A class-selective immunoassay for simultaneous analysis of anilinopyrimidine fungicides using a rationally designed hapten.

The development of multianalyte immunoassays constitutes a main research issue in the field of bioanalytical techniques. In the present study, class-specific antibodies against the three members of the anilinopyrimidine family of fungicides (pyrimethanil, cyprodinil and mepanipyrim) were raised by using a bioconjugate of a rationally designed hapten [5-(6-methyl-2-(phenylamino)pyrimidin-4-yl)pentanoic acid]. Highly sensitive immunoassays were developed for the generic determination of these compounds, using the competitive enzyme-linked immunosorbent assay (ELISA). Particularly, a direct antibody-coated competitive ELISA afforded identical sensitivity for the three anilinopyrimidines, with IC50 values of 0.26, 0.27 and 0.25 µg L-1 for pyrimethanil, cyprodinil and mepanipyrim, respectively. This immunoassay was fully characterized and applied to the multianalyte determination of anilinopyrimidine fungicides in white and red wines, with a limit of quantification of 1 µg L-1, average recoveries from 93.1 to 114.4%, and relative standard deviations lower than 20%. Commercial wine samples were analyzed and those containing detectable anilinopyrimide residues were verified by a reference chromatographic technique.

Direct surface plasmon resonance immunosensing of pyraclostrobin residues in untreated fruit juices.

A surface plasmon resonance (SPR) immunoassay for on-line detection of the strobilurin fungicide pyraclostrobin in untreated fruit juices is presented. The analysis of pyraclostrobin residues is accomplished in apple, grape, and cranberry samples by monitoring the recognition events occurring separately in a two-channel home-made SPR biosensor. Covalent coupling of the analyte derivative results in a reversible method, enabling more than 80 measurements on the same sensor surface. Optimization of the immunoassay conditions provides limits of detection as low as 0.16 µg L(-1). The selectivity and reproducibility of the analysis is ensured by studying both non-specific interactions with unrelated compounds and inter-assay coefficients of variation. Excellent recovery ranging from 98 to 103% was achieved by a simple 1:5 dilution of fruit juice with assay buffer before the analysis. The lack of previous cleaning and homogenization procedures reduces the analysis time of a single food sample to only 25 min, including the regeneration cycle.

Development of monoclonal ELISAs for azinphos-methyl. 1. Hapten synthesis and antibody production.

The development of monoclonal antibody-based enzyme-linked immunosorbent assays for azinphos-methyl is described. A panel of haptens was synthesized for immunoconjugate preparation, and a series of haptens for heterologous, coating or tracer, conjugates was also prepared. Hapten synthesis was based on a strategy in which only a fragment of the whole target molecule was present (fragmentary haptens). From immunized mice, a set of monoclonal antibodies was obtained and ELISA sensitivities were assayed in different formats. Affinities estimated as I(50) values in the low nanomolar range for azinphos-methyl and phosmet were observed for several monoclonal antibodies in the conjugate-coated format and in the antibody-coated format under nonoptimized assay conditions.

Development of monoclonal ELISAs for azinphos-methyl. 2. Assay optimization and water sample analysis.

Two enzyme-linked immunosorbent assays (ELISA) for the insecticide azinphos-methyl have been optimized and characterized. Both ELISAs are based on monoclonal antibodies produced from an immunogen with a hapten containing a phthalimido moiety and on protein conjugates of heterologous ligands containing a 1,2,3-benzotriazine group. Assay I was performed in the conjugate-coated ELISA format and assay II in the antibody-coated format. Several physicochemical factors (ionic strength, pH, incubation times, and Tween 20 and BSA concentrations) that influence assay performance were studied and optimized. Regarding specificity, both monoclonal immunoassays highly cross-reacted with azinphos-ethyl and phosmet. Finally, both assays were applied to the analysis of azinphos-methyl in spiked real water samples. For assay I the sensitivity, estimated as the I(50) value, was 0.40 nM, with a practical working range between 0.10 and 1.75 ng/mL and a limit of detection of 0.05 ng/mL. For assay II the sensitivity was 1.01 nM, with a practical working range between 0.32 and 2.54 ng/mL and a limit of detection of 0.08 ng/mL.