Homogeneous stopped-flow fluorimmunoassay for gliadin determination in food samples

Abstract

A homogeneous competitive immunoassay for gliadin determination is described for the first time. Gliadins are labeled with a terbium(III) chelate to form the tracer which competes with unlabeled gliadins for the active sites of anti-gliadin antibodies. The terbium(III) is released from the tracer by means of an “acidic enhancement solution” and the initial rate of the dissociation reaction is monitored using the stopped-flow mixing technique. Initial rate values obtained are different depending upon whether tracer is free or bound to antibody which can be correlated with the analyte concentration. The use of the fixed-time method has been also assayed, but it is not suitable as the results depend on the sample matrix. The detection limit of the assay using standards is 4 ng ml −1, which is equivalent to 4 μg g −1 in food samples, and the dynamic range of the method is 0.008–0.36 μg ml −1. The precision, expressed as the percentage of relative standard deviation, was 5–6.2%. The method has been applied to the analysis of gluten-containing and gluten-free food samples, with recoveries ranging from 83.8 to 115.1%.