Homogeneous stopped-flow fluoroimmunoassay using europium as label

Abstract

A competitive homogeneous fluoroimmunoassay (FIA) based on the use of europium(III) as label together with kinetic methodology, is described for the first time. This approach has been applied to the determination of skatole, a compound causing boar-taint, in pig back fat samples. The analytical signal was obtained by measuring the initial rate of the dissociation reaction of europium(III) from the tracer through the formation of an intense luminescent chelate. This initial rate is higher for the free tracer than for that corresponding to the tracer bound to the antibody, and it is directly related to the analyte concentration. Each kinetic measurement was obtained in less than 1 s by using stopped-flow (SF) mixing technique. Benzoyltrifluoroacetone, tri- n-octylphosphine and Triton X-100 were used to obtain an adequate luminescent signal. The dynamic range of the calibration graph of the method is 30–300 ng ml −1 and the calculated detection limit is 11 ng ml −1. The precision, obtained for two skatole concentrations, 50 and 150 ng ml −1, and expressed as relative standard deviation ( n=11), was lower than 6.5%. The method was applied to the analysis of pig back fat samples with recoveries ranging from 92 to 108%.