First, the immunoconjugate, which is a polyclonal antibody specific for FXIII-A coupled to
horse radish peroxidase (HRP), is introduced into the microwells coated with a polyclonal
antibody specific for FXIII-A. Then, the diluted tested sample is immediately introduced,
and the immunological reaction starts. When present, FXIII-A 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 FXIII-A in the tested sample.
