Enzyme immunoassay with amperometric flow-injection analysis using horseradish peroxidase as a label. Application to the determination of polychlorinated biphenyls

An amperometric detection system for horseradish peroxidase (HRP) activity was optimized using flow injection analysis (FIA) with glassy carbon as a working electrode. Ferroceneacetic acid was investigated as a co-substrate for the electrochemical detection of HRP. The calculated detection limit for HRP was 2.6 × 10−1M with incubation of 30 min. The substrate was used in an electrochemical enzyme immunoassay for polychlorinated biphenyls (PCB). We used a competitive assay, where PCB-protein conjugate (gelatin) was immobilized to the solid phase (microtitre assay plate) and the competition was carried out with PCB standards using a limiting amount of anti-PCB IgG. The extent of the competition was evaluated using a secondary, HRP labelled, IgG; the amount of the enzyme label was detected after 30 min of incubation with the substrate. The PCB range was 0.1–50 μg ml−1. The overall assay time was 2h and 30 min. The within assay precision, over 6 measurements, was lower than 10% for the entire range.