A new type of protein chip to detect hepatocellular carcinoma-related autoimmune antibodies in the sera of hepatitis C virus-positive patients

Proteome Science

Kazuyuki Nakamura³  Yasuhiro Kuramitsu³  Masaaki Oka¹  Hirofumi Yamano²  Mutsunori Shirai⁴  Motonari Takashima¹  Michiko Tamesa¹  Yukari Kato³  Hiroko Furumoto³  Takao Kitagawa³  Moe Ito³  Akane Ito²  Shuichi Kamei²  Junko Akada³ 

[1] Department of Digestive Surgery and Surgical Oncology, Yamaguchi University Graduate School of Medicine, Ube, Yamaguchi, Japan;Technical Research Laboratory, Toyo Kohan Company. Ltd, Kudamatsu, Yamaguchi, Japan;Department of Biochemistry and Functional Proteomics, Yamaguchi University Graduate School of Medicine, 1-1-1, Minami-kogushi, Ube, Yamaguchi 755-8505, Japan;Department of Genome and Infectious Disease Control and Prevention, Yamaguchi University Graduate School of Medicine, Ube, Yamaguchi, Japan

关键词: Autoantibody;    Tumor-associated antigen;    Hepatocellular carcinoma;    Hepatitis C virus;    Maleimide;    Cysteine-tag;    Antibody profiling;    Antigen chip;   

Others  :  816837
DOI  :  10.1186/1477-5956-11-33

 received in 2012-09-13, accepted in 2013-07-14,  发布年份 2013

【 摘 要 】

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.

【 授权许可】

   
2013 Akada et al.; licensee BioMed Central Ltd.

【 图 表 】

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