First, the immunoconjugate, which is a polyclonal antibody specific for PZ coupled to
horse radish peroxidase (HRP), is introduced into the microwells coated with a polyclonal
antibody specific for PZ. Then, the diluted tested sample is immediately introduced, and
the immunological reaction starts. When present, PZ binds onto the polyclonal antibody
coated solid phase through one epitope, and fixes the polyclonal antibody coupled to HRP
through free epitopes. Following a washing step, the peroxidase substrate, 3,3',5,5' –
Tetramethylbenzidine (TMB), in presence of hydrogen peroxide (H2O2), is introduced and
a blue colour develops. When the reaction is stopped with Sulfuric Acid, a yellow colour is
obtained. The amount of colour developed is directly proportional to the concentration of
human PZ in the tested sample.
