An electrochemical immunoassay for Clostridium perfringens phosholipase C

A sensitive two‐site, enzyme‐linked immunoassay utilizing electrochemical detection has been developed for Clostridium perfringens phospholipase C (α toxin). Alkaline phosphatase conjugated to a mouse monoclonal antibody is used as the enzyme label. The enzyme activity is measured using 1‐naphthyl phosphate as the enzyme substrate. The enzyme‐generated naphthol is detected amperometrically in a thin‐layer cell at a glassy carbon electrode after chromatography on a 50 mm C8 reverse phase column. Two assay formats are described, based on either microtiter plates or polydispersed polymeric microbeads. The limits of detection of the two assay formats were 67.1 and 13.0 ng/mL, respectively, with a total assay time of less than one hour. The performance characteristics of three possible substrates for the alkaline phosphatase catalyzed reaction are also described.