10 - Immunoassay configurations

Abstract

Most immunoassay configurations are divided into two large groups, the “limited reagent” methods (competitive immunoassays) and the “reagent excess” methods (noncompetitive immunoassays). In competitive immunoassays, the analyte and the labeled analyte (tracer) are mixed with a limited amount of anti-analyte antibody. After incubation for a certain period, the bound or the free fraction of the tracer is measured and related to the concentration of the analyte in the sample. In noncompetitive immunoassays, an excess of immunoreactant is added, so that all the analyte is practically in the form of an immunocomplex. Then the immunocomplex is quantified and related to the analyte concentration in the sample. Noncompetitive assays offer higher sensitivity than competitive ones. Both competitive and noncompetitive immunoassays require the measurement of immunocomplexes in the presence of free antibodies and/or antigens. In “heterogeneous” immunoassays, this is accomplished by first separating the immunocomplex from the free immunoreactants. In “homogeneous” immunoassays, a modulation of the signal occurs as a result of the immunoreaction. Therefore the immunocomplex formation can be monitored directly without prior separation of the bound and free tracer. Homogeneous assays employ the same general configurations.