Instability of the ABTS/peroxidase reaction product in biological buffers

The commonly used horseradish peroxidase dye substrate, 2,2'-azino-bis(3-ethyl-benzthiazoline-6-sulphonic acid) (ABTS), was found to be unstable under certain conditions after reaction with the peroxidase. A survey of several buffers and pH values showed that the oxidized dye was destabilized by raising the pH or by the use of "Good" buffers. It is recommended that use of this dye in a peroxidase-based detection system be confined to acetate buffer at low pH, if possible, and that phosphate or Tris buffers be used if a pH near neutrality is required.