Accurate quantitation of salivary and pancreatic amylase activities in human plasma by microchip electrophoretic separation of the substrates and hydrolysates coupled with immunoinhibition
Accurate quantitation of salivary and pancreatic amylase activities in human plasma by microchip electrophoretic separation of the substrates and hydrolysates coupled with immunoinhibition
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DOI：
10.1002/elps.200700688
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发表时间：
2008-05-01
期刊：
影响因子：
2.9
通讯作者：
Baba, Yoshinobu
中科院分区：
文献类型：
--
作者：
Maeda, Eiki;Kataoka, Masatoshi;Baba, Yoshinobu
建立了一种高效的人血浆α-淀粉酶同功酶活性芯片电泳检测系统。结合微芯片电泳底物和水解产物分离和免疫抑制方法的同工酶活性的分化，使用抗人唾液淀粉酶（S-AMY）单克隆抗体允许高选择性测定淀粉酶同工酶（S-AMY和胰淀粉酶（P-AMY））的活动，即使在复杂的基质，如粗血浆样品。我们使用8-氨基芘-1，3，6-三磺酸（APTS）标记的麦芽六糖（G6）作为底物。人血浆样品中的淀粉酶将APTS-G6水解为APTS-麦芽三糖（G3）和G3，其在微芯片电泳上测量为APTS-G3的荧光强度。双对数曲线显示淀粉酶活性与荧光强度在5-500 U/L范围内呈线性关系，检出限为4.38 U/L。用本法测定了13个受试者的淀粉酶活性（r（2）= 0.9995，p < 0.01），并分别与以硝基苯寡糖为底物的常规方法测定的结果进行了比较。在简单线性回归分析中，每种方法均观察到良好的相关性（均p < 0.01）。总淀粉酶和P-AMY的日内重复性分别为2.98- 6.27%和3.83- 6.39%，日间重复性分别为2.88- 5.66%和3.64- 5.63%。该系统能以较高的灵敏度和准确度测定人血浆中淀粉酶同工酶的活性，可应用于临床诊断。
A high-performance determination system for alpha-amylase isoenzyme activities in human plasma involving microchip electrophoresis with a plastic chip was developed. The combination of microchip electrophoresis for substrate and hydrolysate separation and an immunoinhibition method for the differentiation of isoenzyme activities using antihuman salivary amylase (S-AMY) mAb allowed the highly selective determination of amylase isoenzyme (S-AMY and pancreatic amylase (P-AMY)) activities even in a complex matrix such as a crude plasma sample. We used 8-aminopyrene-1,3,6-trisulfonic acid (APTS)-labeled maltohexaose (G6) as a substrate. Amylase in a human plasma sample hydrolyzed APTS-G6 into APTS-maltotriose (G3) and G3, which was measured as the fluorescence intensity of APTS-G3 on microchip electrophoresis. A double logarithm plot revealed a linear relationship between amylase activity and fluorescence intensity in the range of 5-500 U/L of and the LOD was 4.38 U/L. Amylase activities in 13 amylase activity (r(2) = 0.9995 p < 0.01), subjects determined by the present method were compared with the results obtained by conventional methods with nitrophenylated oligosaccharides as substrates, respectively. Good correlations were observed for each method on simple linear regression analysis (both p < 0.01). The reproducibilities of within-days for total amylase and P-AMY were 2.98-6.27 and 3.83-6.39%, respectively, and these between-days were 2.88-5.66 and 3.64-5.63%, respectively. This system enables us to determine amylase isoenzyme activities in human plasma with high sensitivity and accuracy, and thus will be applicable to clinical diagnosis.