Enzyme and lateral flow monoclonal antibody-based immunoassays to simultaneously determine spirotetramat and spirotetramat-enol in foodstuffs

Spirotetramat is employed worldwide to fight insect pests due to its high efficiency. This chemical is quickly metabolized by plants into spirotetramat-enol, so current regulations establish that both compounds must be determined in foodstuffs for monitoring purposes. Nowadays, immunochemical methods constitute rapid and cost-effective strategies for chemical contaminant analysis at trace levels. However, high-affinity binders and suitable bioconjugates are required. In this study, haptens with opposite functionalisation sites were synthesized in order to generate high-affinity monoclonal antibodies. A direct competitive enzyme-linked immunosorbent assay with an IC50 value for the sum of spirotetramat and spirotetramat-enol of 0.1 μg/L was developed using selected antibodies and a novel heterologous bioconjugate carrying a rationally-designed hapten. Studies with fortified grape, grape juice, and wine samples showed good precision and accuracy values, with limits of quantification well below the maximum residue limits. Excellent correlation of results was observed with a standard reference chromatographic method. As a step forward, a lateral flow immunoassay was developed for onsite screening analysis of spirotetramat in wine. This assay was successfully validated according to Regulation 519/2014/EU for semi-quantitative methods at concentrations in line with the legal levels of spirotetramat and spirotetramat-enol in grapes, with a satisfactory false suspect rate below 2%.This work was supported by the Spanish Ministerio de Economía y Competitividad (AGL2015-64488-C2 and RTI2018-096121-B-C21) and cofinanced by European Regional Development Funds. R.E.C.-C. was the recipient of a fellowship (SENESCYT-DMPF-2016-0289-CO) from Secretaría de Educación Superior, Ciencia, Tecnología e Innovación of the Republic of Ecuador, under the programme “Becas para doctorado (PhD) para docentes de universidades y de escuelas politécnicas 2015”. The proteomic analysis was performed at the Proteomics Section of SCSIE of the University of Valencia which belongs to ProteoRed, PRB2-3, and was supported by grant PT17/0019, of the PE I+D+i 2013‒2016, and funded by ISCIII and ERDF.Peer reviewe