Description
In a first incubation step, the α2-macroglobulin in the samples is bound to polyclonal rabbit antibodies (in excess), which are immobilised to the microtiter wells. To remo-ve all unbound substances, a washing step is carried out. In a second incubation step, a peroxidase-labelled anti α2-macroglobulin antibody (POD-antibody) is added. Af-ter another washing step to remove all unbound substances, the solid phase is incu-bated with the substrate, tetramethylbenzidine (TMB). An acidic stop solution is then added to stop the reaction. The colour converts from blue to yellow. The intensity of the yellow colour is directly proportional to the concentration of α2-macroglobulin in the sample. A dose response curve of the absorbance unit (optical density, OD) vs. concentration is generated, using the results obtained from the calibrators. α2-macroglobulin, present in the patient samples, is determined directly from this curve