Spectrophotometric determination of alkaline phosphatase and α-fetoprotein in human serum with 5,10,15,20-tetrakis (4-phosphonooxyphenyl)porphine

Abstract

A highly sensitive kinetic spectrophotometric determination for alkaline phosphatase (ALP) using 5,10,15,20-tetrakis (4-phosphonooxyphenyl)porphine (approximately Δε≈ 3.0 × 10⁵ l mol^–1 cm^–1) was studied. With a measuring time of 30 min, the determination range was 2.0 × 10^–12–8.0 × 10^–9 mol l^–1 and the detection limit (3s) was 1.0 × 10^–12 mol l^–1. The detection limit became lower on increasing the measuring time. For example, the detection limit was up to 10^–15 mol l^–1 for 12h. This method was applied to the assay of ALP in human serum. Using 0.50 ml of sample without any pre-treatment, a good correlation was obtained with the p-nitrophenyl phosphate method, and the relative standard deviation was 4.7%. The method was also applied to the enzyme immunoassay of α-fetoprotein in human serum and provided a good correlation with the 4-methylumbelliferone derivative (r= 0.999) with approximately half the relative standard deviations (1.3–2.7%) obtained with a commercial kit. The proposed method requires very small sample volumes, is highly sensitive, highly accurate, and shows good stability of the reagent; it will be applied to various assays using ALP as a label.