Development and optimization of an antibody array method for potential cancer biomarker detection

作     者：Shuangshuang Wang Ping Zhao Brian Cao 

作者机构：Key Laboratory of Antibody Technology of Ministry of Health Nanjing Medical University Nanjing Jiangsu Province 210029 China Antibody Technology Laboratory Van Andel Research Institute Grand Rapids MI 49503 USA 

出 版 物：《The Journal of Biomedical Research》 (生物医学研究杂志（英文版）)

年 卷 期：2011年第25卷第1期

页      面：63-70页

学科分类：0710[理学-生物学] 071010[理学-生物化学与分子生物学] 081704[工学-应用化学] 081803[工学-地质工程] 07[理学] 08[工学] 0817[工学-化学工程与技术] 0818[工学-地质资源与地质工程] 

基　　金：supported by the Lustgarten Foundation for Pancreatic Cancer Research (No. RFA-08-003) 

主　　题：cancer biomarker HPCAL1 PEBP1 LGALS7 SERPINE2 antibody microarray 

摘      要：Biomarkers play an important role in the detection at an early stage of pancreatic cancer. The aim of the present study was to optimize the conditions of antibody arrays for detecting Hippocalcin-like 1 （HPCAL1）, phosphatidylethanolamine binding protein 1 （PEBP1）, lectin galactoside-binding soluble 7 （LGALS7）, and serpin peptidase inhibitor clade E member 2 （SERPINE2） as biomarkers for pancreatic cancer detection in a single assay and to investigate antibodies’ specificity and cross-reactivity. Capture antibodies against HPCAL1, PEBP1, LGALS7 and SERPINE2 were printed on nitrocellulose coated glass slides. HPCAL1, PEBP1, LGALS7 and SERPINE2 proteins with different concentrations were incubated with the capture antibodies at different temperatures for different time periods. Biotinylated detection antibodies recognizing a different epitope on the captured proteins and a secondary detection molecule （Streptavidin-PE） were used to detect fluorescent signals. The arrays showed the strongest signals when the concentration of the capture antibodies was at 500 μg/mL in PBST0.05 （PBS with 0.05% Tween-20）, and the slides were incubated overnight at 4°C. The lowest protein concentration for detection was 2 ng/mL. Each antibody demonstrated high specificity to the corresponding antigen in detecting a mixture of 4 proteins without significant cross-reactivity. The fluorescence and biomarker concentration displayed a linear correlation. The antibody microarray system could be a useful tool for potential biomarker detection for pancreatic cancer.