Detection of pneumolysin in human urine using an immunosensor on screen-printed carbon electrodes

A voltammetric immunosensor for pneumolysin (PLY), based on the electrochemical detection of indigo blue, produced by alkaline phosphatase (AP) when 3-indoxyl phosphate (3-IP) is used as enzymatic substrate, is prepared and evaluated for measuring this toxin in human urine samples. The single-use immunosensor is fabricated by deposition of biotinylated anti-PLY monoclonal antibodies onto pre-oxidised streptavidin coated screen-printed carbon electrodes (SPCEs). Rabbit polyclonal IgGs anti-PLY are used in combination with an anti-rabbit IgG alkaline phosphatase conjugate as detection antibodies. The estimated limit of detection of the purified recombinant toxin is 0.05 ng/ml in Tris-buffer and 0.12 ng/ml in undiluted urine samples, but the same linear range, between 0.25 and 5.0 ng/ml, is obtained in both cases. The immunosensor is also applied to detect pneumolysin in urine samples from two pneumococcal pneumonia patients. (c) 2005 Elsevier B.V. All rights reserved.