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12/12/2014; 14:02
File: cbm455.tex; BOKCTP/wyn p. 1
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Cancer Biomarkers 00 (2014) 1–7 DOI 10.3233/CBM-140455 IOS Press
Evaluation of Annexin A2 as a novel diagnostic serum biomarker for lung cancer Jie Yanga,1, Fan Yangb,1 , Jiqin Niea , Xiaohua Zoua, Huiqun Tiana, Yu’e Qina and Chaoqi Liua,∗ a
Institute of Molecular Biology, Yichang, Hubei, China The Second People’s Hospital of China Three Gorges University, Yichang, Hubei, China
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Keywords: Annexin A2, serum biomarker, lung cancer, ELISA
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Abstract. Annexin A2 (ANXA2) is a 36 kDa protein which orchestrates multiple biologic processes and clinical associations, especially in cancer progression. It is important to establish a specific and sensitive ANXA2 enzyme-linked immunosorbent assay (ELISA) for the study of ANXA2 functions and its clinical application. Therefore, we prepared a polyclonal antibody (PAb) in rabbits and a monoclonal antibody (MAb) in mices immunized with a recombinant ANXA2 protein. Based on our self-made MAb and PAb, highly specific and sensitive ELISA was developed. The detection limitation of ANXA2 was 10 ng/mL and the linear dynamic range was between 10 and 500 ng/mL. Using the established ELISA, we detected ANXA2 protein in human serum. It was found that soluble ANXA2 concentration in serum samples from 42 lung cancer patients was significantly higher than that from 43 healthy individuals (p < 0.01). Our data provides a new approach for detecting soluble ANXA2, especially in large ongoing and future clinical studies.
1. Introduction
2
Annexin A2 (p36, annexin II, ANXA2) is a 36 kDa protein that is located on chromosome 15q22.2 in human [1,2]. ANXA2 is mainly expressed in endothelial cells, macrophages, mononuclear cells. Intracellular ANXA2 has been reported to play roles in exocytosis, endocytosis, and membrane trafficking [3,4]. Recently many researchers have shown that ANXA2 was overexpressed in many tumors [5]. However, the overexpression of ANXA2 in different tumors or different periods of disease may also reflect differing clinical significance. At the early stage of tumors, such as in lung and liver cancer, ANXA2 was recognized as a biomarker of early diagnosis [6–8]. Lung cancer has the highest incidence and mortality worldwide [9]. Non-small cell lung cancer (NSCLC), which includes adenocarcinoma, squamous cell carcinoma, and large cell carcinoma, accounts for 80% of all lung cancers [10]. Unfortunately, the majority of pa-
6 7 8 9 10 11 12 13 14 15 16 17 18 19
rre
5
co
4
un
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ct
1
1 These
authors acontributed equally to this work. author: Chaoqi Liu, Institute of Molecular Biology CTGU, Yichang 443002, Hubei, China. Tel.: +86 13581499903; Fax: +86 0717 6397179; E-mail: [email protected]. ∗ Corresponding
tients with NSCLC often do not exhibit specific symptoms, particularly in early stages [11,12]. Currently, conventional diagnostic tests such as chest radiograph, computed tomography (CT) and bronchoscopy are not sensitive enough for effective early detection. Studies have illustrated the significance of serum tumor markers in the detection, prognosis, and follow-up of lung cancer [13]. As the ANXA2 is widely overexpressed in tumor and can secrete into the serum, here we developed an enzyme-linked immunosorbent assay (ELISA) based on monoclonal antibody (MAb) and polyclonal antibody (PAb) to determine the ANXA2 in the serum of cancer patients. The results show that ANXA2 levels in serum samples were significantly higher in lung cancer patients than that from healthy individuals. Therefore, we successfully established the ELISA assay for detection of ANXA2 in lung cancer.
20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37
2. Materials and methods
38
2.1. Reagents and materials
39
Plasmid pGEX4T1-ANXA2 was constructed by our research group. Freund’s adjuvant, 3, 3’, 5, 5’-
40
c 2014 – IOS Press and the authors. All rights reserved ISSN 1574-0153/14/$27.50
41
12/12/2014; 14:02
File: cbm455.tex; BOKCTP/wyn p. 1
1
Cancer Biomarkers 00 (2014) 1–7 DOI 10.3233/CBM-140455 IOS Press
Evaluation of Annexin A2 as a novel diagnostic serum biomarker for lung cancer Jie Yanga,1, Fan Yangb,1 , Jiqin Niea , Xiaohua Zoua, Huiqun Tiana, Yu’e Qina and Chaoqi Liua,∗ a
Institute of Molecular Biology, Yichang, Hubei, China The Second People’s Hospital of China Three Gorges University, Yichang, Hubei, China
n
b
ed
pr
Keywords: Annexin A2, serum biomarker, lung cancer, ELISA
oo
fv
er
si o
Abstract. Annexin A2 (ANXA2) is a 36 kDa protein which orchestrates multiple biologic processes and clinical associations, especially in cancer progression. It is important to establish a specific and sensitive ANXA2 enzyme-linked immunosorbent assay (ELISA) for the study of ANXA2 functions and its clinical application. Therefore, we prepared a polyclonal antibody (PAb) in rabbits and a monoclonal antibody (MAb) in mices immunized with a recombinant ANXA2 protein. Based on our self-made MAb and PAb, highly specific and sensitive ELISA was developed. The detection limitation of ANXA2 was 10 ng/mL and the linear dynamic range was between 10 and 500 ng/mL. Using the established ELISA, we detected ANXA2 protein in human serum. It was found that soluble ANXA2 concentration in serum samples from 42 lung cancer patients was significantly higher than that from 43 healthy individuals (p < 0.01). Our data provides a new approach for detecting soluble ANXA2, especially in large ongoing and future clinical studies.
1. Introduction
2
Annexin A2 (p36, annexin II, ANXA2) is a 36 kDa protein that is located on chromosome 15q22.2 in human [1,2]. ANXA2 is mainly expressed in endothelial cells, macrophages, mononuclear cells. Intracellular ANXA2 has been reported to play roles in exocytosis, endocytosis, and membrane trafficking [3,4]. Recently many researchers have shown that ANXA2 was overexpressed in many tumors [5]. However, the overexpression of ANXA2 in different tumors or different periods of disease may also reflect differing clinical significance. At the early stage of tumors, such as in lung and liver cancer, ANXA2 was recognized as a biomarker of early diagnosis [6–8]. Lung cancer has the highest incidence and mortality worldwide [9]. Non-small cell lung cancer (NSCLC), which includes adenocarcinoma, squamous cell carcinoma, and large cell carcinoma, accounts for 80% of all lung cancers [10]. Unfortunately, the majority of pa-
6 7 8 9 10 11 12 13 14 15 16 17 18 19
rre
5
co
4
un
3
ct
1
1 These
authors acontributed equally to this work. author: Chaoqi Liu, Institute of Molecular Biology CTGU, Yichang 443002, Hubei, China. Tel.: +86 13581499903; Fax: +86 0717 6397179; E-mail: [email protected]. ∗ Corresponding
tients with NSCLC often do not exhibit specific symptoms, particularly in early stages [11,12]. Currently, conventional diagnostic tests such as chest radiograph, computed tomography (CT) and bronchoscopy are not sensitive enough for effective early detection. Studies have illustrated the significance of serum tumor markers in the detection, prognosis, and follow-up of lung cancer [13]. As the ANXA2 is widely overexpressed in tumor and can secrete into the serum, here we developed an enzyme-linked immunosorbent assay (ELISA) based on monoclonal antibody (MAb) and polyclonal antibody (PAb) to determine the ANXA2 in the serum of cancer patients. The results show that ANXA2 levels in serum samples were significantly higher in lung cancer patients than that from healthy individuals. Therefore, we successfully established the ELISA assay for detection of ANXA2 in lung cancer.
20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37
2. Materials and methods
38
2.1. Reagents and materials
39
Plasmid pGEX4T1-ANXA2 was constructed by our research group. Freund’s adjuvant, 3, 3’, 5, 5’-
40
c 2014 – IOS Press and the authors. All rights reserved ISSN 1574-0153/14/$27.50
41
