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

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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