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Basic Study on the Development of Novel and Ultrasensitive Immunoassay for Biological Substances

生物物质新型超灵敏免疫分析技术发展的基础研究

基本信息

批准号：
01580168
负责人：
ISHIKAWA Eiji
金额：
$ 1.47万
依托单位：
Miyazaki Medical College
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1989
资助国家：
日本
起止时间：
1989 至 1990
项目状态：
已结题

项目摘要

Ultrasensitive enzyme immunoassay methods not only for antigens but also for antibodies and haptens have been developed. These methods are based on a noncompetitive type of assay using solid phase rather than a competitive one. One of the greatest obstacles limiting the sensitivity of noncompetitive immunoassay methods using solid phase is the nonspecific binding of labeled reactants to solid phase. A novel method (immune complex transfer immunoassay method) to overcome this difficulty has been developed. In the initially developed method, antibodies in test serum were reacted with dinitrophenylated biotinylated antigen. The complex formed was trapped onto (anti-dinitrophenyl group) IgG-coated solid phase. The solid phase was washed to eliminate nonspecific immunoglobulins. The complex was eluted from the solid phase with dinitrophenyl-L-lysine, again trapped onto avidin (streptavidin) -coated solid phase and finally measured by reaction with (anti-immunoglobulin) Fab' -enzyme conjugat … More e. Namely, the complex of antibodies and dinitrophenylated biotinylated antigen was transferred from one solid to another, effectively eliminating nonspecific immunoglobulins nonspecifically adsorbed onto the first solid phase. By this method with and without further modifications, the sensitivity for antibodies in serum has been considerably improved, and applications were made to measure low levels of antibodies against human T-cell leukemia virus and human immunodeficiency virus, which are not detectable by conventional methods. The same principle of immune complex transfer has been applied to the detection of antigens, and one milliattomole (600 molecules) of human ferritin has been detected. Ultrasensitive, noncompetitive immunoassay methods to measure attomole amounts of haptens with amino groups have also been developed. Haptens, especially peptides, were biotinylated by using N-hydroxysuccinimidobiotin, and measured by using enzyme-labeled anti-peptide Feb' and streptavidin- coated solid phase (hetero-two-site enzyme immunoassay). Less

超灵敏的酶免疫分析方法不仅适用于抗原，而且适用于抗体和半抗原。这些方法是基于使用固相的非竞争性类型的测定，而不是竞争性的。限制使用固相的非竞争性免疫测定方法的灵敏度的最大障碍之一是标记反应物与固相的非特异性结合。一种新的方法（免疫复合物转移免疫测定法），以克服这一困难已被开发。在最初开发的方法中，测试血清中的抗体与二硝基苯化生物素化抗原反应。将形成的复合物捕获到（抗二硝基苯基）IgG包被的固相上。洗涤固相以除去非特异性免疫球蛋白。用二硝基苯基-L-赖氨酸从固相上洗脱复合物，再次捕获到抗生物素蛋白（链霉抗生物素蛋白）包被的固相上，最后通过与（抗免疫球蛋白）Fab' -酶缀合物反应进行测量。 ...更多信息 e.即，抗体和二硝基苯基化生物素化抗原的复合物从一种固体转移到另一种固体，有效地消除了非特异性吸附在第一固相上的非特异性免疫球蛋白。通过该方法，无论是否进一步修改，血清中抗体的灵敏度都得到了显著提高，并应用于测量常规方法检测不到的抗人T细胞白血病病毒和人免疫缺陷病毒的低水平抗体。免疫复合物转移的相同原理已被应用于抗原的检测，并已检测到1毫阿摩尔（600分子）的人铁蛋白。超灵敏，非竞争性免疫测定方法来测量阿托摩尔量的半抗原与氨基也已开发。半抗原，特别是肽，通过使用N-羟基琥珀酰亚胺生物素进行生物素化，并通过使用酶标记的抗肽Feb'和链霉亲和素包被的固相（异源双位点酶免疫测定）进行测量。少