Ultrasensitive enzyme immunoassay.

Ultrasensitive enzyme immunoassay methods not only for antigens but also for antibodies and haptens are reviewed. 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 conjugate. 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. 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 haptens with amino groups have also been developed to measure attomole amounts of haptens.