Competitive assays enzyme-labeled antigen conjugate

In this assay, marker-labeled antigen itself acts as substrate. Kohen et al. (K9) developed a homogeneous immunoassay for steroid using a steroid-fluorescent dye conjugate that yields fluorescent products upon hydrolysis with enzyme. The steroid-fluorescent dye conjugate is inactive as a substrate when bound to the antibody to steroid [F-Ag Ab]. But when unlabeled steroid [Ag] is added, it binds competitively to the antibody [Ab Ag],. and the free form of steroid-fluorescent dye conjugate [F-Ag], which is... 

In a direct immunoassay the immobilized antibody binds to the corresponding antigen. The competitive immunoassay relies upon the competition of the analyte with a labelled analyte for antibody binding. These formats are widely used for high throughput affinity arrays. A sandwich immunoassay is based on the trapping or capture of the analyte by another antibody. In ELISA (enzyme linked immunosorbent assays) the second antibody is conjugated with an enzyme. The bound enzyme labelled antibody is detected by its ability to break down its substrate to a colored product. 

Given this information, researchers have devised donor and acceptor components of the p-galactosidase subunit. The enzyme donor is conjugated to antigen, and becomes the indirect label in a competitive immunoassay. If this labeled antigen is not bound to antibody, then the combination of donor and acceptor result in a complete subunit that can combine with three other complete subunits to form an active tetramer. If the labeled antigen is antibody-bound, steric effects prevent donor-acceptor combination, so that the generation of active enzyme is not possible from this potential subunit. The concepts in this assay are shown in Eq. 6.18 ... 

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