New electrochemical assay of alkaline phosphatase using ascorbic acid 2-phosphate and its application to enzyme immunoassay Original Research Article

An alternative substrate is described for an enzyme immunoassay with electrochemical detection. Alkaline phosphatase ⊙ALP) activity is determined by using ascorbic acid 2-phosphate ⊙AsA-P) as substrate. ALP-generated-AsA is detected amperometrically at a glassy carbon electrode in a flow injection system at +400 mV. The optimum assay conditions ⊙pH, incubation time and concentration of reagent) are examined for the ALP assay. The detection limit of ALP was 160 amol per assay ⊙7 amol per injection). On electrochemical detection, many ALP assays using p-aminophenyl phosphate or phenyl phosphate as substrate have been reported. The sensitivity for ALP by the proposed method is almost the same as those of the methods for ALP using p-aminophenyl phosphate or phenyl phosphate. The proposed method was applied to the enzyme immunoassay of human chorionic gonadotropin ⊙hCG) using ALP as a label enzyme. The detection limit of hCG was 2 mIU ml−1. Comparison of the results obtained by the proposed electrochemical EIA and time-resolved fluoroimmunoassay showed excellent agreement ⊙r = 0.997, n = 50). The proposed electrochemical EIA could be performed within 4 h, and could be useful for routine assay in clinical diagnosis.