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Development of Automatic Method for Determining Enzyme Activities in Serum Using Bioreactor System

利用生物反应器系统自动测定血清酶活性方法的开发

基本信息

批准号：
62870108
负责人：
MURACHI Takashi
金额：
$ 10.24万
依托单位：
Kyoto University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Developmental Scientific Research
财政年份：
1987
资助国家：
日本
起止时间：
1987 至 1988
项目状态：
已结题

项目摘要

Enzyme activities in serum have been determined widely using an automated analyzer in clinical analysis. Recently, the automated analysis of nonprotein organic compounds in serum by a bioreactor with immobilized wnzyme in column form has been put to practical use. For the purpose of applying the bioreactor to the determination of enzyme activities in serum and increasing the range of its use, we studied as follows, and obtained a lot of results.(1) Study of the incubation system for the reaction of enzyme. Since the system putting a reaction coil for the reaction of enzyme as a bypath in the flow injection analysis (FIA) system was simple and easier to remove sample blanks, we chose the system.(2) Development of the automatic analysis of enzyme activities in serum using a bioreactor. 1. Determination of lactate dehydrogenase (LDH) activities in serum. LDH reaction was taken place using pyruvate as the substrate in the reaction coil put as a bypath in the FIA system, and lactate produced by LDH reaction was oxidized to pyruvate and hydrogen peroxide by the lactate oxidase bioreactor. Hydrogen peroxide produced was determined by chemiluminescence method. Endogenous lactate was removed by lactate oxidase・catalase precolumn. The results obtained were excellent, and correlated satisfactorily with those by a Hitachi Model 726 automatic analyzer. The present method did not need any sample blank.2. Determination of amylase activity in serum. Maltopentaose was used as the substrate for amylase reaction. Maltose produced by amylase reaction was catalyzed to hydrogen peroxide by the maltose phosphorylase・pyranose oxidase bioreactor. Glucokinase column was used to remove endogenous glucose in serum. The results obtained was good.

血清酶活性的测定已广泛应用于临床分析。近年来，固定化酶柱状生物反应器对血清中非蛋白质有机化合物的自动分析已投入实际应用。为了将生物反应器应用于血清酶活性的测定，扩大其使用范围，我们进行了以下研究，并获得了很多结果。(1)酶反应培养体系的研究。由于在流动注射分析（FIA）系统中采用酶反应线圈作为旁路的系统简单且易于去除样品空白，因此我们选择了该系统。(2)利用生物反应器自动分析血清酶活性的发展。1. 血清乳酸脱氢酶（LDH）活性测定。以丙酮酸盐为底物，在FIA体系中作为旁路的反应线圈中进行乳酸脱氢反应，乳酸氧化酶生物反应器将乳酸脱氢反应产生的乳酸氧化为丙酮酸盐和过氧化氢。用化学发光法测定过氧化氢的生成。内源性乳酸通过乳酸氧化酶·过氧化氢酶柱前去除。所得结果良好，与日立726型自动分析仪的测定结果吻合良好。本方法不需要任何样本空白。血清淀粉酶活性测定。以麦芽糖糖为底物进行淀粉酶反应。采用麦芽糖磷酸化酶·吡喃糖氧化酶生物反应器将淀粉酶反应产生的麦芽糖催化生成过氧化氢。葡萄糖激酶柱去除血清中的内源性葡萄糖。所得结果良好。