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Sensitive electrochemical detection in a flow system is applied to the determination of pneumolysin by an ELISA procedure. Alkaline phosphatase is the enzyme used in the immunoassay and 3‐indoxyl phosphate acts as substrate. The attractive properties of the indigo electrochemical detection, product of the enzymatic hydrolysis, such as sensitive response by alternating current voltammetry, low potential detection and adsorption on carbon paste electrodes are exploited with analytical purposes. The flow system, provided with a sample aspiration mechanism, automates the methodology and decreases analysis time. Parameters related either to the electrochemical technique, to the flow system or to the enzymatic reaction are optimized. Calibration plots obtained under different conditions are reported.

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