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Flow injection analysis using specific biochemical reaction on latex particles and liposome

利用乳胶颗粒和脂质体上的特定生化反应进行流动注射分析

基本信息

批准号：
02650541
负责人：
MORISHITA Fujio
金额：
$ 1.15万
依托单位：
KYOTO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1990
资助国家：
日本
起止时间：
1990 至 1991
项目状态：
已结题

项目摘要

A New Flow injection method (FIA) was proposed in order to improve the reaction efficiency, in which enzyme was immobilized on the surface of synthetic polymer latex. Stirene-acrylic acid copolymer latex synthesized was spherical and had homogeneous size of about 0.5 mm diameter. The enzymatic activity of enzyme-immobilized latex was dependent on the molar fraction of acrylic acid. Lactate dehydrogenase immobilized on the latex particles kept the activity higher than 50% of original one for two months or longer. The results obtained in FIA using latex suspended in the carrier solution were comparable to those in the conventional FIA using enzyme-immobilized reactors. Difficulties in recovering and reusing the used latex were the serious problem.Another new FIA method (flow injection liposome immunoassay : FILIA) was discussed, in which antigen-immobilized liposome having marker molecules in its inner space was used and a competitive immunoassay was carried out in a continuous flow system. The sufficient activity of antibody immobilized on the wall of a reactor was confirmed by observing satisfactory results in a flow injection enzyme immunoassay using POD-conjugated antigen. However, any explicit proof for the immunoreaction could not be found in FILIA because of steric hindrance by bulky liposome and wide wall of the reactor. By inserting a hydrophillic polymer spacer between antigen and liposome or between antibody and the reactor wall, a sigmoid response was obtained for the free antigen injected as a sample. But the response was insufficient for the determination of low concentration of the free analyte.

为了提高反应效率，提出了一种将酶固定在合成聚合物胶乳表面的流动注射方法(FIA)。合成的苯乙烯-丙烯酸共聚乳液为球形，粒径均一，直径约0.5 mm。酶固定化胶乳的酶活性与丙烯酸的摩尔分数有关。乳胶粒子固定化乳酸脱氢酶可使乳酸脱氢酶活性在2个月或更长时间内保持在原来的50%以上。使用悬浮在载体溶液中的胶乳进行流动注射分析的结果与使用酶固定化反应器的传统流动注射分析结果相当。对另一种新的流动注射免疫分析方法(FIIA)进行了探讨，该方法采用内含标记分子的抗原固定化脂质体，并在连续流动系统中进行竞争免疫分析。在使用POD标记抗原的流动注射酶免疫分析中观察到令人满意的结果，证实了固定在反应器壁上的抗体具有足够的活性。然而，由于庞大的脂质体和较宽的反应器壁的空间位阻，在Filia中找不到任何明确的免疫反应证据。通过在抗原和脂质体之间或抗体和反应器壁之间插入亲水性聚合物间隔物，可以获得作为样本注射的游离抗原的S型反应。但对于低浓度的游离分析物的测定，反应不够灵敏。