A new type of protein chip to detect hepatocellular carcinoma-related autoimmune antibodies in the sera of hepatitis C virus-positive patients.
A new type of protein chip to detect hepatocellular carcinoma-related autoimmune antibodies in the sera of hepatitis C virus-positive patients.
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DOI：
10.1186/1477-5956-11-33
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发表时间：
2013-07-19
期刊：
影响因子：
2
通讯作者：
Nakamura K
中科院分区：
文献类型：
--
作者：
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
本文报道一种新型的检测血清抗体的蛋白芯片。该芯片方法用于检测丙型肝炎病毒（HCV）感染者血清中肝细胞癌（HCC）相关自身抗体的原型。将5个半胱氨酸标记（Cys-tag）和绿色荧光蛋白（GFP）融合的重组热休克蛋白70（HSP 70）、超氧化物歧化酶2（SOD 2）和过氧化物氧还蛋白6（PRDX 6）点样并固定在马来酰亚胺掺入的类金刚石碳（DLC）基底上。稀释血清中的抗体在芯片上的每个点处被这些蛋白质捕获，并通过荧光缀合的抗人IgG可视化。用抗GFP小鼠IgG和荧光缀合的第二抗小鼠IgG检测每个斑点的总固定蛋白水平。两种荧光强度之间的比率用于定量每个血清样品中的自身抗体水平。在血清孵育之前，在变性剂和还原剂的溶液中对芯片进行热处理，改善了自身抗体检测。我们使用芯片测试了来自健康个体和HCC患者的血清样本。HSP 70自身抗体在HCV阳性的HCC患者血清中呈高水平，但在HCV阴性的血清中没有。这种蛋白质芯片系统可能具有有用的特性，可以捕获一组特定的抗体，用于预测HCV感染个体中特定癌症（如HCC）的发作。
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. 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. 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.
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