Author:
Akada Junko,Kamei Shuichi,Ito Akane,Ito Moe,Kitagawa Takao,Furumoto Hiroko,Kato Yukari,Tamesa Michiko,Takashima Motonari,Shirai Mutsunori,Yamano Hirofumi,Oka Masaaki,Kuramitsu Yasuhiro,Nakamura Kazuyuki
Abstract
Abstract
Background
We report here a new type of protein chip to detect antibodies in sera. This chip method was used to a prototype created to detect hepatocellular carcinoma (HCC) -related autoantibodies in the sera of hepatitis C virus (HCV) infected individuals.
Results
Five cysteine-tagged (Cys-tag) and green fluorescent protein (GFP)-fused recombinant heat shock protein 70 (HSP70), superoxide dismutase 2 (SOD2), and peroxiredoxin 6 (PRDX6), were spotted and immobilized on maleimide-incorporated diamond-like carbon (DLC) substrates. The antibodies in diluted sera were trapped by these proteins at each spot on the chip, and visualized by a fluorescence-conjugated anti-human IgG. The total immobilized protein level of each spot was detected with anti-GFP mouse IgG and a fluorescence-conjugated secondary anti-mouse IgG. The ratio between the two fluorescence intensities was used to quantify autoantibody levels in each serum sample. Heat treatment of the chip in a solution of denaturing and reducing agents, before serum-incubation, improved autoantibody detection. We tested serum samples from healthy individuals and HCC patients using the chips. The HSP70 autoantibodies were found at high levels in sera from HCV-positive HCC patients, but not in HCV-negative sera.
Conclusion
This protein chip system may have useful properties to capture a specific set of antibodies for predicting the onset of particular cancers such as HCC in HCV-infected individuals.
Publisher
Springer Science and Business Media LLC
Subject
Molecular Biology,Biochemistry
Cited by
14 articles.
订阅此论文施引文献
订阅此论文施引文献,注册后可以免费订阅5篇论文的施引文献,订阅后可以查看论文全部施引文献