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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

Effect of pre-treatment for autoantibody detection on chip. Shown are GFP fluorescence images from a non-pre-treated chip (upper row) and a SDS/2ME/heat pre-treated chip (lower row) incubated with serum no. 150. Shown are fluorescence microscopic images before (prior to step 1 in Figure 1) and after pre-treatment (after step 2) and following incubation with serum (step 10). The Alexa555 images taken at step 10 show the total amount of protein bound to the plate, and the Alexa633 images show the amount of serum autoantibodies present against the C-terminus of HSP70 (HC). Pre-treatment with SDS/2ME improved the Alexa555 signal and reduced the Alexa633 background. Abbreviations for proteins are same as Figure 2.
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Figure 3: Effect of pre-treatment for autoantibody detection on chip. Shown are GFP fluorescence images from a non-pre-treated chip (upper row) and a SDS/2ME/heat pre-treated chip (lower row) incubated with serum no. 150. Shown are fluorescence microscopic images before (prior to step 1 in Figure 1) and after pre-treatment (after step 2) and following incubation with serum (step 10). The Alexa555 images taken at step 10 show the total amount of protein bound to the plate, and the Alexa633 images show the amount of serum autoantibodies present against the C-terminus of HSP70 (HC). Pre-treatment with SDS/2ME improved the Alexa555 signal and reduced the Alexa633 background. Abbreviations for proteins are same as Figure 2.

Mentions: To check the effect of the heat-denaturing step in this protocol, GFP fluorescence was checked at each step by fluorescent microscopy. GFP fluorescence disappeared after treatment with SDS/2ME (Figure 3, lower line). In addition, Alexa555 fluorescence from the GFP tag increased for all protein spots. Alexa633 fluorescence from HSP70C remained the same, but the background was lower (Figure 3, lower line) than on non-denatured chips (Figure 3, upper line). Figure 4 shows Alexa555 fluorescence from the GFP tag and Alexa633 fluorescence from human IgG for each spot using denatured or non-denatured chip (closed or open circles, respectively). Detection of HSP70 using serum No. 193 showed that the denaturation (bold line in Figure 4) increased the linearity between Alexa555 and Alexa633 fluorescence compared to non-denatured chips (dotted line in Figure 4). It was observed that the GFP left-over fluorescence in some spots in the non-denatured chip could hardly be detected by the GFP antibody signal, and it caused lower detection in Alexa555 GFP cannels, but not Alexa633 autoantibody cannels. Carrying out denaturation as the first step mainly improved the quantitation of the GFP-tagged antigenic proteins by GFP antibody, and this in turn improved the specific quantitation of autoantibodies, which bind to the antigenic parts of the same proteins on the chips (Figures 3 and4).


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)

Effect of pre-treatment for autoantibody detection on chip. Shown are GFP fluorescence images from a non-pre-treated chip (upper row) and a SDS/2ME/heat pre-treated chip (lower row) incubated with serum no. 150. Shown are fluorescence microscopic images before (prior to step 1 in Figure 1) and after pre-treatment (after step 2) and following incubation with serum (step 10). The Alexa555 images taken at step 10 show the total amount of protein bound to the plate, and the Alexa633 images show the amount of serum autoantibodies present against the C-terminus of HSP70 (HC). Pre-treatment with SDS/2ME improved the Alexa555 signal and reduced the Alexa633 background. Abbreviations for proteins are same as Figure 2.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Effect of pre-treatment for autoantibody detection on chip. Shown are GFP fluorescence images from a non-pre-treated chip (upper row) and a SDS/2ME/heat pre-treated chip (lower row) incubated with serum no. 150. Shown are fluorescence microscopic images before (prior to step 1 in Figure 1) and after pre-treatment (after step 2) and following incubation with serum (step 10). The Alexa555 images taken at step 10 show the total amount of protein bound to the plate, and the Alexa633 images show the amount of serum autoantibodies present against the C-terminus of HSP70 (HC). Pre-treatment with SDS/2ME improved the Alexa555 signal and reduced the Alexa633 background. Abbreviations for proteins are same as Figure 2.
Mentions: To check the effect of the heat-denaturing step in this protocol, GFP fluorescence was checked at each step by fluorescent microscopy. GFP fluorescence disappeared after treatment with SDS/2ME (Figure 3, lower line). In addition, Alexa555 fluorescence from the GFP tag increased for all protein spots. Alexa633 fluorescence from HSP70C remained the same, but the background was lower (Figure 3, lower line) than on non-denatured chips (Figure 3, upper line). Figure 4 shows Alexa555 fluorescence from the GFP tag and Alexa633 fluorescence from human IgG for each spot using denatured or non-denatured chip (closed or open circles, respectively). Detection of HSP70 using serum No. 193 showed that the denaturation (bold line in Figure 4) increased the linearity between Alexa555 and Alexa633 fluorescence compared to non-denatured chips (dotted line in Figure 4). It was observed that the GFP left-over fluorescence in some spots in the non-denatured chip could hardly be detected by the GFP antibody signal, and it caused lower detection in Alexa555 GFP cannels, but not Alexa633 autoantibody cannels. Carrying out denaturation as the first step mainly improved the quantitation of the GFP-tagged antigenic proteins by GFP antibody, and this in turn improved the specific quantitation of autoantibodies, which bind to the antigenic parts of the same proteins on the chips (Figures 3 and4).

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