Chemiluminescent assay of alkaline phosphatase using dihydroxyacetone phosphate as substrate detected with lucigenin

A sensitive and simple chemiluminescent assay (CL) for alkaline phosphatase (ALP) using dihydroxyacetone phosphate or its ketal (DHAP or DHAP-ketal) was developed. New substrates were transformed to dihydroxyacetone (DHA) after they were hydrolysed by ALP, which reacts with lucigenin and produces strong chemiluminescence. Under the optimum assay condition, the detection limits were 3.8 x 10(-19) and 1.5 x 10(-18) moles of ALP, respectively. The coefficients of variation (CV) at each points on the standard curve were 0.8-5.4% and 1.8-7.1% (n = 6), respectively. The mechanism of lucigenin CL with DHA was investigated by ESR spectrometry using the spin-trapping method. The mechanism was speculated as follows: the O2(-) generated by the reaction of DHA and O2 in alkaline solution reacts with lucigenin, and then emit light. The proposed CL assay was applied to the enzyme immunoassay of 17beta-oestradiol, using ALP as a label enzyme. The measurable range of 17beta-oestradiol was 15-4000-pg/mL, and the proposed method was four times more sensitive than the colorimetric assay for ALP by using 4-nitrophenyl phosphate as substrate.