TY - JOUR
T1 - Molecular super-gluing
T2 - a straightforward tool for antibody labelling and its application to mycotoxin biosensing
AU - Pradanas-González, Fernando
AU - Glahn-Martínez, Bettina
AU - Benito-Peña, Elena
AU - Arola, Henri O.
AU - Nevanen, Tarja K.
AU - Moreno-Bondi, María C.
N1 - Funding Information:
F.P. acknowledges the MSIU for an FPU contract. Recombinant antibodies were developed in the Mycodetect project funded by Business Finland (TEKES) 40305/2011
PY - 2022/1/3
Y1 - 2022/1/3
N2 - 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: [Figure not available: see fulltext.].
AB - 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: [Figure not available: see fulltext.].
KW - Fluorescent recombinant fusion proteins
KW - Food safety
KW - HT-2 toxin
KW - Non-competitive immunoassay
KW - SpyTag/SpyCatcher
UR - http://www.scopus.com/inward/record.url?scp=85122237429&partnerID=8YFLogxK
U2 - 10.1007/s00216-021-03841-3
DO - 10.1007/s00216-021-03841-3
M3 - Article
C2 - 34978587
AN - SCOPUS:85122237429
SN - 1618-2642
VL - 414
SP - 5373
EP - 5384
JO - Analytical and Bioanalytical Chemistry
JF - Analytical and Bioanalytical Chemistry
IS - 18
ER -