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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

The first-designed HCV-related HCC patient autoantigen protein chip. (A) The five recombinant proteins confirmed by SDS-PAGE. The proteins were 6×His-GFP-5×Cys (G), 6×His-GFP-HSP70-C-terminal domain-5×Cys (HC), 6×His-GFP-HSP70-N-terminal domain-5×Cys (HN), 6× His-GFP-SOD2-5×Cys (S), and 6×His-GFP-PRDX6-5×Cys (P). (B-G) The first-designed protein chip and its certification. (B) GFP fluorescence from the protein chip as detected by fluorescence microscopy. The proteins (128 fmol per spot) were covalently cross-linked to maleimide-incorporated diamond-like carbon chip. Spots were 200 μm in diameter. Abbreviations for proteins are same as in A. (C-G) Full color fluorescence images of immobilized protein by ProscanArray. The protein chips were detected after denaturation and incubation of primary anti-His antibody (C), anti-GFP (D), anti-HSP70 (E), anti-SOD2 (F), and anti-PRDX6 (G), visualized using fluorescent secondary antibodies.
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Figure 2: The first-designed HCV-related HCC patient autoantigen protein chip. (A) The five recombinant proteins confirmed by SDS-PAGE. The proteins were 6×His-GFP-5×Cys (G), 6×His-GFP-HSP70-C-terminal domain-5×Cys (HC), 6×His-GFP-HSP70-N-terminal domain-5×Cys (HN), 6× His-GFP-SOD2-5×Cys (S), and 6×His-GFP-PRDX6-5×Cys (P). (B-G) The first-designed protein chip and its certification. (B) GFP fluorescence from the protein chip as detected by fluorescence microscopy. The proteins (128 fmol per spot) were covalently cross-linked to maleimide-incorporated diamond-like carbon chip. Spots were 200 μm in diameter. Abbreviations for proteins are same as in A. (C-G) Full color fluorescence images of immobilized protein by ProscanArray. The protein chips were detected after denaturation and incubation of primary anti-His antibody (C), anti-GFP (D), anti-HSP70 (E), anti-SOD2 (F), and anti-PRDX6 (G), visualized using fluorescent secondary antibodies.

Mentions: The expression of recombinant GFP-tagged antigenic proteins were confirmed by SDS-PAGE after purification using Ni-affinity gel (Figure 2A). The proteins were printed on 3 × 3 mm maleimide-coated substrate chips as 10 nL, 200 μm spots containing 128 fmol protein each. The amount of antigenic proteins on each chip was verified by measuring GFP fluorescence with a fluorescent microscope (Figure 2B), and representative chips were validated by fluorescent immunodetection using specific antibodies against 6×His (Figure 2C), GFP (Figure 2D), HSP70C (Figure 2E), SOD2 (Figure 2F), or PRDX6 (Figure 2G), visualized fluorescent secondary antibodies. HSP70N (lane HN in Figure 2B) was not detected by polyclonal antibodies against HSP70 on this first-designed chip (Figure 2E), suggesting that the ATPase domain of HSP70, a functionally important conserved domain, could not be recognized by commercially available antibodies on this chip.


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)

The first-designed HCV-related HCC patient autoantigen protein chip. (A) The five recombinant proteins confirmed by SDS-PAGE. The proteins were 6×His-GFP-5×Cys (G), 6×His-GFP-HSP70-C-terminal domain-5×Cys (HC), 6×His-GFP-HSP70-N-terminal domain-5×Cys (HN), 6× His-GFP-SOD2-5×Cys (S), and 6×His-GFP-PRDX6-5×Cys (P). (B-G) The first-designed protein chip and its certification. (B) GFP fluorescence from the protein chip as detected by fluorescence microscopy. The proteins (128 fmol per spot) were covalently cross-linked to maleimide-incorporated diamond-like carbon chip. Spots were 200 μm in diameter. Abbreviations for proteins are same as in A. (C-G) Full color fluorescence images of immobilized protein by ProscanArray. The protein chips were detected after denaturation and incubation of primary anti-His antibody (C), anti-GFP (D), anti-HSP70 (E), anti-SOD2 (F), and anti-PRDX6 (G), visualized using fluorescent secondary antibodies.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: The first-designed HCV-related HCC patient autoantigen protein chip. (A) The five recombinant proteins confirmed by SDS-PAGE. The proteins were 6×His-GFP-5×Cys (G), 6×His-GFP-HSP70-C-terminal domain-5×Cys (HC), 6×His-GFP-HSP70-N-terminal domain-5×Cys (HN), 6× His-GFP-SOD2-5×Cys (S), and 6×His-GFP-PRDX6-5×Cys (P). (B-G) The first-designed protein chip and its certification. (B) GFP fluorescence from the protein chip as detected by fluorescence microscopy. The proteins (128 fmol per spot) were covalently cross-linked to maleimide-incorporated diamond-like carbon chip. Spots were 200 μm in diameter. Abbreviations for proteins are same as in A. (C-G) Full color fluorescence images of immobilized protein by ProscanArray. The protein chips were detected after denaturation and incubation of primary anti-His antibody (C), anti-GFP (D), anti-HSP70 (E), anti-SOD2 (F), and anti-PRDX6 (G), visualized using fluorescent secondary antibodies.
Mentions: The expression of recombinant GFP-tagged antigenic proteins were confirmed by SDS-PAGE after purification using Ni-affinity gel (Figure 2A). The proteins were printed on 3 × 3 mm maleimide-coated substrate chips as 10 nL, 200 μm spots containing 128 fmol protein each. The amount of antigenic proteins on each chip was verified by measuring GFP fluorescence with a fluorescent microscope (Figure 2B), and representative chips were validated by fluorescent immunodetection using specific antibodies against 6×His (Figure 2C), GFP (Figure 2D), HSP70C (Figure 2E), SOD2 (Figure 2F), or PRDX6 (Figure 2G), visualized fluorescent secondary antibodies. HSP70N (lane HN in Figure 2B) was not detected by polyclonal antibodies against HSP70 on this first-designed chip (Figure 2E), suggesting that the ATPase domain of HSP70, a functionally important conserved domain, could not be recognized by commercially available antibodies on this chip.

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