Molecular super-gluing: a straightforward tool for antibody labelling and its application to mycotoxin biosensing



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Pradanas González, Fernando and Glahn Martínez, Bettina and Benito Peña, Elena and Arola, Henri O. and Nevanen, Tarja K. and Moreno Bondi, María C. (2022) Molecular super-gluing: a straightforward tool for antibody labelling and its application to mycotoxin biosensing. Analytical and Bioanalytical Chemistry . ISSN 1618-2642

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Mycotoxins are low molecular weight toxic compounds, which can cause severe health problems in animals and humans. Immunoassays allow rapid, simple and cost-efective screening of mycotoxins. Sandwich assays with a direct readout provide great improvement in terms of selectivity and sensitivity, compared to the widely used competitive assay formats, for the analysis of low molecular weight molecules. In this work, we report a non-competitive fuorescence anti-immune complex (IC) immunoassay, based on the specifc recognition of HT-2 toxin with a pair of recombinant antibody fragments, namely antigen-binding fragment (Fab) (anti-HT-2 (10) Fab) and single-chain variable fragment (scFv) (anti-IC HT-2 (10) scFv). The SpyTag and SpyCatcher glue proteins were applied for the frst time as a bioconjugation tool for the analysis of mycotoxins. To this aim, a SpyTag-mScarlet-I (fuorescent protein) and scFv-SpyCatcher fusion proteins were constructed, produced and fused in situ during the assay by spontaneous Tag-Catcher binding. The assay showed an excellent sensitivity with an EC50 of 4.8±0.4 ng mL−1 and a dynamic range from 1.7±0.3 to 13±2 ng mL−1, an inter-day reproducibility of 8.5% and a high selectivity towards HT-2 toxin without cross-reactivity with other Fusarium toxins. The bioassay was applied to the analysis of the toxin in an oat reference material and in oat samples, with a LOD of 0.6 µg kg−1, and the results were validated by analysing a certifcate reference material and by HPLC–MS/MS.

Item Type:Article
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CRUE-CSIC (Acuerdos Transformativos 2021)

Uncontrolled Keywords:SpyTag/SpyCatcher, Fluorescent recombinant fusion proteins, Non-competitive immunoassay, HT-2 toxin, Food safety
Subjects:Sciences > Chemistry > Analytic chemistry
ID Code:70321
Deposited On:16 Feb 2022 14:23
Last Modified:22 Feb 2022 09:58

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