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Method and reagent for detecting ovarian clear cell adenocarcinoma

Inactive Publication Date: 2017-11-09
PUBLIC UNIV CORP YOKOHAMA CITY UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention introduces a new way to detect ovarian clear cell adenocarcinoma. This method can accurately and high sensitivity and specificity recognize this cancer among other benign and malignant ovarian tumors, while avoiding false positives or negatives.

Problems solved by technology

However, the positive rate of CA125 in ovarian cancer is generally about 80%, and false-negative results are obtained in some cases.
Thus, judgment by CA125 is impossible in about 20% of ovarian cancer.
CA125 is also utilized as an auxiliary marker for endometriosis, which is a benign tumor, but CA125 cannot clearly distinguish between benign ovarian tumors and malignant ovarian tumors, and identification of the tissue types of malignant tumors is difficult therewith.
However, the presence of the processed TFPI2 polypeptide has not been known to date.
Furthermore, detection of ovarian clear cell adenocarcinoma by measurement of the processed polypeptide, and the effect of the processed polypeptide detection, have of course been unknown.

Method used

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  • Method and reagent for detecting ovarian clear cell adenocarcinoma
  • Method and reagent for detecting ovarian clear cell adenocarcinoma
  • Method and reagent for detecting ovarian clear cell adenocarcinoma

Examples

Experimental program
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Effect test

example 1

Construction of Vector for DNA Immunization

[0130]For efficient induction of humoral immunity by DNA immunization, it is preferred to localize the subject antigen protein on the cell surface as a membrane-bound protein. Since TFPI2 is originally a secretory protein, a plasmid vector that can express a protein in which a GPI (glycosylphosphatidylinositol) anchor is attached to the C-terminal side of TFPI2 (hereinafter referred to as GPI-anchor type TFPI2) was constructed for allowing localization of TFPI2 on the cell surface.[0131](1) Using the following primers (a), a polynucleotide composed of the 73rd to 705th bases of TFPI2 cDNA (GenBank No. NM_006528) was amplified by RT-PCR according to a conventional method.[0132](a) Primers for GPI-anchor Type TFPI2 Expression Plasmid Forward:

[0133]5′-cgatgacgacaagettgetcaggagccaaca-3′ (SEQ ID NO:2; wherein the 15 bases in the 3′-end side correspond to the base sequence from position 73 to position 87 in GenBank No. NM_006528)

[0134]Reverse:

[01...

example 2

Immunization and Blood Collection

[0143]Immunization of mice was carried out by administering, to four Balb / c mice, 100 μL of a PBS solution prepared such that the solution contains 40 μg of the GPI-anchor type TFPI2 expression plasmid constructed in Example 1 (2) in terms of the amount of DNA. On Day 7, Day 14, Day 21, Day 28, and Day 35 after the first immunization, additional administration was carried out. On Day 42 after the first immunization, blood was collected to prepare antiserum to provide antisera A-1 to A-4.

example 3

Preparation of GPI-Anchor Type TFPI2 Constantly Expressing Cells

[0144]For evaluation of the antisera, a Chinese-hamster-ovary-derived cell line CHO-K1 that can constantly express the GPI-anchor type TFPI2 was prepared by the following method.[0145](1) The TFPI2 expression plasmid constructed in Example 1[0146](2) was introduced into the CHO-K1 cell line by gene transfer according to a conventional method. Thereafter, the cells were cultured in a 5% CO2 incubator using Hams F12 medium supplemented with 10% FBS (manufactured by Wako Pure Chemical Industries, Ltd.) for 24 hours at 37° C.[0147](2) Thereafter, a solution of the antibiotic Geneticin (manufactured by Invitrogen) was added to the culture at 250 μg / mL, and culture was carried out for additional three weeks.[0148](3) CHO-K1 cells that constantly express the GPI-anchor type TFPI2 were obtained with an anti FLAG antibody using a cell sorter.

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Abstract

The present invention aims to provide a method for detecting, with high sensitivity and specificity, ovarian clear cell adenocarcinoma, which is highly malignant, among benign and malignant ovarian tumors having various tissue types, and a reagent that can be used for the method. The present invention provides NT-TFPI2, which is a novel processed tissue factor pathway inhibitor 2 polypeptide, as a new detection marker for ovarian clear cell adenocarcinoma. The detection of ovarian clear cell adenocarcinoma is carried out by measuring the amount of NT-TFPI2, or the total amount of NT-TFPI2 and intact TFPI2. The reagent for detecting ovarian clear cell adenocarcinoma contains an antibody that specifically recognizes NT-TFPI2 and intact TFPI2.

Description

TECHNICAL FIELD[0001]The present invention relates to a novel processed polypeptide of tissue factor pathway inhibitor 2 (TFPI2) protein (hereinafter referred to as “NT-TFPI2”) to be used for detection of ovarian clear cell adenocarcinoma, which is highly malignant among ovarian tumors, and a method for detecting ovarian clear cell adenocarcinoma based on measurement of NT-TFPI2. More specifically, the present invention relates to a method for detecting ovarian clear cell adenocarcinoma using a measurement method in which the total amount of NT-TFPI2 and intact TFPI2 is calculated, and a reagent for detecting ovarian clear cell adenocarcinoma.BACKGROUND ART[0002]Ovarian cancer is the tumor with the highest mortality rate among gynecological malignancies. In Japan, its annual incidence is about 7,000 to 8,000, and its annual mortality is about 4,000. These numbers are expected to increase year by year. Ovarian surface epithelial malignant tumors account for about 85% of ovarian cance...

Claims

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Application Information

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IPC IPC(8): G01N33/574C07K14/81C07K16/38
CPCG01N33/57449G01N2333/8114C07K16/38C07K14/8114G01N33/6848C07K2317/30C07K14/47C12N15/09G01N33/574
Inventor ARAKAWA, NORIAKIHIRANO, HISASHIMIYAGI, ETSUKOOHTAKE, NORIHISA
Owner PUBLIC UNIV CORP YOKOHAMA CITY UNIV
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