Electrochemical studies of o-tilidine as a hydrogen donor substrate for peroxidase and its application in enzyme immunoassay

A highly sensitive electrochemical method is described for assay of horseradish peroxidase (HRP) using o-tilidine as substrate. Under the optimal conditions, the detection limit for HRP is 2.5 mU/l and the calibration range is from 5.0 to 1000 mU/l. The relative standard deviation of 11 measurements is 6.3% for 10.0 mU/l HRP. This new electrochemical system was further combined with an indirect enzyme immunoassay using direct antigen-coating format for the detection of cucumber mosaic virus (CMV). The results show an improved sensitivity over the traditional o-phenylenediamine (OPD) spectrophotometric enzyme-linked immunosorbent assay (ELISA) method. Using this technique, a minimum detectable level of 2.0 ng/ml of purified CMV and 1:12500 dilution of CMV in infected leaf extractions can be achieved. (C) 2002 Elsevier Science B.V. All rights reserved.