Development and validation of a direct competitive monoclonal antibody-based immunoassay for the sensitive and selective analysis of the phytoregulator forchlorfenuron

Forchlorfenuron is a synthetic phytohormone with cytokinin-like activity used worldwide as a plant growth regulator to increase fruit size in a number of crops, mostly in kiwifruit and grape vines. A monoclonal antibody-based enzyme-linked immunosorbent assay (ELISA) for the determination of forchlorfenuron has been characterized and optimized. The selected immunoreagents afforded a highly selective assay with a limit of detection of 10 ng L−1 in buffer. This direct competitive ELISA was validated in terms of trueness, precision, and robustness using both commercial juice and whole fruit samples. Recoveries from fortified kiwifruit juices and white and red musts were between 97 % and 131 %, with relative standard deviations below 16 %. When homogenized whole fruits were analysed after acetonitrile extraction, recoveries between 96 % and 113 % were found, with a limit of quantification of 5 μg kg−1. The proposed immunoassay was validated by comparison with a reference chromatographic method using fruits from in-field treated grape and kiwifruit vines. Linear regression analysis of ELISA and HPLC–UV determinations showed an excellent correlation (r 2 = 0.998), whereas analysis of the slope (0.99 ± 0.01) and of the intercept (−1 ± 3) clearly proved that the developed competitive immunoassay provided results that were statistically comparable to those obtained by the instrumental method for the analysis of forchlorfenuron in fruits at trace levels.This work was supported by Ministerio de Educación y Ciencia (PET2006_0423_00 and AGL2009-12940-C02/01/02/ALI), Ecología y Protección Agricola S.L., and FEDER Funds. C.S.-P., F.A.E.-T., and J.V.M. were hired by the CSIC under a predoctoral I3P contract, a postdoctoral JAE contract, and a Ramón y Cajal contract, respectively. All contracts were cofinanced by Ministerio de Ciencia e Innovación and the European Social Fund.Peer Reviewe