ABTS diammonium salt is a substrate for horseradish peroxidase (HRP) conjugate. In Vitro: A micro-technique of enzyme-linked immunosorbent assay (ELISA) using ABTS, as a substrate for HRP conjugate is studied. In a comparative study among 4 substrates, namely; 5-aminosalicylic acid (5AS), O-phenylenediamine (OPD), O-tolidine (OT) and ABTS, for HRP in terms of sensitivity, ABTS is the most sensitive, stable and the best in visuality by its bluish-green color^[1]. ABTS is a typical peroxidase substrate. For purification and characterization peroxidase positive transformants are cultivated in large scale (XL) under conditions that yield active protein in the culture supernatant. After 160 h cultivation an activity of 55,000 U/L in relation to the substrate ABTS is achieved and the supernatant containing the peroxidase is harvested. With ABTS as substrate the peroxidase activity falls significantly when the H₂O₂ concentration rose above 0.125 mM, indicating that the enzyme is inhibited by H₂O₂. Maximum reaction rates depending upon substrate tested reache values between 31.2 and 125 µM^[2].