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 ΔIµ≈ 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.