Antibody generation and immunoassay development in diverse formats for pyrimethanil specific and sensitive analysis

Immunochemical techniques are complementary tools to modern analytical requirements. These methods rely on the production of immunoreagents with adequate binding properties. In the present study, a rationally designed and functionalized derivative of pyrimethanil-a modern anilinopyrimidine fungicide-was synthesized in order to generate for the first time high-affinity and selective antibodies to this xenobiotic. A single coupling procedure-based on hapten activation using N,N′-disuccinimidyl carbonate and purification of the active ester-was followed to prepare both immunizing and assay conjugates. Polyclonal antibodies were produced and characterized by enzyme-linked immunosorbent assay (ELISA) in four alternative formats: one indirect and three direct competitive procedures. The selected immunoassay displayed a limit of detection of 0.024 μg L-1, far lower than the official maximum residue limits and close to the sensitivity of regular instrumental assays. This ELISA was shown to be robust to buffer changes and tolerant to the presence of little amounts of methanol, ethanol and acetonitrile. Finally, the developed assay was applied to the analysis of pyrimethanil in carrot juice samples, and a limit of quantification of 0.040 mg L-1 was determined.This work was supported by the Spanish Ministerio de Ciencia e Innovación (AGL2009-12940-C02-01-02/ALI) and cofinanced by FEDER funds. J.V.M. and F.A.E.-T. were hired by the Consejo Superior de Investigaciones Científicas (CSIC), the 60 former under a Ramón y Cajal contract and the latter under a JAE-doc contract, both of them financed by Ministerio de Ciencia e Innovación and the European Social Fund.Peer Reviewe