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

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. (C)2000 Elsevier Science B.V. All rights reserved.