be performed, since small cancers often invade and metastasize to other organs, pancreatic cancer often recurs, and the 5-year survival rate is 9%. The prognosis is very poor. In addition, gemcitabine, which is one of anticancer agents, is used for the purpose of preventing recurrence after surgery. However, the effect of gemcitabine administration is mainly analgesic, and its tumor shrinking effect and / or life prolonging effect cannot basically be expected. In addition, there are also hospitals using TS-1, an anticancer agent currently used in gastric cancer, but the si...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/574C12Q1/68G01N33/53C07K14/47C07K16/18C12N15/02C12P21/08
CPCC07K14/4738C07K16/303C12Q1/6886G01N33/57438C07K2317/34C07K2317/56G01N2333/705C12Q2600/158C07K16/30C07K14/47C07K16/18
Inventor 井户隆喜冈野文义
Owner TORAY IND INC
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