4.4.2. 8-Isoprostane (8-Iso)
To measure the concentration of 8-isoprostane, we used plasma samples obtained from blood with EDTA. We also used the 8-isoprostane EIA kit (Cayman Chemical, Ann Arbor, MI, USA). The basis of this measurement is the competition between 8-isoprostane and an 8-isoprostane–acetylcholinesterase conjugate for the binding of a certain number of 8-isoprostane-specific antibodies. The amount of 8-isoprostane tracer remains constant, but the levels of 8-isoprostane in the sample vary such that the amount of tracer that binds to the specific antibody of the 8-isoprostane is inversely proportional to the concentration of 8-isoprostane in the sample. The antibody–8-isoprostane complex binds to a second antibody, and upon the addition of the corresponding substrate, a yellow-colored compound is formed, which is measured spectrophotometrically at 412 nm. The intensity of the color is inversely proportional to the amount of 8-isoprostane present in the analyzed sample.