KR20090029868A - Annexin 2 as a liver cancer marker in body fluids and liver cancer diagnosis kit using the same - Google Patents
Annexin 2 as a liver cancer marker in body fluids and liver cancer diagnosis kit using the same Download PDFInfo
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- KR20090029868A KR20090029868A KR1020070095017A KR20070095017A KR20090029868A KR 20090029868 A KR20090029868 A KR 20090029868A KR 1020070095017 A KR1020070095017 A KR 1020070095017A KR 20070095017 A KR20070095017 A KR 20070095017A KR 20090029868 A KR20090029868 A KR 20090029868A
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- South Korea
- Prior art keywords
- liver cancer
- annexin
- protein
- antibody
- leu
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Abstract
본 발명은 간암 환자의 체액에서 아넥신 2의 과발현 현상을 이용하여 간단한 분석을 통해 간암 진단의 정확성을 높인 간암 마커 및 이를 이용한 간암 진단 키트에 관한 발명이다.The present invention relates to a liver cancer marker that improves the accuracy of liver cancer diagnosis through a simple analysis using an overexpression phenomenon of Annexin 2 in the body fluid of a liver cancer patient, and a liver cancer diagnostic kit using the same.
Description
본 발명은 간암 환자의 혈청에서 아넥신 2의 과발현 현상을 이용하여 혈액 분석을 통해 간암 진단의 정확성을 높인 간암 마커 및 간암 진단 키트에 관한 발명이다.The present invention relates to a liver cancer marker and a liver cancer diagnostic kit that improve the accuracy of liver cancer diagnosis through blood analysis using an overexpression of Annexin 2 in serum of liver cancer patients.
간염, 간경변, 간암 등을 포함한 간질환은 한국, 일본, 대만, 중국, 대부분의 동남아 국가에서 단일 질병으로는 가장 많은 환자가 발생하고 있다. 간암으로 인한 사망률은 장기별 기준으로 보면 세계적으로 네 번째 (50만 5천명)이며(세계보건기구 1997년도 자료), 국내에 있어서는 세 번째(11.5%)를 차지하고 있다(한국 암 발생 통계 2002 자료).Liver diseases, including hepatitis, cirrhosis and liver cancer, are the most common single disease in Korea, Japan, Taiwan, China, and most Southeast Asian countries. The mortality rate from liver cancer is the fourth largest in the world (505,000) by long-term basis (World Health Organization 1997 data) and the third in Korea (11.5%) (Korea Cancer Incident Statistics 2002 data). .
간암은 조직 검사를 실시하거나 혈중 알파 태아 단백(alpha-fetoprotein, 이하 "AFP")과 같은 간암표지 단백질 검사를 실시하여 간암 여부를 진단하여 왔다.Liver cancer has been diagnosed as liver cancer by performing a biopsy or by testing a liver cancer marker protein such as blood alpha-fetoprotein (AFP).
현재 간암 관련 진단, 예후 판정, 치료평가 바이오 마커로 가장 잘 알려진 것은 AFP, PIVKA(Protein Induced by Vitamin K Absence)-II 등이 있으나 특이도, 민감도가 부족한 면이 있다. 간세포암 진단에 있어서 AFP의 유용성은 잘 알려져 있다. 진행된 간세포암의 진단뿐만 아니라 간경변증의 자연경과 중 매년 3-10%의 환자에서 간세포암이 발병하기 때문에 간암 조기발견을 위한 정기적인 AFP 측정이 필요하다. 그러나 AFP는 간세포암뿐만 아니라 알콜성 간염, 만성간염이나 간경변증과 같은 양성질환에서도 고농도로 상승하기 때문에 위양성이 많고 실제 양성율이 50-60%에 지나지 않는다(민감도는 20ng/㎖ 및 400ng/㎖에서 각각 29.9% 및 65.8%). PIVKA-II는 DCP(des-r-carboxyprothrombin) 즉 응고작용이 없는 비정상 프로트롬빈으로서 혈청 AFP와는 독립적으로 간세포암의 진단에서 각각 48.2% 및 95.9%의 민감도와 특이도가 보고되고 있다. 이러한 생물학적 지표들은 현재 임상적으로 이용되고 있으나 모든 간암의 생물학적 특성을 반영하지 못하고 제한적으로 이용되고 있다. 따라서, 현재의 AFP나 PIVKAII보다 간암을 특이적이고 효과적으로 진단할 수 있는 marker 마커를 발굴하고 이를 이용하여 간암을 조기 진단할 수 있는 검사 시약의 개발이 필요하다.At present, AFP and PIVKA (Protein Induced by Vitamin K Absence) -II are the most well-known biomarkers for liver cancer diagnosis, prognosis and treatment evaluation. However, they lack specificity and sensitivity. The usefulness of AFP in the diagnosis of hepatocellular carcinoma is well known. In addition to the diagnosis of advanced hepatocellular carcinoma, hepatocellular carcinoma develops in 3-10% of patients every year during the natural course of cirrhosis, so regular AFP measurements are necessary for early detection of liver cancer. However, AFP is highly false positive because of its elevated concentration in benign diseases such as alcoholic hepatitis, chronic hepatitis, or cirrhosis, as well as hepatocellular carcinoma, and the actual positive rate is only 50-60% (sensitivity is 20ng / ml and 400ng / ml, respectively). 29.9% and 65.8%). PIVKA-II is a DCP (des-r-carboxyprothrombin), a non-coagulant abnormal prothrombin, and independent of serum AFP, 48.2% and 95.9%, respectively, have been reported in the diagnosis of hepatocellular carcinoma. Although these biological indicators are currently used clinically, they do not reflect the biological characteristics of all liver cancers and are used in a limited manner. Therefore, it is necessary to find a marker that can diagnose liver cancer more specifically and effectively than current AFP or PIVKAII, and to develop a test reagent capable of early diagnosis of liver cancer.
현재 혈액, 조직, 배설물로부터 종양 마커가 개발되어 있으나 많은 종류의 종양 마커는 암이 없어도 증가하거나 검출되는 경우가 있다. 종양 마커는 암을 사전에 예방하거나 혹은 조기발견, 치료 중인 경우는 그 예후를 관찰하기 위한 목적으로 활용되고 있다. 최근에 제노믹스, 프로테오믹스 연구에 의해 몇 가지 종류의 간암 마커 후보 단백질과 유전자가 보고되고 있다. 현재 대부분의 종양 마커는 조직의 유전자 발현 정도를 중심으로 발굴되고 있으나, 바이오마커의 성질상 조직에서 유전자 발현이 높다고 해서 뇨 또는 혈청으로 진단 가능한 것은 아니다. 그러므 로 진단의 편의성을 위하여 혈액이나 뇨에서 측정 가능한 종양 마커 발굴이 요구되고 있다.Currently, tumor markers have been developed from blood, tissue, and excreta, but many types of tumor markers are sometimes increased or detected even without cancer. Tumor markers are used to prevent cancer in advance, or to monitor the prognosis in early detection and treatment. Recently, several types of liver cancer marker candidate proteins and genes have been reported by genomes and proteomics studies. Currently, most tumor markers have been discovered based on the degree of gene expression in tissues, but high gene expression in tissues does not mean that the tumor marker can be diagnosed with urine or serum. Therefore, it is required to discover tumor markers that can be measured in blood or urine for the convenience of diagnosis.
아넥신 2(본 발명의 상세한 설명 및 특허청구범위, 도면에서 "ANXA2"와 혼용함)는 아넥신(Annexin) II, 아넥신 A2라고도 불리며, 아넥신 패밀리의 한 멤버이다. 아넥신 2는 일종의 칼슘-의존적 인지질-결합 단백질(calcium-dependent phospholipid-binding protein)로서, 세포 성장과 신호전달을 조절하는 기능을 지니며, 용골세포(osteoclast) 형성과 골질(bone) 재흡수를 높이는 자가분비 인자(autocrine factor)이다. 관련 질환으로는 진주종(cholesteatoma), 대장암(Guzman-AA et.al., J Cell Biochem.2005.94(1):178-93), 신장암(Domoto T et.al., Cancer Sci.2007;98(1)77-82), 췌장암(Roda O, Proteomics.2006 Suppl 1:S36-41), 백혈병(Olwill SA et al., Thromb Res.2005:115(1-2):109-14)), 위암(Emoto K wet.al., Anticancer Res.2001:21(2B0: 1339-45), 유방암(Chuthapisith S et. al., Int J Oncol.2007; 30(6);1545-51), 전립선암(Yee DS et.al., Arch Pathol Lab Med. 2007:131(6) 902-8) 등이 있다. 김남순 등에 의해 간암조직에서 정상조직과 비교하여 발현율 차이를 보이는 14종의 유전자를 발굴, 이들 유전자의 발현 차이를 이용하여 간암 진단에 활용할 수 있다는 논문과 특허가 발표된바 있다(대한민국 특허공개 10-2005-0076876: International J. of oncology, 29 :315-327, 2006). 상기 특허와 논문에서는 14종 유전자에 대하여 경쟁적 RT-PCR을 실시하여 간암 진단의 유용성을 밝혔으며, 항체를 이용한다면 단백질 측정도 가능할 것 이라는 가능성을 제시하였다. 이 14종 유전자에는 ANXA2 유전자도 포함되어 있으나 이는 조직을 대상으로 한 유전자 진단법이다. 그러나 혈액 등 체액분석을 통한 간암 진단에 대한 보고는 현재까지 발표된 바 없다.Annexin 2 (combined with "ANXA2" in the description and claims of the invention, in the figures) is also called Annexin II, Annexin A2, and is a member of the Annexin family. Annexin 2 is a type of calcium-dependent phospholipid-binding protein that regulates cell growth and signal transduction, and is responsible for osteoclast formation and bone resorption. Height is an autocrine factor. Related diseases include cholesteatoma, colorectal cancer (Guzman-AA et.al., J Cell Biochem. 2005.94 (1): 178-93), kidney cancer (Domoto T et.al., Cancer Sci. 2007; 98 (1) 77-82), pancreatic cancer (Roda O, Proteomics. 2006 Suppl 1: S36-41), leukemia (Olwill SA et al., Thromb Res. 2005: 115 (1-2): 109-14)) , Gastric cancer (Emoto K wet.al., Anticancer Res. 2001: 21 (2B0: 1339-45), breast cancer (Chuthapisith S et. Al., Int J Oncol. 2007; 30 (6); 1545-51), prostate Cancer (Yee DS et.al., Arch Pathol Lab Med. 2007: 131 (6) 902-8), etc. Discovered 14 genes with different expression rates in liver cancer tissues compared to normal tissues there have been articles and patents that can be utilized in liver cancer diagnosis published by the use of the differentially expressed genes (Republic of Korea Patent Publication 10-2005-0076876: International J. of oncology , 29: 315-327, 2006). The patents and papers revealed the usefulness of diagnosing liver cancer by performing competitive RT-PCR on 14 genes and suggested the possibility of protein measurement using antibodies. The 14 genes also include the ANXA2 gene, but this is a gene diagnostic method for tissues. However, no report on the diagnosis of liver cancer through analysis of body fluids such as blood has been published.
따라서, 본 발명은 체액을 통하여 간단한 방법으로 간암의 발병, 예후 진단이 가능한 진단 키트를 제공하려는 것을 목적으로 한다.Accordingly, an object of the present invention is to provide a diagnostic kit capable of diagnosing the onset and prognosis of liver cancer in a simple manner through body fluids.
본 발명에서는 아넥신 2의 생물학적 기능을 밝히고 임상적 의의를 알아보기 위하여 간암(hepatocellular carcinoma, HCC) 조직과 간암 세포주에서 RNA, 단백질 발현 정도를 근거로 혈액에서의 분비차이를 밝히고 간암의 진단, 예후 마커로서의 가능성을 평가하였다.In the present invention, in order to clarify the biological function and clinical significance of Annexin 2, the secretion of blood secretion is revealed on the basis of RNA and protein expression levels in hepatocellular carcinoma (HCC) tissues and liver cancer cell lines, and the diagnosis and prognosis of liver cancer. The possibility as a marker was evaluated.
이를 위하여 간암조직, 정상인의 간 조직, 간암환자의 정상 간조직을 대상으로 cDNA 마이크로어레이, RT-PCR, 웨스턴 블랏을 실시하였으며 실험 결과 아넥신 2는 비암(non-tumor) 조직 또는 정상 간조직보다 간암 조직에서 높은 RNA 및 단백질 발현을 보였다. 또한 간암 세포주를 대상으로 웨스턴 블랏을 실시한 결과 Huh-7, SK-Hep1에서 아넥신 2 발현을 보였으며, 이뮤노닷(Immunodot), ELISA 와 같은 방법을 이용하여 임상혈청 시료 내 아넥신 2의 분포도를 조사해 본 결과 간암에서 높은 농도의 아넥신 2가 분비됨을 볼 수 있었다. 이를 바탕으로 아넥신 2를 이용한 간암진단용 ELISA 키트를 구성하였다.To this end, cDNA microarray, RT-PCR, and Western blot were performed on liver cancer tissues, liver tissues of normal humans, and liver tissues of liver cancer patients. Hepatic cancer tissues showed high RNA and protein expression. In addition, Western blot analysis of liver cancer cell lines showed annexin 2 expression in Huh-7 and SK-Hep1, and the distribution of annexin 2 in clinical serum samples was measured using immunodot and ELISA. The results showed that high concentrations of Annexin 2 were secreted from liver cancer. Based on this, an ELISA kit for liver cancer diagnosis using Annexin 2 was constructed.
본 발명에 의하면, ELISA 법 등의 정량분석법을 이용하여 혈액에서 아넥신 2를 분석함으로써 간암의 조기 진단, 예후, 치료 모니터링에 효과적으로 사용할 수 있다.According to the present invention, by analyzing annexin 2 in the blood using a quantitative analysis method such as ELISA method, it can be effectively used for early diagnosis, prognosis and treatment monitoring of liver cancer.
또한, 본 발명은 간암 치료 연구 및 암화 특성을 연구하는데도 사용될 수 있다.The present invention can also be used to study liver cancer treatment and to study the cancerous properties.
본 발명은 ANXA2 유전자의 mRNA 또는 이의 단백질 수준을 측정하는 제제를 포함하는 간암 진단 키트에 관한 것이다.The present invention relates to a liver cancer diagnostic kit comprising an agent for measuring mRNA or protein level of ANXA2 gene.
또한, 본 발명은 ANXA2 유전자의 mRNA 또는 이의 단백질 수준을 측정하는 제제를 생물학적 시료와 접촉시키는 단계를 포함하는 간암 마커 검출 방법에 관한 것이다.The present invention also relates to a method for detecting liver cancer markers comprising contacting a biological sample with an agent that measures the mRNA or protein level of the ANXA2 gene.
본 발명에서 "마커"는 간암 세포를 정상 세포와 구분하여 진단할 수 있는 물질로, ANXA2 유전자에 대한 핵산 마커 및 ANXA2 단백질에 대한 폴리펩타이드 마커일 수 있으며, 이들 마커들은 정상 세포 및 비간암 세포에 비해 간암 세포에서 발현이 증가하는 특징을 나타낸다.In the present invention, "marker" is a substance capable of diagnosing liver cancer cells from normal cells, and may be a nucleic acid marker for ANXA2 gene and a polypeptide marker for ANXA2 protein, and these markers may be used for normal and non-hepatic cancer cells. In comparison, hepatic cancer cells exhibit increased expression.
본 발명에서 "진단"은 간암 상태의 존재 또는 특징을 확인하는 것을 의미한다."Diagnosis" in the present invention means identifying the presence or characteristics of liver cancer conditions.
본 발명은 인간 아넥신 2의 간암에서의 생물학적 특징과 임상의학적 의의를 제공한다. 또한 이를 이용하여 간암세포와 조직, 혈청에서 아넥신 2의 RNA, 단백질의 발현을 비교하는 것을 그 특징으로 한다. 아넥신 2의 발현은 간암 발병 원인 규 명, 진단 및 치료에 이용할 수 있을 것으로 기대한다.The present invention provides the biological characteristics and clinical significance of human Annexin 2 in liver cancer. In addition, it is characterized by comparing the expression of RNA, protein of Annexin 2 in liver cancer cells and tissues, serum. The expression of Annexin 2 is expected to be used for the identification, diagnosis and treatment of liver cancer.
본 발명을 더욱 상세히 설명하면 다음과 같다.The present invention is described in more detail as follows.
본 발명에서는 아넥신 2의 생물학적 기능을 밝히고 임상적 의의를 알아보기 위하여 간암조직에서 RNA, 단백질 발현 정도를 밝혀 간암의 진단, 예후 마커로서의 가능성을 평가해 보았다. 이를 위하여 전북대학교 병원의 간암환자로부터 원발성 간암 조직을 얻어 양성시료로 사용하였다. 조직 시료로부터 RNA를 분리하여 cDNA 마이크로어레이, RT-PCR을 실시하여 RNA 발현분포를 보았다. 단백질 발현을 보기 위하여 웨스턴 블랏을 실시하였으며 혈청에서의 분포를 알아보기 위하여 단국대학교와 전북대학교에서 간암으로 판정된 환자로부터 혈액을 채취, ELISA(Enzyme linked immunosorbent assay)법을 이용하여 혈청 중 아넥신 2의 양을 분석하였다. cDNA 마이크로어레이, RT-PCR, 웨스턴 블랏 실험결과 아넥신 2는 간암조직에서 비암 조직(non-tumor tissue) 또는 정상 간과 비교했을 때보다 높은 RNA 및 단백질 발현을 나타내었다. 이뮤노닷, ELISA 실험 결과 아넥신 2는 간암환자의 혈청에서 정상인과 비교했을 때보다 높은 단백질 분비를 보였다.In the present invention, in order to clarify the biological function and clinical significance of Annexin 2, the expression level of RNA and protein in liver cancer tissues was examined to evaluate the possibility as a diagnostic and prognostic marker of liver cancer. For this purpose, primary liver cancer tissue was obtained from liver cancer patients at Chonbuk National University Hospital and used as a positive sample. RNA was isolated from tissue samples, and cDNA microarray and RT-PCR were performed to examine RNA expression distribution. Western blot was performed to examine protein expression. Blood samples were collected from patients diagnosed as liver cancer at Dankook University and Chonbuk National University to determine the distribution in serum. Annexin 2 in serum using ELISA (Enzyme linked immunosorbent assay) The amount of was analyzed. In the cDNA microarray, RT-PCR, Western blot experiments, Annexin 2 showed higher RNA and protein expression in liver cancer tissues than in non-tumor tissues or normal livers. Immunod and ELISA showed that annexin 2 produced higher protein secretion in liver cancer patients compared to normal patients.
생물학적 시료 중의 ANXA2 간암 마커 유전자의 발현 수준은 mRNA 또는 이의 단백질 양을 확인함으로써 알 수 있으며, 생물학적 시료에서 mRNA와 단백질을 분리하는 과정은 공지의 공정을 이용하여 수행할 수 있으며, mRNA와 단백질의 양은 다양한 방법으로 측정할 수 있다.The expression level of the ANXA2 liver cancer marker gene in the biological sample can be determined by checking the mRNA or its protein amount. The process of separating the mRNA and the protein from the biological sample can be performed using a known process. It can be measured in various ways.
본 발명에서 "생물학적 시료"란 간단히 채취하여 ANXA2 간암 마커 유전자의 mRNA 또는 이의 단백질 수준의 차이를 검출할 수 있는 체액 즉, 뇨, 타액, 혈액, 혈장, 혈청 등을 포함한다.As used herein, the term "biological sample" includes body fluids, ie, urine, saliva, blood, plasma, serum, and the like, which can be simply collected to detect differences in mRNA or protein levels of the ANXA2 liver cancer marker gene.
본 발명에서 "mRNA 수준 측정"이란 ANXA2 간암 마커 유전자를 검출하기 위하여 생물학적 시료에서 ANXA2 마커 유전자들의 mRNA 존재 여부와 발현 정도를 확인하는 과정으로 mRNA의 양을 측정한다. 이를 위한 분석 방법으로는 RT-PCR, 경쟁적 RT-PCR, 실시간 RT-PCR, RNase 보호 분석법(RNase protection assay), 노던블랏팅(Nothern blotting), DNA 칩 등이 있으나 이로 제한되는 것은 아니다.In the present invention, "mRNA level measurement" refers to the process of confirming the presence and expression level of mRNA of ANXA2 marker genes in a biological sample in order to detect ANXA2 liver cancer marker gene. Analytical methods for this purpose include, but are not limited to, RT-PCR, competitive RT-PCR, real-time RT-PCR, RNase protection assay, Northern blotting, DNA chip, and the like.
상기 검출 방법들을 통하여 정상 대조군에서의 mRNA 발현량과 간암 의심환자에서의 mRNA 발현량을 비교할 수 있고, ANXA2 마커 유전자에서 mRNA로의 유의한 발현량의 증가여부를 판단하여 간암 의심 환자의 실제 간암 발명 여부를 진단할 수 있다.Through the above detection methods, it is possible to compare the mRNA expression level in the normal control group and the mRNA expression level in the suspected liver cancer patient, and to determine whether the significant expression level of the ANXA2 marker gene is increased in the mRNA to determine whether the actual liver cancer of the suspected liver cancer patient was invented. Diagnosis can be made.
mRNA 수준을 RT-PCR로 측정하기 위한 키트는 ANXA2 위암 마커 유전자에 대한 특이적인 각각의 프라이머 쌍을 포함한다. 프라이머는 각 마커 유전자의 핵산서열에 특이적인 서열을 가지는 뉴클레오타이드로서, 약 7 내지 50bp의 길이, 좀더 바람직하게는 약 10 내지 30bp의 길이이다. 또한, 대조군 유전자의 핵산서열에 특이적인 프라이머를 포함할 수 있다. 그 외 RT-PCR 키트는 테스트 튜브 또는 다른 적절한 컨테이너, 반응 완충액, 데옥시뉴클레오타이드(dNTPs), Taq-폴리머레이즈 및 역전사효소와 같은 효소, DNase, RNase 억제제 DEPC수(DEPC water), 멸균수 등을 포함할 수 있다. "프라이머"는 짧은 자유 3' 말단 수산화기(free 3' hydroxyl group)를 가지는 핵산 서열로 상보적인 템플레이트와 염기쌍을 형성할 수 있고 템플레이트 가닥 복사를 위한 시작 지점으로 기능하는 짧은 핵산 서열을 의미한다. 프라이머는 적절한 완충용액 및 온도에서 중합반응을 위한 시약 및 상이한 네 가지 뉴클레오사이드 3인산의 존재 하에서 DNA 합성을 개시할 수 있다. 프라이머는 DNA 합성의 개시점으로 작용하는 프라이머의 기본 성질을 변화시키지 않는 추가의 특징을 혼입할 수 있다. 프라이머는 포스포르아미다이트 고체 지지체 방법 또는 기타 널리 공지된 방법을 사용하여 화학적으로 합성할 수 있다. 이러한 핵산 서열은 또한 당해 분야에 공지된 많은 수단을 이용하여 변형시킬 수 있다.Kits for measuring mRNA levels by RT-PCR include each primer pair specific for the ANXA2 gastric cancer marker gene. The primer is a nucleotide having a sequence specific to the nucleic acid sequence of each marker gene, and is about 7 to 50 bp in length, more preferably about 10 to 30 bp in length. In addition, a primer specific for the nucleic acid sequence of the control gene may be included. Other RT-PCR kits include test tubes or other suitable containers, reaction buffers, deoxynucleotides (dNTPs), enzymes such as Taq-polymerase and reverse transcriptase, DNase, RNase inhibitor DEPC water, sterile water, and the like. It may include. "Primer" refers to a short nucleic acid sequence that is capable of forming base pairs with complementary templates with nucleic acid sequences having short free 3 'hydroxyl groups and that serves as a starting point for template strand copying. Primers can initiate DNA synthesis in the presence of four different nucleoside triphosphates and reagents for polymerization in the appropriate buffer and temperature. Primers can incorporate additional features that do not change the basic properties of the primers that serve as a starting point for DNA synthesis. Primers can be synthesized chemically using phosphoramidite solid support methods or other well known methods. Such nucleic acid sequences can also be modified using many means known in the art.
본 발명에서 "단백질 수준 측정"이란 생물학적 시료에서 ANXA2 간암 마커 유전자에서 발현된 단백질의 존재 여부와 발현 정도를 확인하는 과정으로서 바람직하게는 상기 유전자의 단백질에 대하여 특이적으로 결합하는 항체를 이용하여 단백질의 양을 확인한다.In the present invention, "protein level measurement" is a process of confirming the presence and expression of the protein expressed in the ANXA2 liver cancer marker gene in a biological sample, preferably using an antibody that specifically binds to the protein of the gene. Check the amount of.
"항체"란 항원성 부위에 대해서 지시되는 특이적인 단백질 분자를 의미한다. 본 발명의 목적상 항체는 ANXA2 간암 마커 단백질에 대해 특이적으로 결합하는 항체를 의미하며, 폴리클론 항체, 모노클론 항체 및 재조합 항체를 모두 포함한다."Antibody" means a specific protein molecule directed against an antigenic site. For the purposes of the present invention, an antibody means an antibody that specifically binds to ANXA2 liver cancer marker protein, and includes all polyclonal antibodies, monoclonal antibodies, and recombinant antibodies.
항체를 이용하여 단백질 수준을 측정하는 분석방법으로는 웨스턴 블랏, ELISA(Enzyme linked immunosorbent assay), RIA(Radioimmunoassay), 방사면역 확산법(Radioimmunodiffusion), 오크털로니(Ouchterlony) 면역확산법, 로켓 면역전기영동, 조직면역염색, 면역침전분석(Immunoprecipitation), 보체고정분석(Complement fixation assay), FACS, 단백질 칩 등이 있으며, 기재된 방법으로 제한되는 것은 아니다.Assays for measuring protein levels using antibodies include Western blot, Enzyme linked immunosorbent assay (ELISA), Radioimmunoassay (RIA), Radioimmunodiffusion, Ouchterlony immunodiffusion, Rocket immunoelectrophoresis, Tissue immunostaining, immunoprecipitation, complement fixation assay, FACS, protein chips, and the like, are not limited to the described methods.
위와 같은 분석방법을 통하여 정상 대조군의 항원-항체 복합체의 형성량과 간암 의심환자의 항원-항체 복합체의 형성량을 비교할 수 있고, ANXA2 단백질의 유의한 발현량 증가 여부를 판단하여 간암 의심환자의 실제 간암 여부를 진단할 수 있다.Through the above analysis method, the amount of antigen-antibody complex formation in the normal control group and the amount of antigen-antibody complex formation in suspected liver cancer patients can be compared, and whether the significant expression level of ANXA2 protein is increased is determined by the actual liver cancer suspected patient. Can diagnose liver cancer.
"항원-항체 복합체"란 ANXA2 단백질과 이에 특이적인 항체의 결합물을 의미하고, 항원-항체 복합체의 형성량은 검출 라벨(detection label)의 신호 크기를 통하여 정량적으로 측정할 수 있다. 검출 라벨은 효소, 형광물질, 리간드, 발광물질, 미소입자(microparticle), 레독스 분자 및 방사선 동위원소로 이루어진 그룹 중에서 선택할 수 있으며, 상기 기재된 물질로 제한되는 것은 아니다. 검출 라벨로 효소를 사용하는 경우 이용할 수 있는 효소로는 β-글루쿠로니다제, β-D-글루코시다제, β-D-갈락토시다제, 우레아제, 퍼옥시다아제 또는 알칼라인 포스파타아제, 아세틸콜린에스터라아제, 글루코스 옥시다아제, 헥소키나아제 등이 있으며, 상기 기재된 범위로 제한되지 않는다. 형광물질로는 플루오레신, 피코시아닌, 플루오레스카민 등이 있고, 상기 기재된 물질로 제한되는 것은 아니다. 리간드로는 바이오틴 유도체 등이 있고, 이에 제한되지는 않는다. 발광물질로는 루시페린 등이 있고, 이에 제한되지는 않는다. 미소입자로는 콜로이드 금 등이 있고, 이에 제한되는 것은 아니다. 레독스 분자로는 퀴논, 1,4-벤조퀴논, 하이드로퀴논 등이 있고, 이에 제한되는 것은 아니다. 방사선 동위원소에는 3H,14C 등이 포함되며, 이에 제한되는 것은 아니다.By "antigen-antibody complex" is meant a combination of ANXA2 protein and an antibody specific to it, and the amount of antigen-antibody complex formation can be quantitatively determined through the signal size of the detection label. The detection label can be selected from the group consisting of enzymes, fluorescent materials, ligands, luminescent materials, microparticles, redox molecules and radioisotopes, but is not limited to the materials described above. When enzymes are used as detection labels, the available enzymes include β-glucuronidase, β-D-glucosidase, β-D-galactosidase, urease, peroxidase or alkaline phosphatase, acetyl Cholinesterase, glucose oxidase, hexokinase and the like, and are not limited to the ranges described above. Fluorescent materials include fluorescein, phycocyanin, fluorescarmine and the like, and are not limited to the above-described materials. Ligands include, but are not limited to, biotin derivatives. Luminescent materials include luciferin and the like, but are not limited thereto. The microparticles include colloidal gold and the like, but are not limited thereto. Redox molecules include, but are not limited to, quinone, 1,4-benzoquinone, hydroquinone, and the like. Radioisotopes include, but are not limited to, 3 H, 14 C, and the like.
단백질 발현수준 측정은 바람직하게는 ELISA를 이용한다. ELISA로는 고체 지 지체에 부착된 항체와 항원의 복합체에서 항원을 인지하는 표지된 또다른 항체를 이용하는 직접적 샌드위치 ELISA, 고체 지지체에 부착된 항체와 항원의 복합체에서 항원을 인지하는 또 다른 항체와 반응시킨 후 이 항체를 인지하는 표지된 2차 항체를 이용하는 간접적 샌드위치 ELISA 등 다양한 ELISA 방법을 포함한다. 좀더 바람직하게는 고체 지지체에 항체를 부착시키고 시료를 반응시킨 후 항원-항체 복합체의 항원을 인지하는 표지된 항체를 부착시켜 효소적으로 발색시키거나, 항원-항체 복합체의 항원을 인지하는 항체에 대해 표지된 2차 항체를 부착시켜 효소적으로 발색시키는 샌드위치 ELISA 방법에 의해서 검출한다. ANXA2 간암 마커 단백질과 항체의 복합체 형성 정도를 확인하여 간암 발병 여부를 확인할 수 있는 것이다.Protein expression level measurement is preferably by using an ELISA. ELISA includes a direct sandwich ELISA using another labeled antibody that recognizes the antigen in the complex of the antibody with the antibody attached to the solid retard, and reacting with another antibody that recognizes the antigen in the complex of the antibody with the antibody attached to the solid support. And various ELISA methods including indirect sandwich ELISA using a labeled secondary antibody which then recognizes this antibody. More preferably, the antibody is attached to a solid support and reacted with a sample, followed by enzymatic color development by attaching a labeled antibody that recognizes the antigen of the antigen-antibody complex, or an antibody that recognizes the antigen of the antigen-antibody complex. It is detected by the sandwich ELISA method which attaches a labeled secondary antibody and enzymatically develops. ANXA2 liver cancer marker protein and antibody can be confirmed by determining the degree of complex formation of liver cancer.
또 다른 방법으로 ANXA2 간암 마커에 대한 하나 이상의 항체가 기판 위 정해진 위치에 배열되어 고밀도로 고정화되어 있는 단백질 칩을 이용할 수 있다. 단백질 칩을 이용하여 시료를 분석하는 방법은 시료에서 단백질을 분리하고, 분리한 단백질을 단백질 칩과 혼성화시켜 항원-항체 복합체를 형성시키고 이를 판독하여 단백질의 존재 또는 발현 정도를 확인하여 간암 발병 여부를 확인할 수 있다.Alternatively, one or more antibodies against ANXA2 liver cancer markers may be arranged in a predetermined position on a substrate, and used in a high density immobilized protein chip. In the method of analyzing a sample using a protein chip, the protein is separated from the sample, and the separated protein is hybridized with the protein chip to form an antigen-antibody complex. You can check it.
또 다른 방법으로 ANXA2에 대한 항체를 이용하여 웨스턴 블랏팅을 하는 것이다. 시료에서 전체 단백질을 분리하고, 이것을 전기영동하여 단백질을 크기에 따라 분리한 다음 나이트로셀룰로스 막으로 이동시켜 항체와 반응시킨다. 생성된 항원-항체 복합체의 양을 표지된 항체를 이용하여 확인함으로써 간암 발병 여부를 확인할 수 있다.Another method is western blotting using an antibody against ANXA2. The whole protein is isolated from the sample, which is electrophoresed to separate the protein according to size and then transferred to the nitrocellulose membrane to react with the antibody. The amount of the generated antigen-antibody complex can be confirmed by using a labeled antibody to determine the onset of liver cancer.
위와 같은 검출방법들은 정상 대조군의 마커 유전자 발현량과 간암 발병 세 포에서의 마커 유전자의 발현량을 비교하는 단계를 포함한다. mRNA 또는 단백질의 수준은 ANXA2 마커 단백질의 절대적 또는 상대적 차이로 나타낼 수 있다.Such detection methods include comparing the expression levels of marker genes in liver cancer-causing cells with the expression levels of marker genes in the normal control group. The level of mRNA or protein can be expressed as an absolute or relative difference between ANXA2 marker proteins.
본 발명은 ANXA2 단백질에 특이적으로 결합하는 항체를 포함하는 간암 진단 키트에 관한 것이다.The present invention relates to a liver cancer diagnostic kit comprising an antibody that specifically binds to ANXA2 protein.
또한, 본 발명은 ANXA2 단백질에 특이적으로 결합하는 항체를 생물학적 시료와 접촉시키는 단계를 포함하는 간암 마커 검출 방법에 관한 것이다.The present invention also relates to a method for detecting liver cancer markers comprising contacting an antibody that specifically binds an ANXA2 protein with a biological sample.
본 발명에서 ANXA2 단백질이 간암 마커로 규명되었으므로, 이를 이용하여 항체를 생성하는 것은 당업계에 널리 공지된 기술을 이용하여 용이하게 제조할 수 있다. 본 발명이 속하는 기술분야에 널리 알려진 바에 따라, 폴리클론 항체는 ANXA2 단백질 항원을 동물에 주사하여 동물로부터 채혈하여 항체를 포함하는 혈청을 얻는다. 동물로는 염소, 토끼, 돼지 등 임의의 동물 숙주를 이용하여 제조할 수 있다. 모노클론 항체는 본 발명이 속하는 기술분야에 널리 알려진 대로 하이브리도마 방법(hybridoma method)(Kohler & Milstein(1976) European J. Immunology 6:511-519 참조) 또는 파지 항체 라이브러리(Clackson et al., Nature, 352:624-628, 1991; Marks et al, J. Mol . Biol ., 222:58, 1-597, 1991) 기술을 이용하여 제조할 수 있다.Since the ANXA2 protein has been identified as a liver cancer marker in the present invention, the production of antibodies using the same can be easily prepared using techniques well known in the art. As is well known in the art, polyclonal antibodies are injected from an animal with ANXA2 protein antigen to be collected from the animal to obtain a serum comprising the antibody. Animals can be prepared using any animal host such as goat, rabbit, pig. Monoclonal antibodies are known in the art to which the present invention pertains, such as the hybridoma method (see Kohler & Milstein (1976) European J. Immunology 6 : 511-519) or phage antibody libraries (Clackson et al., Nature , 352 : 624-628, 1991; Marks et al, J. Mol . Biol . , 222 : 58, 1-597, 1991).
하이브리도마 방법은 ANXA2 단백질 항원을 주사한 마우스와 같은 면역학적으로 적합한 숙주동물의 세포를 이용하고, 다른 하나는 암 또는 골수종 세포주를 이용한다. 이러한 두 종류의 세포들을 폴리에틸렌글라이콜과 같은 본 발명이 속하는 기술분야에 널리 공지된 방법으로 융합시킨 후 항체생산 세포를 표준적인 조직 배 양방법으로 증식시킨다. 한계 희석법(limited dilution technique)에 의한 서브클로닝에 의해 균일한 세포 집단을 얻은 후 ANXA2 단백질에 대해 특이적인 항체를 생산할 수 있는 하이브리도마를 표준 기술에 따라 시험관 내 또는 생체 내에서 대량 배양한다.The hybridoma method utilizes cells from an immunologically suitable host animal, such as a mouse injected with ANXA2 protein antigen, and the other uses cancer or myeloma cell lines. These two kinds of cells are fused by methods well known in the art, such as polyethyleneglycol, and then antibody-producing cells are propagated by standard tissue culture methods. After obtaining a uniform cell population by subcloning by the limited dilution technique, hybridomas capable of producing antibodies specific for ANXA2 protein are mass cultured in vitro or in vivo according to standard techniques.
파지 항체 라이브러리 방법은 ANXA2 단백질에 대한 항체 유전자를 획득하여 이를 파지(phage)의 표면에 융합 단백질 형태로 발현하여 항체 라이브러리를 시험관 내에서 제작하고, 이 라이브러리로부터 ANXA2 단백질과 결합하는 모노클론 항체를 분리, 제작하는 방법이다.The phage antibody library method obtains an antibody gene for ANXA2 protein and expresses it in the form of a fusion protein on the surface of the phage to prepare an antibody library in vitro, and isolates the monoclonal antibody binding to the ANXA2 protein from this library. , How to make.
상기 방법에 의하여 제조된 항체는 전기영동, 투석, 이온교환 크로마토그래피, 친화 크로마토그래피 등의 방법으로 분리할 수 있다.Antibodies prepared by the above method can be separated by electrophoresis, dialysis, ion exchange chromatography, affinity chromatography and the like.
본 발명의 키트에 포함되는 항체는 2개의 전체 길이 경쇄(light chain) 및 2개의 전체 길이 중쇄(heavy chain)를 가지는 완전한 형태뿐만 아니라, 항체 분자의 기능적인 단편을 포함한다. 항체 분자의 기능적 단편이란 적어도 항원 결합기능을 보유하고 있는 단편을 뜻하며, Fab, F(ab'), F(ab')2, F(ab)2 및 Fv 등이 있다.Antibodies included in the kits of the present invention include functional fragments of antibody molecules, as well as complete forms having two full length light chains and two full length heavy chains. The functional fragment of an antibody molecule means a fragment which retains at least antigen binding function, and includes Fab, F (ab '), F (ab') 2 , F (ab) 2 and Fv.
ANXA2 단백질에 특이적으로 결합하는 항체를 포함하는 본 발명의 진단 키트는 바람직하게는 ELISA용 진단키트로서, 대조군 단백질에 특이적인 항체를 포함할 수 있으며, 그 밖에도 항원-항체 복합체를 검출할 수 있는 시약, 예컨대 표지된 2차 항체, 발색단, 항체와 컨쥬게이트된 효소 및 그 기질 또는 항체와 결합할 수 있는 다른 물질 등을 포함할 수 있다.The diagnostic kit of the present invention comprising an antibody that specifically binds to ANXA2 protein is preferably a diagnostic kit for ELISA, which may include an antibody specific for a control protein, in addition to detecting an antigen-antibody complex. Reagents such as labeled secondary antibodies, chromophores, enzymes conjugated with the antibody and its substrate or other substance capable of binding to the antibody, and the like.
이하, 다음 실시예를 들어 본 발명의 구성을 좀더 자세히 설명한다. 그러나, 본 발명의 범위가 아래 실시예의 기재범위 내로 한정되는 것이 아님은 본 발명의 기술분야에서 통상의 지식을 가진 자에게 자명하다.Hereinafter, the configuration of the present invention in more detail with reference to the following examples. However, it will be apparent to those skilled in the art that the scope of the present invention is not limited to the scope of the following examples.
<< 실시예Example 1> 조직 시료 처리 1> tissue sample processing
가톨릭대학교 병원으로부터 간암 환자 40명의 원발성 간암 조직과 주위 비간암 조직을 얻었다. 조직 시료는 액체 질소에서 동결시켰다. RNeasy midi kit(Qiagen, Hilden, Germany)을 이용하여 총 RNA를 분리하였다.Primary liver cancer tissues and 40 non-hepatic cancer tissues were obtained from the Catholic University Hospital. Tissue samples were frozen in liquid nitrogen. Total RNA was isolated using RNeasy midi kit (Qiagen, Hilden, Germany).
<< 실시예Example 2> 간암조직에서 2> in liver cancer tissue 아넥신Annexin 2의 발현 조사 Expression survey of 2
간암관련 유전자를 발굴하기 위해 cDNA microarrayer를 이용하여 40명 간암환자의 암 조직과 인접 조직을 대상으로 ANXA2 유전자 발현을 조사, 비교한 결과 ANXA2 유전자가 간암조직에서 중요한 발현 차이를 보였다. 이를 근거로 RT-PCR을 실시하였다(도 1). 정상 간 조직에 비해 간암 환자에서 ANXA2의 RNA 발현이 높게 나타났다.In order to identify genes related to liver cancer, the ANXA2 gene expression in cancer tissues and adjacent tissues of 40 liver cancer patients using cDNA microarrayer was compared. RT-PCR was performed based on this (FIG. 1). Compared to normal liver tissue, ANXA2 RNA expression was higher in liver cancer patients.
<< 실시예Example 3> 간암 세포주에서의 3> in liver cancer cell lines 아넥신Annexin 2 발현 2 expressions
간암 세포주에서 단백질 발현을 비교하기 위하여 다클론 항체를 이용하여 웨스턴 블랏 분석을 실시하였다(도 2). 이를 위하여 간암 세포주를 배양한 후 세포를 용출용액 (1% Triton X-100, 150 mM NaCl, 100 mM KCl, 20 mM HEPES (pH 7.9), 10 mM EDTA, 1 mM sodium orthovanadate, 10 ㎍/㎖ aprotinin, 10 ㎍/㎖ leupeptin, 1M PMSF)에 용출시킨 후 세포 용균액(cell lysate)을 전기영동하고 나이트로셀룰로스 막(Bio-Rad, CA, USA)에 블랏팅하였다. 이뮤노블랏팅 막(immunoblotting membrane)을 5% 탈지분유, 0.1% Tween 20이 포함된 PBS(phosphate buffered saline) 용액에 넣고 실온에서 2시간 동안 블로킹한 후 아넥신 2에 대한 항체 (1:5,000)로 두 시간 동안 반응시킨 후, 5,000배 희석된 호스래디쉬 퍼옥시데이즈가 접합된 2차 항체로 1시간 동안 반응시켰다. 마지막으로 enhanced chemiluminescence assay kit(Pierce, IL, USA)로 전개시켜 분석하였다. 도 3에서 보여주듯이 Huh-7, SKHep-1에서 아넥신 2가 많이 발현됨을 보였다. 상등액에서도 ANXA2가 발현됨을 알 수 있었고, 이와 같은 결과로부터 ANXA2가 세포 외로 분비되는 것을 알 수 있었다.Western blot analysis was performed using polyclonal antibodies to compare protein expression in liver cancer cell lines (FIG. 2). To this end, after culturing the liver cancer cell line, cells were eluted (1% Triton X-100, 150 mM NaCl, 100 mM KCl, 20 mM HEPES (pH 7.9), 10 mM EDTA, 1 mM sodium orthovanadate, 10 ㎍ / ml aprotinin). , 10 μg / ml leupeptin, 1M PMSF), and then cell lysate were electrophoresed and blotted onto nitrocellulose membranes (Bio-Rad, CA, USA). Immunoblotting membranes were placed in a phosphate buffered saline (PBS) solution containing 5% skim milk powder, 0.1% Tween 20 and blocked for 2 hours at room temperature, followed by antibody to Annexin 2 (1: 5,000). After the reaction for 2 hours, the reaction was performed with a secondary antibody conjugated with 5,000-fold diluted horseradish peroxidase for 1 hour. Finally, the analysis was performed using an enhanced chemiluminescence assay kit (Pierce, IL, USA). As shown in FIG. 3, annexin 2 was expressed in Huh-7 and SKHep-1. ANXA2 was also expressed in the supernatant, and from these results, it was found that ANXA2 was secreted out of the cells.
<< 실시예Example 4> 간 조직에서 4> in liver tissue 아넥신Annexin 2 단백질 발현 2 protein expression
간암조직에서의 단백질 발현을 비교하기 위하여 다클론 항체를 이용하여 웨스턴 블랏 분석을 실시하였다(도 3). 이를 위하여 간암조직을 희석하여 전기영동하고 나이트로셀룰로스 막(Bio-Rad, CA, USA)에 블랏팅하였다. 이뮤노블랏팅 막을 5% 탈지분유, 0.1% Tween 20이 포함된 PBS용액에 넣고 실온에서 2시간 동안 블로킹한 후 아넥신 2에 대한 항체 (1:5,000)로 2시간 동안 반응시킨 후, 5,000배 희석된 호스래디쉬 퍼옥시데이즈가 접합된 2차 항체로 1시간 동안 반응시켰다. 마지막으로 enhanced chemiluminescence assay kit (Pierce, IL, USA)로 전개시켜 분석하였다. 도 3에서 보여주듯이 간암조직과 혈청에서 아넥신 2가 많이 발현됨을 보였다. 이는 아넥신 2의 발현증가와 간암의 발전이 관련이 있음을 암시한다.Western blot analysis was performed using polyclonal antibodies to compare protein expression in liver cancer tissues (FIG. 3). To this end, liver cancer tissues were diluted and electrophoresed and blotted onto nitrocellulose membranes (Bio-Rad, CA, USA). The immunoblotting membrane was placed in a PBS solution containing 5% skim milk powder and 0.1% Tween 20, blocked for 2 hours at room temperature, and then reacted with an antibody to Annexin 2 (1: 5,000) for 2 hours, followed by diluting 5,000 times. 1 hour was reacted with the secondary horseradish peroxidase conjugated secondary antibody. Finally, the analysis was performed using an enhanced chemiluminescence assay kit (Pierce, IL, USA). As shown in FIG. 3, annexin 2 was expressed in liver cancer tissues and serum. This suggests that the expression of annexin 2 is related to the development of liver cancer.
<< 실시예Example 5> 5> 이뮤노닷Immunodot 분석에 의한 환자 혈청 중 In patient serum by analysis 아넥신Annexin 2 단백질 측정 2 Protein Measurement
다클론 항체를 이용하여 이뮤노닷 방법을 확립하여 혈청 중 아넥신 2의 분비정도를 비교하였다. 나이트로 셀룰로즈 멤브레인에 5배, 10배 희석한 혈청시료 (각 환자당 10개체)를 2㎕씩 점을 찍은 후 상온에서 건조시키고, 1% BSAT(bovine serum albumin in Tris-buffered saline)로 블로킹하였다. 아넥신 2에 대한 다클론 항체(1:1000)를 반응시킨 후 2차 항체 접합 호스래디쉬 퍼옥시데이즈(1:2000)를 가하고 DAB 용액(0.5㎎/㎖diaminobenzidine in PBT)으로 발색시켰다. 스캔하여 발색 정도를 비교하였다. 간암의 경우 정상보다 많이 분비되는 것을 알 수 있었다(도 4).An immunodot method was established using polyclonal antibodies to compare the secretion levels of Annexin 2 in serum. Two- and 5-fold diluted serum samples (10 for each patient) were spotted on nitrocellulose membranes, dried at room temperature, and blocked with 1% BSAT (bovine serum albumin in Tris-buffered saline). . After reacting the polyclonal antibody (1: 1000) to Annexin 2, secondary antibody conjugated horseradish peroxidases (1: 2000) were added and developed with DAB solution (0.5 mg / ml diaminobenzidine in PBT). Scanning was performed to compare the degree of color development. In the case of liver cancer was found to be secreted more than normal (Fig. 4).
<< 실시예Example 6> 6> ELISAELISA 를 이용한 간암 환자 혈 중 Blood of Liver Cancer Patients 아넥신Annexin 2 단백질 농도 측정 2 protein concentration measurement
단일클론 항체와 다클론 항체를 이용하여 ELISA 방법을 확립하였다. 토끼에서 얻은 다클론 항체를 1㎍/㎖로 코팅한 후 1% BSAT(bovine serum albumin in Tris-buffered saline)로 블로킹하였다. 아넥신 2 표준액 및 희석된 환자 혈청을 가한 후 아넥신 2에 대한 단일클론 항체를 가하여 2시간 동안 반응시킨 후 세척하였다. 적당한 농도로 희석된 2차 항체 접합 호스래디쉬 퍼옥시데이즈를 가하고 1시간 후 세척하고 TMB(Tetramethylbenzidine)로 발색하였다. 표준액의 검정곡선에 따 라 ELISA 방법에 의하여 환자 혈청 중 아넥신 2 단백질 농도를 측정하였다. 정상인과 간암환자의 혈청을 각각 희석하여 흡광도를 측정해 본 결과 간암의 경우 정상치보다 상승된 아넥신 2 농도를 보였다(도 5). 이 결과는 혈청 내 아넥신 2 농도의 상승이 간암 발생과 연관이 있음을 뜻한다.The ELISA method was established using monoclonal and polyclonal antibodies. Polyclonal antibodies obtained from rabbits were coated with 1 μg / ml and blocked with 1% BSAT (bovine serum albumin in Tris-buffered saline). Annexin 2 standard solution and diluted patient serum were added and then monoclonal antibodies against Annexin 2 were added for reaction for 2 hours and then washed. Secondary antibody conjugated horseradish peroxidase diluted to an appropriate concentration was added, washed after 1 hour, and developed with TMB (Tetramethylbenzidine). According to the calibration curve of the standard solution, the concentration of Annexin 2 protein in patient serum was measured by ELISA method. As a result of measuring the absorbance by diluting the serum of normal and liver cancer patients, hepatic cancer showed higher concentration of Annexin 2 than normal (FIG. 5). These results indicate that elevated serum annexin 2 levels are associated with liver cancer development.
결론적으로 간암에서 아넥신 2 수준은 간암 진단, 예후 인자로서 가능성과 효용성이 크다.In conclusion, annexin 2 level in liver cancer is highly useful and useful as a diagnostic and prognostic factor for liver cancer.
<< 실시예Example 7> 7> 샌드위치형Sandwich Type ELISAELISA 키트Kit
다음의 구성요소들을 사용하여 아넥신 2 농도 측정용 키트를 제조하였다.The kit for measuring annexin 2 concentration was prepared using the following components.
A. 고상형 항체: 항체가 흡착된 마이크로타이터 플레이트로서, 아넥신 2 에 대한 다클론 항체 100㎕를 마이크로타이터 플레이트에 가하고 4℃에서 하룻밤 동안 방치한 후 고상체 표면의 공간에 알부민을 흡착시켜 제조하였다.A. Solid antibody: Antibody-adsorbed microtiter plate, in which 100 μl of polyclonal antibody against annexin 2 is added to the microtiter plate and allowed to stand overnight at 4 ° C., followed by adsorption of albumin to the space on the solid surface. It was prepared by.
B. 검출용 항체 : 아넥신 2 에 대한 단일클론항체B. Detection antibody: monoclonal antibody against Annexin 2
C. 효소결합 항체: 호스래디쉬 퍼옥시다제가 결합된 2차 항체 용액(anti-mouse-IgG-peroxidase)C. Enzyme-Binding Antibodies: Secondary Antibody Solution with Horseradish Peroxidase (anti-mouse-IgG-peroxidase)
D. 혈청 희석용액: 1% BSA, 0.02% Tween 20, 1mM EDTA in PBSD. Serum Dilutions: 1% BSA, 0.02% Tween 20, 1 mM EDTA in PBS
E. 기질액: TMB 용액(R&D Systems, Inc., Minneapolis, USA)E. Substrate solution: TMB solution (R & D Systems, Inc., Minneapolis, USA)
F. 세척액: 0.05% 트윈이 포함된 인산염 완충액F. Wash Solution: Phosphate Buffer with 0.05% Tween
G. 표준용액: 아넥신 2 표준액G. Standard Solution: Annexin 2 Standard Solution
상기 키트를 이용하여 간암 환자 중 아넥신 2의 혈청희석 반응을 다음과 같 이 검사하였다. A의 고상형 항체, 즉 마이크로타이터 플레이트에 각 혈청 시료를 혈청 희석용액(D)을 사용, 적절히 희석하여 웰당 100㎕씩 가한 후 B, C, E의 구성요소들을 사용하여 실시예 6과 같은 샌드위치형 측정방법으로 아넥신 2 농도를 검사하였다. 도 6은 샌드위치형 키트로 수행한 실험결과를 나타낸 표준액 반응곡선이다. 도 7은 임상시료를 대상으로 샌드위치형 키트로 ANXA2를 분석한 결과를 나타낸 그림이다.Serine dilution of Annexin 2 in liver cancer patients was examined using the kit as follows. After diluting each serum sample to the solid-state antibody of A, that is, the microtiter plate, using a serum dilution solution (D), and diluting 100 μl per well, the components of B, C, and E were used as in Example 6. Annexin 2 concentration was examined by a sandwich measuring method. Figure 6 is a standard reaction curve showing the results of the experiment performed with the sandwich kit. Figure 7 is a diagram showing the results of the analysis of ANXA2 in a sandwich kit for clinical samples.
도 1은 간암 조직에서 아넥신 2(ANXA2)의 발현 정도를 나타낸 그림이다.1 is a diagram showing the expression level of Annexin 2 (ANXA2) in liver cancer tissue.
RT-PCR 방법으로 간암 환자의 비암 조직과 암 조직에서 ANXA2 발현을 비교하였다.RT-PCR method was used to compare ANXA2 expression in non-cancerous and cancerous tissues of liver cancer patients.
도 2는 웨스턴 블랏 방법으로 간암 세포주에서 ANXA2의 발현 정도를 비교한 그림이다.Figure 2 is a figure comparing the expression level of ANXA2 in liver cancer cell line by Western blot method.
도 3은 조직과 혈청 내 ANXA2 단백질의 발현 정도를 웨스턴 블랏을 실시하여 비교한 그림이다.Figure 3 is a comparison of the expression level of the tissue and serum ANXA2 protein by Western blot.
도 4는 이뮤노닷 분석에 의한 ANXA2 분비 차이를 나타낸 그림이다.Figure 4 is a diagram showing the difference between ANXA2 secretion by immunodot analysis.
도 5는 ELISA에 의한 간암시료와 정상시료의 희석곡선을 나타낸 그림이다.Figure 5 is a diagram showing the dilution curve of liver cancer sample and normal sample by ELISA.
도 6은 샌드위치형 ELISA 키트로 수행한 표준액 반응곡선이다.6 is a standard reaction curve performed with a sandwich ELISA kit.
도 7은 샌드위치형 ELISA 키트를 이용하여 임상시료 내 ANXA2를 측정한 결과이다.7 shows the results of measuring ANXA2 in clinical samples using a sandwich type ELISA kit.
<110> Korea Research Institute of Bioscience and Biotechnology Industrial coorperation foundation Chonbul national university <120> Annexin 2 as a tumor associated marker of hepatocellular carcinoma in human serum and a hepatocellular carcinoma diagnostic kit using thereof <130> KRIBB-EYSONG-ANXA <160> 4 <170> KopatentIn 1.71 <210> 1 <211> 1074 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (1)..(1071) <400> 1 atg ggc cgc cag cta gcg ggg tgc gga gac gct ggg aag aag gct tcc 48 Met Gly Arg Gln Leu Ala Gly Cys Gly Asp Ala Gly Lys Lys Ala Ser 1 5 10 15 ttc aaa atg tct act gtt cac gaa atc ctg tgc aag ctc agc ttg gag 96 Phe Lys Met Ser Thr Val His Glu Ile Leu Cys Lys Leu Ser Leu Glu 20 25 30 ggt gat cac tct aca ccc cca agt gca tat ggg tct gtc aaa gcc tat 144 Gly Asp His Ser Thr Pro Pro Ser Ala Tyr Gly Ser Val Lys Ala Tyr 35 40 45 act aac ttt gat gct gag cgg gat gct ttg aac att gaa aca gcc atc 192 Thr Asn Phe Asp Ala Glu Arg Asp Ala Leu Asn Ile Glu Thr Ala Ile 50 55 60 aag acc aaa ggt gtg gat gag gtc acc att gtc aac att ttg acc aac 240 Lys Thr Lys Gly Val Asp Glu Val Thr Ile Val Asn Ile Leu Thr Asn 65 70 75 80 cgc agc aat gca cag aga cag gat att gcc ttc gcc tac cag aga agg 288 Arg Ser Asn Ala Gln Arg Gln Asp Ile Ala Phe Ala Tyr Gln Arg Arg 85 90 95 acc aaa aag gaa ctt gca tca gca ctg aag tca gcc tta tct ggc cac 336 Thr Lys Lys Glu Leu Ala Ser Ala Leu Lys Ser Ala Leu Ser Gly His 100 105 110 ctg gag acg gtg att ttg ggc cta ttg aag aca cct gct cag tat gac 384 Leu Glu Thr Val Ile Leu Gly Leu Leu Lys Thr Pro Ala Gln Tyr Asp 115 120 125 gct tct gag cta aaa gct tcc atg aag ggg ctg gga acc gac gag gac 432 Ala Ser Glu Leu Lys Ala Ser Met Lys Gly Leu Gly Thr Asp Glu Asp 130 135 140 tct ctc att gag atc atc tgc tcc aga acc aac cag gag ctg cag gaa 480 Ser Leu Ile Glu Ile Ile Cys Ser Arg Thr Asn Gln Glu Leu Gln Glu 145 150 155 160 att aac aga gtc tac aag gaa atg tac aag act gat ctg gag aag gac 528 Ile Asn Arg Val Tyr Lys Glu Met Tyr Lys Thr Asp Leu Glu Lys Asp 165 170 175 att att tcg gac aca tct ggt gac ttc cgc aag ctg atg gtt gcc ctg 576 Ile Ile Ser Asp Thr Ser Gly Asp Phe Arg Lys Leu Met Val Ala Leu 180 185 190 gca aag ggt aga aga gca gag gat ggc tct gtc att gat tat gaa ctg 624 Ala Lys Gly Arg Arg Ala Glu Asp Gly Ser Val Ile Asp Tyr Glu Leu 195 200 205 att gac caa gat gct cgg gat ctc tat gac gct gga gtg aag agg aaa 672 Ile Asp Gln Asp Ala Arg Asp Leu Tyr Asp Ala Gly Val Lys Arg Lys 210 215 220 gga act gat gtt ccc aag tgg atc agc atc atg acc gag cgg agc gtg 720 Gly Thr Asp Val Pro Lys Trp Ile Ser Ile Met Thr Glu Arg Ser Val 225 230 235 240 ccc cac ctc cag aaa gta ttt gat agg tac aag agt tac agc cct tat 768 Pro His Leu Gln Lys Val Phe Asp Arg Tyr Lys Ser Tyr Ser Pro Tyr 245 250 255 gac atg ttg gaa agc atc agg aaa gag gtt aaa gga gac ctg gaa aat 816 Asp Met Leu Glu Ser Ile Arg Lys Glu Val Lys Gly Asp Leu Glu Asn 260 265 270 gct ttc ctg aac ctg gtt cag tgc att cag aac aag ccc ctg tat ttt 864 Ala Phe Leu Asn Leu Val Gln Cys Ile Gln Asn Lys Pro Leu Tyr Phe 275 280 285 gct gat cgg ctg tat gac tcc atg aag ggc aag ggg acg cga gat aag 912 Ala Asp Arg Leu Tyr Asp Ser Met Lys Gly Lys Gly Thr Arg Asp Lys 290 295 300 gtc ctg atc aga atc atg gtc tcc cgc agt gaa gtg gac atg ttg aaa 960 Val Leu Ile Arg Ile Met Val Ser Arg Ser Glu Val Asp Met Leu Lys 305 310 315 320 att agg tct gaa ttc aag aga aag tac ggc aag tcc ctg tac tat tat 1008 Ile Arg Ser Glu Phe Lys Arg Lys Tyr Gly Lys Ser Leu Tyr Tyr Tyr 325 330 335 atc cag caa gac act aag ggc gac tac cag aaa gcg ctg ctg tac ctg 1056 Ile Gln Gln Asp Thr Lys Gly Asp Tyr Gln Lys Ala Leu Leu Tyr Leu 340 345 350 tgt ggt gga gat gac tga 1074 Cys Gly Gly Asp Asp 355 <210> 2 <211> 357 <212> PRT <213> Homo sapiens <400> 2 Met Gly Arg Gln Leu Ala Gly Cys Gly Asp Ala Gly Lys Lys Ala Ser 1 5 10 15 Phe Lys Met Ser Thr Val His Glu Ile Leu Cys Lys Leu Ser Leu Glu 20 25 30 Gly Asp His Ser Thr Pro Pro Ser Ala Tyr Gly Ser Val Lys Ala Tyr 35 40 45 Thr Asn Phe Asp Ala Glu Arg Asp Ala Leu Asn Ile Glu Thr Ala Ile 50 55 60 Lys Thr Lys Gly Val Asp Glu Val Thr Ile Val Asn Ile Leu Thr Asn 65 70 75 80 Arg Ser Asn Ala Gln Arg Gln Asp Ile Ala Phe Ala Tyr Gln Arg Arg 85 90 95 Thr Lys Lys Glu Leu Ala Ser Ala Leu Lys Ser Ala Leu Ser Gly His 100 105 110 Leu Glu Thr Val Ile Leu Gly Leu Leu Lys Thr Pro Ala Gln Tyr Asp 115 120 125 Ala Ser Glu Leu Lys Ala Ser Met Lys Gly Leu Gly Thr Asp Glu Asp 130 135 140 Ser Leu Ile Glu Ile Ile Cys Ser Arg Thr Asn Gln Glu Leu Gln Glu 145 150 155 160 Ile Asn Arg Val Tyr Lys Glu Met Tyr Lys Thr Asp Leu Glu Lys Asp 165 170 175 Ile Ile Ser Asp Thr Ser Gly Asp Phe Arg Lys Leu Met Val Ala Leu 180 185 190 Ala Lys Gly Arg Arg Ala Glu Asp Gly Ser Val Ile Asp Tyr Glu Leu 195 200 205 Ile Asp Gln Asp Ala Arg Asp Leu Tyr Asp Ala Gly Val Lys Arg Lys 210 215 220 Gly Thr Asp Val Pro Lys Trp Ile Ser Ile Met Thr Glu Arg Ser Val 225 230 235 240 Pro His Leu Gln Lys Val Phe Asp Arg Tyr Lys Ser Tyr Ser Pro Tyr 245 250 255 Asp Met Leu Glu Ser Ile Arg Lys Glu Val Lys Gly Asp Leu Glu Asn 260 265 270 Ala Phe Leu Asn Leu Val Gln Cys Ile Gln Asn Lys Pro Leu Tyr Phe 275 280 285 Ala Asp Arg Leu Tyr Asp Ser Met Lys Gly Lys Gly Thr Arg Asp Lys 290 295 300 Val Leu Ile Arg Ile Met Val Ser Arg Ser Glu Val Asp Met Leu Lys 305 310 315 320 Ile Arg Ser Glu Phe Lys Arg Lys Tyr Gly Lys Ser Leu Tyr Tyr Tyr 325 330 335 Ile Gln Gln Asp Thr Lys Gly Asp Tyr Gln Lys Ala Leu Leu Tyr Leu 340 345 350 Cys Gly Gly Asp Asp 355 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 ctctacaccc ccaagtgcat 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 tcagtgctga tgcaagttcc 20 <110> Korea Research Institute of Bioscience and Biotechnology Industrial coorperation foundation Chonbul national university <120> Annexin 2 as a tumor associated marker of hepatocellular carcinoma in human serum and a hepatocellular carcinoma diagnostic kit using <130> KRIBB-EYSONG-ANXA <160> 4 <170> KopatentIn 1.71 <210> 1 <211> 1074 <212> DNA <213> Homo sapiens <220> <221> CDS (222) (1) .. (1071) <400> 1 atg ggc cgc cag cta gcg ggg tgc gga gac gct ggg aag aag gct tcc 48 Met Gly Arg Gln Leu Ala Gly Cys Gly Asp Ala Gly Lys Lys Ala Ser 1 5 10 15 ttc aaa atg tct act gtt cac gaa atc ctg tgc aag ctc agc ttg gag 96 Phe Lys Met Ser Thr Val His Glu Ile Leu Cys Lys Leu Ser Leu Glu 20 25 30 ggt gat cac tct aca ccc cca agt gca tat ggg tct gtc aaa gcc tat 144 Gly Asp His Ser Thr Pro Pro Ser Ala Tyr Gly Ser Val Lys Ala Tyr 35 40 45 act aac ttt gat gct gag cgg gat gct ttg aac att gaa aca gcc atc 192 Thr Asn Phe Asp Ala Glu Arg Asp Ala Leu Asn Ile Glu Thr Ala Ile 50 55 60 aag acc aaa ggt gtg gat gag gtc acc att gtc aac att ttg acc aac 240 Lys Thr Lys Gly Val Asp Glu Val Thr Ile Val Asn Ile Leu Thr Asn 65 70 75 80 cgc agc aat gca cag aga cag gat att gcc ttc gcc tac cag aga agg 288 Arg Ser Asn Ala Gln Arg Gln Asp Ile Ala Phe Ala Tyr Gln Arg Arg 85 90 95 acc aaa aag gaa ctt gca tca gca ctg aag tca gcc tta tct ggc cac 336 Thr Lys Lys Glu Leu Ala Ser Ala Leu Lys Ser Ala Leu Ser Gly His 100 105 110 ctg gag acg gtg att ttg ggc cta ttg aag aca cct gct cag tat gac 384 Leu Glu Thr Val Ile Leu Gly Leu Leu Lys Thr Pro Ala Gln Tyr Asp 115 120 125 gct tct gag cta aaa gct tcc atg aag ggg ctg gga acc gac gag gac 432 Ala Ser Glu Leu Lys Ala Ser Met Lys Gly Leu Gly Thr Asp Glu Asp 130 135 140 tct ctc att gag atc atc tgc tcc aga acc aac cag gag ctg cag gaa 480 Ser Leu Ile Glu Ile Ile Cys Ser Arg Thr Asn Gln Glu Leu Gln Glu 145 150 155 160 att aac aga gtc tac aag gaa atg tac aag act gat ctg gag aag gac 528 Ile Asn Arg Val Tyr Lys Glu Met Tyr Lys Thr Asp Leu Glu Lys Asp 165 170 175 att att tcg gac aca tct ggt gac ttc cgc aag ctg atg gtt gcc ctg 576 Ile Ile Ser Asp Thr Ser Gly Asp Phe Arg Lys Leu Met Val Ala Leu 180 185 190 gca aag ggt aga aga gca gag gat ggc tct gtc att gat tat gaa ctg 624 Ala Lys Gly Arg Arg Ala Glu Asp Gly Ser Val Ile Asp Tyr Glu Leu 195 200 205 att gac caa gat gct cgg gat ctc tat gac gct gga gtg aag agg aaa 672 Ile Asp Gln Asp Ala Arg Asp Leu Tyr Asp Ala Gly Val Lys Arg Lys 210 215 220 gga act gat gtt ccc aag tgg atc agc atc atg acc gag cgg agc gtg 720 Gly Thr Asp Val Pro Lys Trp Ile Ser Ile Met Thr Glu Arg Ser Val 225 230 235 240 ccc cac ctc cag aaa gta ttt gat agg tac aag agt tac agc cct tat 768 Pro His Leu Gln Lys Val Phe Asp Arg Tyr Lys Ser Tyr Ser Pro Tyr 245 250 255 gac atg ttg gaa agc atc agg aaa gag gtt aaa gga gac ctg gaa aat 816 Asp Met Leu Glu Ser Ile Arg Lys Glu Val Lys Gly Asp Leu Glu Asn 260 265 270 gct ttc ctg aac ctg gtt cag tgc att cag aac aag ccc ctg tat ttt 864 Ala Phe Leu Asn Leu Val Gln Cys Ile Gln Asn Lys Pro Leu Tyr Phe 275 280 285 gct gat cgg ctg tat gac tcc atg aag ggc aag ggg acg cga gat aag 912 Ala Asp Arg Leu Tyr Asp Ser Met Lys Gly Lys Gly Thr Arg Asp Lys 290 295 300 gtc ctg atc aga atc atg gtc tcc cgc agt gaa gtg gac atg ttg aaa 960 Val Leu Ile Arg Ile Met Val Ser Arg Ser Glu Val Asp Met Leu Lys 305 310 315 320 att agg tct gaa ttc aag aga aag tac ggc aag tcc ctg tac tat tat 1008 Ile Arg Ser Glu Phe Lys Arg Lys Tyr Gly Lys Ser Leu Tyr Tyr Tyr 325 330 335 atc cag caa gac act aag ggc gac tac cag aaa gcg ctg ctg tac ctg 1056 Ile Gln Gln Asp Thr Lys Gly Asp Tyr Gln Lys Ala Leu Leu Tyr Leu 340 345 350 tgt ggt gga gat gac tga 1074 Cys Gly Gly Asp Asp 355 <210> 2 <211> 357 <212> PRT <213> Homo sapiens <400> 2 Met Gly Arg Gln Leu Ala Gly Cys Gly Asp Ala Gly Lys Lys Ala Ser 1 5 10 15 Phe Lys Met Ser Thr Val His Glu Ile Leu Cys Lys Leu Ser Leu Glu 20 25 30 Gly Asp His Ser Thr Pro Pro Ser Ala Tyr Gly Ser Val Lys Ala Tyr 35 40 45 Thr Asn Phe Asp Ala Glu Arg Asp Ala Leu Asn Ile Glu Thr Ala Ile 50 55 60 Lys Thr Lys Gly Val Asp Glu Val Thr Ile Val Asn Ile Leu Thr Asn 65 70 75 80 Arg Ser Asn Ala Gln Arg Gln Asp Ile Ala Phe Ala Tyr Gln Arg Arg 85 90 95 Thr Lys Lys Glu Leu Ala Ser Ala Leu Lys Ser Ala Leu Ser Gly His 100 105 110 Leu Glu Thr Val Ile Leu Gly Leu Leu Lys Thr Pro Ala Gln Tyr Asp 115 120 125 Ala Ser Glu Leu Lys Ala Ser Met Lys Gly Leu Gly Thr Asp Glu Asp 130 135 140 Ser Leu Ile Glu Ile Ile Cys Ser Arg Thr Asn Gln Glu Leu Gln Glu 145 150 155 160 Ile Asn Arg Val Tyr Lys Glu Met Tyr Lys Thr Asp Leu Glu Lys Asp 165 170 175 Ile Ile Ser Asp Thr Ser Gly Asp Phe Arg Lys Leu Met Val Ala Leu 180 185 190 Ala Lys Gly Arg Arg Ala Glu Asp Gly Ser Val Ile Asp Tyr Glu Leu 195 200 205 Ile Asp Gln Asp Ala Arg Asp Leu Tyr Asp Ala Gly Val Lys Arg Lys 210 215 220 Gly Thr Asp Val Pro Lys Trp Ile Ser Ile Met Thr Glu Arg Ser Val 225 230 235 240 Pro His Leu Gln Lys Val Phe Asp Arg Tyr Lys Ser Tyr Ser Pro Tyr 245 250 255 Asp Met Leu Glu Ser Ile Arg Lys Glu Val Lys Gly Asp Leu Glu Asn 260 265 270 Ala Phe Leu Asn Leu Val Gln Cys Ile Gln Asn Lys Pro Leu Tyr Phe 275 280 285 Ala Asp Arg Leu Tyr Asp Ser Met Lys Gly Lys Gly Thr Arg Asp Lys 290 295 300 Val Leu Ile Arg Ile Met Val Ser Arg Ser Glu Val Asp Met Leu Lys 305 310 315 320 Ile Arg Ser Glu Phe Lys Arg Lys Tyr Gly Lys Ser Leu Tyr Tyr Tyr 325 330 335 Ile Gln Gln Asp Thr Lys Gly Asp Tyr Gln Lys Ala Leu Leu Tyr Leu 340 345 350 Cys Gly Gly Asp Asp 355 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 ctctacaccc ccaagtgcat 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 tcagtgctga tgcaagttcc 20
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| KR1020070095017A KR20090029868A (en) | 2007-09-19 | 2007-09-19 | Annexin 2 as a liver cancer marker in body fluids and liver cancer diagnosis kit using the same |
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| KR1020070095017A KR20090029868A (en) | 2007-09-19 | 2007-09-19 | Annexin 2 as a liver cancer marker in body fluids and liver cancer diagnosis kit using the same |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102428184A (en) * | 2009-04-17 | 2012-04-25 | Cbs生物科学有限公司 | Marker for prognosis of liver cancer |
| CN102809657A (en) * | 2012-08-29 | 2012-12-05 | 沃克(天津)生物科技有限公司 | Food intolerant serological specificity IgG detection kit and preparation method thereof |
| WO2015009082A1 (en) * | 2013-07-19 | 2015-01-22 | 삼성전자 주식회사 | Fusion protein comprising annexin a2 binding protein, p53 protein, and p18 protein, and composition containing same for preventing or treating cancer |
| US9470677B2 (en) | 2013-11-28 | 2016-10-18 | Samsung Electronics Co., Ltd. | Cell with surface coated with ANXA1 and use thereof |
| KR20200082951A (en) * | 2018-12-31 | 2020-07-08 | 충남대학교산학협력단 | Biomarker composition comprising NOX4 for diagnosis or predicting prognosis of hepatocellular carcinoma |
-
2007
- 2007-09-19 KR KR1020070095017A patent/KR20090029868A/en not_active Ceased
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102428184A (en) * | 2009-04-17 | 2012-04-25 | Cbs生物科学有限公司 | Marker for prognosis of liver cancer |
| CN102809657A (en) * | 2012-08-29 | 2012-12-05 | 沃克(天津)生物科技有限公司 | Food intolerant serological specificity IgG detection kit and preparation method thereof |
| CN102809657B (en) * | 2012-08-29 | 2015-07-29 | 沃克(天津)生物科技有限公司 | Food intolerance serological specificity IgG detection kit and preparation method thereof |
| WO2015009082A1 (en) * | 2013-07-19 | 2015-01-22 | 삼성전자 주식회사 | Fusion protein comprising annexin a2 binding protein, p53 protein, and p18 protein, and composition containing same for preventing or treating cancer |
| US9470677B2 (en) | 2013-11-28 | 2016-10-18 | Samsung Electronics Co., Ltd. | Cell with surface coated with ANXA1 and use thereof |
| KR20200082951A (en) * | 2018-12-31 | 2020-07-08 | 충남대학교산학협력단 | Biomarker composition comprising NOX4 for diagnosis or predicting prognosis of hepatocellular carcinoma |
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