Hapten Design and Antibody Generation for Immunoanalysis of Spirotetramat and Spirotetramat-enol

Abstract

Spirotetramat—a tetramic acid insecticide—is rapidly metabolized or degraded to give spirotetramat-enol; so, common residue definitions include the sum of both compounds. In the present study, two spirotetramat-functionalized derivatives (haptens) have been designed to generate immunoreagents to these molecules for rapid immunochemical analysis. Haptens have been synthesized with alternative linker tethering sites and, for the first time, high-affinity antibodies have been generated with different specificities to these active principles. Two sensitive assays have been developed using the same antibody in different formats, and by using linker-site heterologous haptens, the selectivity of the final immunoassay could be improved. A generic immunoassay with sensitivity similar to spirotetramat and spirotetramat-enol and a specific assay of spirotetramat-enol have been developed. The described antibody and bioconjugates showed great potential for sensitive immunosensor development and analysis of this complex analyte.

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

“…Hapten-to-protein molar ratios of ca . 12.9, 3.8, and 0.5 for BSA–SP m , OVA–SP m , and HRP–SP m conjugates, and 17.9, 10.5, and 1.2 for BSA–SP h , OVA–SP h , and HRP–SP h conjugates, respectively, were obtained, which are equivalent to those of the previously published SP o conjugates 19 . MALDI-TOF mass spectra (Figures S2‒S4) of the newly prepared bioconjugates are provided in the Supplementary Information file.…”

“…1 a). In a previous study, the bovine serum albumin (BSA) conjugate of hapten SP o —with the spacer arm at the aliphatic ring, distal to the aromatic ring of the SP molecule—afforded good polyclonal antibodies 19 , so it was used in the present study to generate monoclonals. In that same study, the conjugate BSA‒SP c —with the linker in a central position of the hapten molecule—did not render good antibodies, most probably because of the lability of the carbonate moiety, so it was discarded for immunization of mice.…”

“…Hapten SP m was prepared by total synthesis from aryl iodide 1 , which in turn was obtained, as detailed in the Supplementary Information file (Figure S1 ), from 1,4-cyclohexanedione monoethylene acetal and 2-( o -tolyl) acetic acid. This procedure followed a synthetic route similar to that previously used for the preparation of the methylated analogue at the C-5 position of the phenyl ring 19 , 22 . First, O -methylation of the hydroxyl group at the C-8 position of the 1-azabicyclic moiety was carried out (Fig.…”

“…All reactions involving air-sensitive compounds were conducted in oven-dried glassware under a nitrogen atmosphere. The synthesis of hapten SP o was reported in a previous study 19 . Details of all of the synthetic steps and characterization data of intermediate compounds are provided in the Supplementary Information file.…”

“…The high sensitivity, portability, and friendly use of immunochemical methods at competitive prices are being increasingly appreciated by the industrial sector. Recently, polyclonal antibodies to SP and SP-enol were generated showing the great potential of the studied synthetic strategy to prepare haptens 19 . In the present study, monoclonal antibodies (mAbs) to SP and SP-enol were raised for the first time with the aim to develop alternative immunoassays for simultaneous residue appraisal in food samples.…”