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

超灵敏的酶免疫测定方法不仅用于抗原，而且用于抗体和半抗原。这些方法是基于使用固相而不是竞争相的非竞争类型的分析。限制使用固相的非竞争性免疫测定方法灵敏度的最大障碍之一是标记的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分析法）克服了这一困难。在最初开发的方法中，测试血清中的抗体与二硝基苯基化的生物素化抗原反应。形成的配合物被捕获在（抗二硝基苯基）igg包被的固相上。清洗固相以消除非特异性免疫球蛋白。用二硝基苯赖氨酸从固相中洗脱复合物，再次被捕获到亲和素（链亲和素）包被的固相中，最后与（抗免疫球蛋白）Fab' -酶偶联物反应。更多e.即抗体和二硝基苯化的生物素化抗原的复合物从一个固体转移到另一个固体，有效地消除非特异性吸附在第一个固相上的非特异性免疫球蛋白。该方法对血清抗体的敏感性有了显著提高，可用于检测常规方法无法检测到的抗人类t细胞白血病病毒和人类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应用于抗原的检测，并已检测到1毫微摩尔（600分子）的人铁蛋白。超灵敏的、非竞争性的免疫分析方法也被开发出来用于测量含有氨基的半抗原的原子量。半抗原，尤其是多肽，用n-羟基琥珀酰亚胺生物素进行生物素化，用酶标记的抗肽Feb'和链霉亲和素包被固相法（异位点酶免疫法）进行测定。少

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