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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

Autoantibody detection on chip. (A) Autoantibody detection procedures. Protein chips were immersed in SDS/2ME sample buffer and heated at 95°C for 5 min (Immunodetection step 1) and washed (step 2). The chips were blocked with BSA (step 3), incubated with diluted serum (step 4) or mouse anti-GFP monoclonal IgG (step 5), washed (step 6), incubated with AlexaFluora 633 (Alexa633)-conjugated anti-human IgG to quantify bound serum autoantibodies (step 7), incubated with AlexaFluore 555 (Alexa555)-conjugated anti-mouse IgG antibody to quantify the amount of GFP-tagged protein (step 8), and washed (step 9). The chips were examined for Alexa555 and Alexa633 fluorescence of the same spot in the same chip and Alexa633 fluorescence of each spot was normalized using Alexa555 fluorescence of the same spot (step 10).(B-C) Schema of predicted protein chip surface before step 1 (B) and after step 9 (C). 6H: 6xHis-tag, 5C: 5xCys-tag.
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Figure 1: Autoantibody detection on chip. (A) Autoantibody detection procedures. Protein chips were immersed in SDS/2ME sample buffer and heated at 95°C for 5 min (Immunodetection step 1) and washed (step 2). The chips were blocked with BSA (step 3), incubated with diluted serum (step 4) or mouse anti-GFP monoclonal IgG (step 5), washed (step 6), incubated with AlexaFluora 633 (Alexa633)-conjugated anti-human IgG to quantify bound serum autoantibodies (step 7), incubated with AlexaFluore 555 (Alexa555)-conjugated anti-mouse IgG antibody to quantify the amount of GFP-tagged protein (step 8), and washed (step 9). The chips were examined for Alexa555 and Alexa633 fluorescence of the same spot in the same chip and Alexa633 fluorescence of each spot was normalized using Alexa555 fluorescence of the same spot (step 10).(B-C) Schema of predicted protein chip surface before step 1 (B) and after step 9 (C). 6H: 6xHis-tag, 5C: 5xCys-tag.

Mentions: Here, we describe a new chip-based system for the detection of multiple autoantibodies in sera from HCC patients. Model antigenic proteins to test this prototype chip system were HSP70, SOD2 and PRDX2. These three HCV-related HCC TAAs were selected from a PROTEOMEX study using the sera of Japanese HCC patients by our group, and have also been reported by others[17,18]. Using the characteristic properties of cysteine[22], cysteine-tagged recombinant antigenic proteins were immobilized on maleimide-coated diamond-like carbon (DLC)-coated silicon chips by the covalent bond between cysteine sulfhydryl and maleimide residues[23]. Theoretically, cysteine-tagged proteins on this chip were floating in surrounding solution attached to the chip substrate only at the C-terminus (Figure 1B and C), which is different from proteins bound on conventional nitrocellulose membrane via hydrophobic residues throughout the proteins. This small, hard, DLC silicon chip is stable to heat, is easy to hand for the washing process, provides sensitive detection of antibodies in low backgrounds, and is capable of high-throughput screening of sera. We constructed a procedure for the detection and quantification of antibodies bound on a cysteine-tagged protein array via maleimide residues on a DLC silicon chip. HSP70 antibodies were highly detectable in this protein chip system among HCC patient and HCV infected individual.


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)

Autoantibody detection on chip. (A) Autoantibody detection procedures. Protein chips were immersed in SDS/2ME sample buffer and heated at 95°C for 5 min (Immunodetection step 1) and washed (step 2). The chips were blocked with BSA (step 3), incubated with diluted serum (step 4) or mouse anti-GFP monoclonal IgG (step 5), washed (step 6), incubated with AlexaFluora 633 (Alexa633)-conjugated anti-human IgG to quantify bound serum autoantibodies (step 7), incubated with AlexaFluore 555 (Alexa555)-conjugated anti-mouse IgG antibody to quantify the amount of GFP-tagged protein (step 8), and washed (step 9). The chips were examined for Alexa555 and Alexa633 fluorescence of the same spot in the same chip and Alexa633 fluorescence of each spot was normalized using Alexa555 fluorescence of the same spot (step 10).(B-C) Schema of predicted protein chip surface before step 1 (B) and after step 9 (C). 6H: 6xHis-tag, 5C: 5xCys-tag.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
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Figure 1: Autoantibody detection on chip. (A) Autoantibody detection procedures. Protein chips were immersed in SDS/2ME sample buffer and heated at 95°C for 5 min (Immunodetection step 1) and washed (step 2). The chips were blocked with BSA (step 3), incubated with diluted serum (step 4) or mouse anti-GFP monoclonal IgG (step 5), washed (step 6), incubated with AlexaFluora 633 (Alexa633)-conjugated anti-human IgG to quantify bound serum autoantibodies (step 7), incubated with AlexaFluore 555 (Alexa555)-conjugated anti-mouse IgG antibody to quantify the amount of GFP-tagged protein (step 8), and washed (step 9). The chips were examined for Alexa555 and Alexa633 fluorescence of the same spot in the same chip and Alexa633 fluorescence of each spot was normalized using Alexa555 fluorescence of the same spot (step 10).(B-C) Schema of predicted protein chip surface before step 1 (B) and after step 9 (C). 6H: 6xHis-tag, 5C: 5xCys-tag.
Mentions: Here, we describe a new chip-based system for the detection of multiple autoantibodies in sera from HCC patients. Model antigenic proteins to test this prototype chip system were HSP70, SOD2 and PRDX2. These three HCV-related HCC TAAs were selected from a PROTEOMEX study using the sera of Japanese HCC patients by our group, and have also been reported by others[17,18]. Using the characteristic properties of cysteine[22], cysteine-tagged recombinant antigenic proteins were immobilized on maleimide-coated diamond-like carbon (DLC)-coated silicon chips by the covalent bond between cysteine sulfhydryl and maleimide residues[23]. Theoretically, cysteine-tagged proteins on this chip were floating in surrounding solution attached to the chip substrate only at the C-terminus (Figure 1B and C), which is different from proteins bound on conventional nitrocellulose membrane via hydrophobic residues throughout the proteins. This small, hard, DLC silicon chip is stable to heat, is easy to hand for the washing process, provides sensitive detection of antibodies in low backgrounds, and is capable of high-throughput screening of sera. We constructed a procedure for the detection and quantification of antibodies bound on a cysteine-tagged protein array via maleimide residues on a DLC silicon chip. HSP70 antibodies were highly detectable in this protein chip system among HCC patient and HCV infected individual.

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