Forchlorfenuron-mimicking haptens: From immunogen design to antibody characterization by hierarchical clustering analysis

To obtain highly-specific and selective forchlorfenuron binders, a collection of functionalized derivatives with different spacer arm locations and lengths was prepared. By immunization with target-mimicking haptens, a large battery of monoclonal and polyclonal antibodies against this synthetic cell regulator was produced and exhaustively characterized in two immunoassay formats using homologous and heterologous conjugates. Antibodies with IC50 values lower than 0.3 nM were successfully raised from the prepared immunogens, thus evidencing the efficacy of the explored strategies. In order to identify significant epitopes in the antibody-antigen interaction, a series of new chemical forchlorfenuron analogues, with slight modifications at both rings of the target molecule, were synthesized and evaluated in competitive assays. As a novel approach in hapten recognition studies, data processing was performed by computational classification methods based on hierarchical clustering. This strategy was shown to be highly valuable for a straightforward profiling of antibodies according to analogue recognition patterns. A relationship could be established between the antigen binding properties of antibodies and the structure of the immunogen. Whereas antibodies with equivalent affinities had been obtained from all of the derivatives, their specificity was found to be largely influenced by the differential exposition of the molecule to the immune system.This work was supported by Ministerio de Educación y Ciencia (AGL2009-12940-C02/01/02/ALI), and cofinanced by FEDER Funds. C.S.P. and J.V.M. were hired by the CSIC, the former under a predoctoral I3P contract and the latter under a Ramón y Cajal postdoctoral contract.Peer Reviewe

Development of an immunochromatographic assay based on carbon nanoparticles for the determination of the phytoregulator forchlorfenuron

Rapid analytical methods enabling the determination of diverse targets are essential in a number of research areas, from clinical diagnostics to feed and food quality and safety. Herein, the development of a quantitative immunochromatographic assay for the detection of the synthetic phytoregulator forchlorfenuron (CPPU) is described. The competitive lateral flow immunoassay (LFIA) was based on the immobilization onto a nitrocellulose membrane of an ovalbumin-CPPU conjugate (test line) and on the use of an immunodetection ligand consisting of carbon nanoparticles labeled with an anti-CPPU monoclonal antibody through interaction with a secondary antibody. The presence of CPPU in horticultural samples was visually interpreted by the decrease in the black signal intensity of the test line, according to the competitive character of the format. The quantitative determination of the analyte was easily performed by a two-step procedure consisting of flatbed scanning of the strips followed by computer-based image analysis of the pixel gray volumes of the test lines. Under optimized conditions, the immunochromatographic test afforded a limit of quantification in buffer of 89. ng/L. The accuracy of the strip test was assessed by the analysis of fruit samples with incurred residues, and the obtained results were compared with those derived from two reference methods, ELISA and HPLC. The LOQ of the CPPU-specific LFIA in kiwifruits and grapes was established at 33.4 οg/kg. The excellent analytical performance of the developed strip test demonstrates the potential of immunochromatographic assays for the quantitative monitoring of small organic molecules in complex matrices. © 2012 Elsevier B.V.This work was supported by the Ministerio de Educación y Ciencia (AGL2009-12940-C02/01/02/ALI), and cofinanced by FEDER Funds. Celia Suárez Pantaleón was hired by the CSIC under a predoctoral I3P contract.Peer Reviewe

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