Sensitive detection for enzyme-linked immunosorbent assays based on the adsorptive stripping voltammetry of indigo in a flow system

The suitability of carrying out sensitive electrochemical detection in conv entional enzyme-linked immunosorbent assays (ELISAs) that use alkaline phos phatase (AP) as label was demonstrated. Studies were carried out for pneumo lysin (PLY) and interleukin-10 (IL-10) ELISAs. The supports of this sensiti ve detection were the use of 3-indoxylphosphate (IP) as substrate and the e mploy of both an appropriate electrochemical technique (alternating current adsorptive stripping voltammetry (ACAdSV)) and a flow system. The adsorpti on of the enzymatic hydrolysis product on a carbon paste electrode permitte d its pre-concentration and the combination with a flow system allows perfo rming the measurement in fresh electrolyte, eliminating possible interferen ces. Calibration curves obtained in optimised conditions indicated an incre ase in sensitivity (70 times for PLY and 10 times for IL-10) when compared with conventional spectrophotometric detection. Limits of detection as low as 0.4pg PLY per well and 0.2pg IL-10/ml were obtained. Validation of the m ethodology was made by determining samples of a proteic matrix spiked with standards. (C) 2001 Elsevier Science B.V. All rights reserved.