Rapid Determination of Blood Coagulation Factor XIII Activity Using Protein Arrays for Serodiagnosis of Human Plasma
Rapid Determination of Blood Coagulation Factor XIII Activity Using Protein Arrays for Serodiagnosis of Human Plasma
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DOI：
10.1021/ac1032275
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发表时间：
2011-03-15
影响因子：
7.4
通讯作者：
Ha, Kwon-Soo
中科院分区：
文献类型：
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作者：
Kwon, Mi-Hye;Kong, Deok-Hoon;Ha, Kwon-Soo
我们开发了一种新的芯片上检测使用蛋白质阵列定量和快速分析人血浆中的凝血因子XIII（FXIII）的活性。FXIII通过凝血酶和Ca 2+的协同作用被激活，并且在止血、血管生成和伤口愈合中起重要作用。我们通过将纤维蛋白原固定到井型阵列的3-氨丙基三甲氧基硅烷层上来制造蛋白质阵列，并通过用Cy 3缀合的链霉亲和素分析生物素化的纤维蛋白原来确定FM活性。我们确定了用于芯片活性测定的Ca 2+、凝血酶和S-（生物素酰胺基）戊胺（BAPA）的最佳浓度，检测限为0.01 Lowey U/mL（9.9 pM）。使用芯片活性测定，肝细胞癌患者（n = 24），而不是肝炎（n = 24）或肝硬化患者（n = 41），具有比正常个体（n = 41）显著更低的FXIII活性（p < 0.001），表明FXIII活性是肝细胞癌的可能的诊断标志物。此外，我们已经成功地使用这种活性测定，以揭示个体差异（37- 57%，n = 65）的抑制率的FXIII活性的异烟肼，一线抗结核药物。因此，我们优化的芯片上FXIII活性测定提供了一种定量和高通量的方法来研究FXIII在人类疾病中的作用。此外，它具有很强的潜力，可用于FXIII相关的个性化药物。
We developed a novel on-chip assay using protein arrays for quantitative and rapid analysis of blood coagulation factor XIII (FXIII) activity in human plasma. FXIII is activated by concerted action of thrombin and Ca2+ and plays essential roles in hemostasis, angiogenesis, and wound healing. We fabricated protein arrays by immobilizing fibrinogen onto the 3-aminopropyltrimethoxysilane layer of well-type arrays and determined FM activity by analyzing biotinylated fibrinogen with Cy3-conjugated streptavidin. We determined optimal concentrations of Ca2+, thrombin, and S-(biotinamido)pentylamine (BAPA) for the on-chip activity assay, and the detection limit was 0.01 Lowey U/mL (9.9 pM). Using the on-chip activity assay, hepatocellular carcinoma patients (n = 24), but not hepatitis (n = 24) or liver cirrhosis patients (n = 41), had significantly lower FXIII activities (p < 0.001) than normal individuals (n = 41), indicating that FXIII activity is a possible diagnostic marker for hepatocellular carcinoma. In addition, we have successfully used this activity assay to reveal individual variations (37-57%, n = 65) in the inhibition rate of FXIII activity by isoniazid, the first-line antituberculosis agent. Thus, our optimized on-chip FXIII activity assay provides a quantitative and high-throughput approach to investigating the role(s) of FXIII in human diseases. Moreover, it has a strong potential to be applied toward FXIII-related personalized medicines.