A non-equilibrium flow-injection enzyme immunoassay for thyroxine (model hapten) involving affinity separation of the immunocomplex of horseradish peroxidase-labelled antibodies and antigen from the free labelled antibodies is described. An enhanced chemiluminescence reaction was used for detection of horseradish peroxidase as an antibody marker. The method allows the determination of thyroxine concentrations as low as 10−11 M within 5 min and the precision is 10% (relative standard deviation).

A flow-injection sandwich enzyme immunoassay for human IgG as model antigen by using horseradish peroxidase as label, polystyrene beads as solid support, and the enhanced chemiluminescence reaction for peroxidase quantitation is described. the kinetics of antigen—immobilized antibody interaction has been studied and the quantitative time-concentration ranges of reactions have been estimated. Each of the two immunochemical steps of analysis have been pursued in the kinetic regime. the time for each immunochemical step was reduced to 2–3 min. the enhanced luminescent reaction involving luminol and p-iodophenol as substrates was used to detect the peroxidase label. the conditions for chemiluminescent reaction were optimized. the detection limit for peroxidase in a 3 min assay was 5–10−16 moles/tube. the detection limit for IgG, in the developed immunoassay, is 10−9 M, the overall time of the assay being 5–10 min.