In ELISA, we use anti-mouse HRP as secondary antibody and o-phenylenediamine as substrate. Why we need add OPD to citrate phosphate and H2O2? What are the roles of them individually.
Thanks.
ELISA is a test based on enzyme substrate interaction that finally produced color can be read spectrophotometrically. When we use HRP as enzyme, the specific substrate was peroxide (H2O2) and the result is liberation of free oxygen without color detection. Though OPD (chromogenic material) in ELISA is oxidized by the oxygen liberated from enzymatic reaction on H2O2 lead to formation of 2,3-diaminophenazine, this product has been characterized by melting point, mass spectrometry and can be read spectrophotometrically at 450 nm. For any enzymatic reaction suitable PH is required and this achieved by citrate phosphate
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