4.7 Article

Immunochemical method for penthiopyrad detection through thermodynamic and kinetic characterization of monoclonal antibodies

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TALANTA
卷 226, 期 -, 页码 -

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ELSEVIER
DOI: 10.1016/j.talanta.2021.122123

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Hapten; Equilibrium constant; KD value; Surface plasmon resonance; Langmuir; Competitive ELISA

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Immunoassays are widely used for rapid contaminant analysis in various samples, with the characterization of antibody-antigen interaction being crucial for developing sensitive tests. Competitive strategies are often employed for low molecular weight compounds to estimate apparent affinity values. In this study, monoclonal antibodies to penthiopyrad were successfully raised, with apparent equilibrium constants assessed using competitive enzyme-linked immunosorbent assays. The study showed close agreement between the obtained KD values and those calculated from surface plasmon resonance evaluation, and the developed immunoassay demonstrated good correlation with LC-MS/MS results.
Immunoassays are nowadays being employed for rapid contaminant analysis in clinical, environmental, and agrochemical samples. A thorough characterization of the antibody-antigen interaction can bring light to the immunoreagent selection process in order to develop sensitive and robust tests. Thus, determination of equilibrium and reaction rate constants is usually recommendable. However, this can be quite tricky for low molecular weight compounds, and competitive strategies are commonly followed to estimate apparent affinity values. In the present study, a collection of monoclonal antibodies to penthiopyrad was raised for the first time, and apparent equilibrium constants were assessed by the Langmuir model using three different competitive enzyme-linked immunosorbent assay formats. The obtained K-D values from antibody-coated assays were quite close to the corresponding K-D values calculated from surface plasmon resonance (SPR) evaluation. These studies were employed to select a pair of immunoreagents for immunoassay development. The K-D value for penthiopyrad of the selected antibody obtained by SPR was 0.28 nM. The optimized direct assay showed an IC50 value for penthiopyrad of 0.42 nM (0.15 ng mL(-1)) in buffer. The limit of quantification for grape, must, and wine samples was 10 ng mL(-1). An excellent correlation was found when immunochemical results were compared with those from LC-MS/MS. As an application case, it was determined that 58% of penthiopyrad was still found in wine after fermentation.

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