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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

Autoantibodies detection by the third-designed protein chip. (A-H) Full color autoantibody-detected images by ×10 diluted sera and anti-human IgG-Alexa633. The representative chip images from four serum samples tested in Figure 5, No. 244 (A), No.150 (B), No. 158 (C), No. 164 (D) and three new HCV+/HCC + serum samples, No. 595 (E), No. 657 (F), 679 (G), and a HCV+/HCC- serum sample, No. 643 (H), are shown. (I) Quantitative results of the third-designed chip. Others are shown same as Figure 6.
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Figure 7: Autoantibodies detection by the third-designed protein chip. (A-H) Full color autoantibody-detected images by ×10 diluted sera and anti-human IgG-Alexa633. The representative chip images from four serum samples tested in Figure 5, No. 244 (A), No.150 (B), No. 158 (C), No. 164 (D) and three new HCV+/HCC + serum samples, No. 595 (E), No. 657 (F), 679 (G), and a HCV+/HCC- serum sample, No. 643 (H), are shown. (I) Quantitative results of the third-designed chip. Others are shown same as Figure 6.

Mentions: Furthermore, the detection procedure was modified by adjusting the fold of serum dilution (×10) and GFP antibody dilution (x5000) using the third-designed chip. Re-detection of four tested sera in Figure 5 is shown in Figure 7A-D. Negative sera in Figure 5 (serum No. 244 of HCV-/HCC- patient having some other disease) showed the same negative detection (Figure 7A), serum No. 150 and 164 increased the HSP70C detection (Figure 7B and7D), and No. 158 was slightly positive for HSP70N (Figure 7C). Four newly tested representative HSP70C positive sera, three HCV/HCC (+/+) sera and an HCV/HCC (+/−) serum, are shown in Figure 7E-H. Quantitative analysis of autoantibodies on the third chip showed increased detection of autoantibodies and statistical significance compared with GFP non-specific detection (Figure 7I). SOD2 detection was not significant in the third chip (No. 150 in Figures 6M and7I). PRDX6 detection was not significant except for one serum (serum 643, Figure 7H and I). It became apparent that HSP70C auto-antibody was highly significant among HCV-positive individual and/or HCC patient sera tested.


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)

Autoantibodies detection by the third-designed protein chip. (A-H) Full color autoantibody-detected images by ×10 diluted sera and anti-human IgG-Alexa633. The representative chip images from four serum samples tested in Figure 5, No. 244 (A), No.150 (B), No. 158 (C), No. 164 (D) and three new HCV+/HCC + serum samples, No. 595 (E), No. 657 (F), 679 (G), and a HCV+/HCC- serum sample, No. 643 (H), are shown. (I) Quantitative results of the third-designed chip. Others are shown same as Figure 6.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 7: Autoantibodies detection by the third-designed protein chip. (A-H) Full color autoantibody-detected images by ×10 diluted sera and anti-human IgG-Alexa633. The representative chip images from four serum samples tested in Figure 5, No. 244 (A), No.150 (B), No. 158 (C), No. 164 (D) and three new HCV+/HCC + serum samples, No. 595 (E), No. 657 (F), 679 (G), and a HCV+/HCC- serum sample, No. 643 (H), are shown. (I) Quantitative results of the third-designed chip. Others are shown same as Figure 6.
Mentions: Furthermore, the detection procedure was modified by adjusting the fold of serum dilution (×10) and GFP antibody dilution (x5000) using the third-designed chip. Re-detection of four tested sera in Figure 5 is shown in Figure 7A-D. Negative sera in Figure 5 (serum No. 244 of HCV-/HCC- patient having some other disease) showed the same negative detection (Figure 7A), serum No. 150 and 164 increased the HSP70C detection (Figure 7B and7D), and No. 158 was slightly positive for HSP70N (Figure 7C). Four newly tested representative HSP70C positive sera, three HCV/HCC (+/+) sera and an HCV/HCC (+/−) serum, are shown in Figure 7E-H. Quantitative analysis of autoantibodies on the third chip showed increased detection of autoantibodies and statistical significance compared with GFP non-specific detection (Figure 7I). SOD2 detection was not significant in the third chip (No. 150 in Figures 6M and7I). PRDX6 detection was not significant except for one serum (serum 643, Figure 7H and I). It became apparent that HSP70C auto-antibody was highly significant among HCV-positive individual and/or HCC patient sera tested.

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