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

Akada J, Kamei S, Ito A, Ito M, Kitagawa T, Furumoto H, Kato Y, Tamesa M, Takashima M, Shirai M, Yamano H, Oka M, Kuramitsu Y, Nakamura K - Proteome Sci (2013)

Bottom Line: 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 ratio between the two fluorescence intensities was used to quantify autoantibody levels in each serum sample.We tested serum samples from healthy individuals and HCC patients using the chips.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry and Functional Proteomics, Yamaguchi University Graduate School of Medicine, 1-1-1, Minami-kogushi, Ube, Yamaguchi 755-8505, Japan. nakamura@yamaguchi-u.ac.jp.

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.

No MeSH data available.


Related in: MedlinePlus

Quantification of autoantibodies in human serum samples by the first HCC antigen protein chip. Sera from 6 healthy individuals (Healthy), 8 HCV-/HCC- subjects, 13 HCV+/HCC+, and 4 HCV+/HCC- subjects were tested using the first designed protein chip. Each column shows the average with standard error bar of the ratio amount (Alexa633/555) of autoantibodies detected for five or six spots of GFP, GFP-HSP70-C-terminus, GFP-HSP70-N-terminus, GFP-SOD2, GFP-PRDX6 (from left to right) using one chip for each serum (showing examples in the third row of the box).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3750938&req=5

Figure 5: Quantification of autoantibodies in human serum samples by the first HCC antigen protein chip. Sera from 6 healthy individuals (Healthy), 8 HCV-/HCC- subjects, 13 HCV+/HCC+, and 4 HCV+/HCC- subjects were tested using the first designed protein chip. Each column shows the average with standard error bar of the ratio amount (Alexa633/555) of autoantibodies detected for five or six spots of GFP, GFP-HSP70-C-terminus, GFP-HSP70-N-terminus, GFP-SOD2, GFP-PRDX6 (from left to right) using one chip for each serum (showing examples in the third row of the box).

Mentions: Using these protein chips and the improved detection protocol, 31 sera from four test groups were tested, including: 6 healthy individuals (“Healthy” in Figure 5), 8 subjects that were negative for both HCV and HCC, but having some other disease (HCV-/HCC-), 13 subjects that were positive for HCV and HCC (HCV+/HCC+), and 4 subjects that were positive for HCV, but negative for HCC (HCV+/HCC-). Chips incubated with sera from healthy individuals and HCV-/HCC- subjects had low antibody levels as indicated by low Alexa633 fluorescence despite strong Alexa555 fluorescence (indicating effective immobilization of GFP-tagged protein). The Alexa633/555 ratios of these negative control chips were below 0.8 (Figure 5, upper row). Chips incubated in sera from HCV+/HCC + subjects had high Alexa633 florescence for some antigen spots. The level of Alexa555 fluorescence was constant for all spots, and the Alexa633/555 ratio for the positive spots was very high (above 0.8) in four sera from HCV+/HCC + subjects (Serum No. 150, 193, 222c, 419, middle row in Figure 5), and one serum from HCV+/HCC- group (serum No. 259, bottom row in Figure 5). Thus, the protein chips could detect elevated autoantibody levels in several sera from 13 HCV+/HCC + and HCV+/HCC- subjects (Figure 5).


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

Akada J, Kamei S, Ito A, Ito M, Kitagawa T, Furumoto H, Kato Y, Tamesa M, Takashima M, Shirai M, Yamano H, Oka M, Kuramitsu Y, Nakamura K - Proteome Sci (2013)

Quantification of autoantibodies in human serum samples by the first HCC antigen protein chip. Sera from 6 healthy individuals (Healthy), 8 HCV-/HCC- subjects, 13 HCV+/HCC+, and 4 HCV+/HCC- subjects were tested using the first designed protein chip. Each column shows the average with standard error bar of the ratio amount (Alexa633/555) of autoantibodies detected for five or six spots of GFP, GFP-HSP70-C-terminus, GFP-HSP70-N-terminus, GFP-SOD2, GFP-PRDX6 (from left to right) using one chip for each serum (showing examples in the third row of the box).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3750938&req=5

Figure 5: Quantification of autoantibodies in human serum samples by the first HCC antigen protein chip. Sera from 6 healthy individuals (Healthy), 8 HCV-/HCC- subjects, 13 HCV+/HCC+, and 4 HCV+/HCC- subjects were tested using the first designed protein chip. Each column shows the average with standard error bar of the ratio amount (Alexa633/555) of autoantibodies detected for five or six spots of GFP, GFP-HSP70-C-terminus, GFP-HSP70-N-terminus, GFP-SOD2, GFP-PRDX6 (from left to right) using one chip for each serum (showing examples in the third row of the box).
Mentions: Using these protein chips and the improved detection protocol, 31 sera from four test groups were tested, including: 6 healthy individuals (“Healthy” in Figure 5), 8 subjects that were negative for both HCV and HCC, but having some other disease (HCV-/HCC-), 13 subjects that were positive for HCV and HCC (HCV+/HCC+), and 4 subjects that were positive for HCV, but negative for HCC (HCV+/HCC-). Chips incubated with sera from healthy individuals and HCV-/HCC- subjects had low antibody levels as indicated by low Alexa633 fluorescence despite strong Alexa555 fluorescence (indicating effective immobilization of GFP-tagged protein). The Alexa633/555 ratios of these negative control chips were below 0.8 (Figure 5, upper row). Chips incubated in sera from HCV+/HCC + subjects had high Alexa633 florescence for some antigen spots. The level of Alexa555 fluorescence was constant for all spots, and the Alexa633/555 ratio for the positive spots was very high (above 0.8) in four sera from HCV+/HCC + subjects (Serum No. 150, 193, 222c, 419, middle row in Figure 5), and one serum from HCV+/HCC- group (serum No. 259, bottom row in Figure 5). Thus, the protein chips could detect elevated autoantibody levels in several sera from 13 HCV+/HCC + and HCV+/HCC- subjects (Figure 5).

Bottom Line: 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 ratio between the two fluorescence intensities was used to quantify autoantibody levels in each serum sample.We tested serum samples from healthy individuals and HCC patients using the chips.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry and Functional Proteomics, Yamaguchi University Graduate School of Medicine, 1-1-1, Minami-kogushi, Ube, Yamaguchi 755-8505, Japan. nakamura@yamaguchi-u.ac.jp.

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.

No MeSH data available.


Related in: MedlinePlus