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

Fernando Pradanas-González,Henri O Arola,Elena Benito-Peña,María C Moreno-Bondi,Bettina Glahn-Martínez,Tarja K Nevanen

doi:10.1007/s00216-021-03841-3

Fernando Pradanas-González, Henri O Arola + Show 4 more

Open Access

https://doi.org/10.1007/s00216-021-03841-3

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Abstract

Mycotoxins are low molecular weight toxic compounds, which can cause severe health problems in animals and humans. Immunoassays allow rapid, simple and cost-effective 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 fluorescence anti-immune complex (IC) immunoassay, based on the specific 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 first time as a bioconjugation tool for the analysis of mycotoxins. To this aim, a SpyTag-mScarlet-I (fluorescent 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 certificate reference material and by HPLC–MS/MS.Graphical abstract

Highlights

Mycotoxins are toxic compounds produced naturally by a wide variety of filamentous fungal species
The Food and Agriculture Organization (FAO) has estimated that more than 25% of the crops around the world have been affected by mycotoxins and recent studies revealed that their occurrence above detectable levels is up to 60–80%
We report the application of these “glue proteins” for the generation of the analytical signal upon spontaneous binding of the SpyTag-mScarlet-I fusion protein with the anti-immune complex (IC) HT-2 (10) scFv-Spy Catcher through the formation of an isopeptide bond Tag-Catcher

Summary

Introduction

Mycotoxins are toxic compounds produced naturally by a wide variety of filamentous fungal species. The main mycotoxin-producing moulds include those of the genus. Published in the topical collection featuring Promising Early-Career (Bio)Analytical Researchers with guest editors Antje J. Aspergillus, Fusarium, Penicillium, Aspergillus, Alternaria and Claviceps, which infect a wide variety of food crops. The Food and Agriculture Organization (FAO) has estimated that more than 25% of the crops around the world have been affected by mycotoxins and recent studies revealed that their occurrence above detectable levels is up to 60–80%. Climate change and the availability of increasingly sensitive analytical methods have been suggested to explain such high occurrence [1]

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