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Nanobody-Dependent Detection of Microcystis aeruginosa by ELISA and Thermal Lens Spectrometry.
Folorunsho, Oginni Gbenga; Oloketuyi, Sandra Folarin; Mazzega, Elisa; Budasheva, Hanna; Beran, Alfred; Cabrini, Marina; Korte, Dorota; Franko, Mladen; de Marco, Ario.
Afiliação
  • Folorunsho OG; Laboratory for Environmental and Life Sciences, University of Nova Gorica, Vipavska cesta 13, 5000, Nova Gorica, SI, Slovenia.
  • Oloketuyi SF; Laboratory for Environmental and Life Sciences, University of Nova Gorica, Vipavska cesta 13, 5000, Nova Gorica, SI, Slovenia.
  • Mazzega E; Laboratory for Environmental and Life Sciences, University of Nova Gorica, Vipavska cesta 13, 5000, Nova Gorica, SI, Slovenia.
  • Budasheva H; Laboratory for Environmental and Life Sciences, University of Nova Gorica, Vipavska cesta 13, 5000, Nova Gorica, SI, Slovenia.
  • Beran A; Istituto Nazionale di Oceanografia e di Geofisica Sperimentale (OGS), Trieste, Italy.
  • Cabrini M; Istituto Nazionale di Oceanografia e di Geofisica Sperimentale (OGS), Trieste, Italy.
  • Korte D; Laboratory for Environmental and Life Sciences, University of Nova Gorica, Vipavska cesta 13, 5000, Nova Gorica, SI, Slovenia.
  • Franko M; Laboratory for Environmental and Life Sciences, University of Nova Gorica, Vipavska cesta 13, 5000, Nova Gorica, SI, Slovenia.
  • de Marco A; Laboratory for Environmental and Life Sciences, University of Nova Gorica, Vipavska cesta 13, 5000, Nova Gorica, SI, Slovenia. ario.demarco@ung.si.
Appl Biochem Biotechnol ; 193(9): 2729-2741, 2021 Sep.
Article em En | MEDLINE | ID: mdl-33871768
ABSTRACT
Nanobodies against cell surface antigens of toxic cyanobacteria Microcystis aeruginosa were recovered by whole-cell biopanning of a naïve phage display library of nanobodies. Six unique sequences were identified and three sub-cloned and purified as fusion immunoreagents together with either green fluorescent protein or AviTag to be used for diagnostics. The yields of nanobody constructs were in the range of 5-10 mg/l and their specificity and sensitivity was initially evaluated by immunofluorescence and by fluorescent enzyme-linked immunosorbent assay (ELISA) using fluorescent nanobodies. The ELISA data confirmed the nanobody specificity but showed that the saturation of the fluorescence signal already in the presence of few hundreds of cells limited the dynamic range of the method. As an alternative, Avi-tagged nanobodies were used in combination with streptavidin-linked horseradish peroxidase for developing a diagnostic colorimetric cell ELISA, the limit-of-detection of which was 3.2 and 4.5 cells/ml for the two tested cyanobacteria strains, whereas the linear range of the assay was expanded from 10 to 10,000 cells. The fluorescent nanobodies were finally exploited for quantifying cyanobacteria by thermal lens spectrometry (TLS) that enabled to reach a limit-of-detection of 1.2 cells/ml and provided a linear range of measurement between 0 and 10,000 cells. No cross-reactivity with unrelated microalgae was detected and both colorimetric ELISA and TLS provided a linear range of detection of few logs. The data indicate that nanobodies are suitable capture reagents and that both TLS and colorimetric ELISA are reliable to monitor variations of cyanobacteria populations.
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Texto completo: 1 Base de dados: MEDLINE Assunto principal: Microcystis / Anticorpos de Domínio Único / Anticorpos Antibacterianos Tipo de estudo: Diagnostic_studies Idioma: En Ano de publicação: 2021 Tipo de documento: Article

Texto completo: 1 Base de dados: MEDLINE Assunto principal: Microcystis / Anticorpos de Domínio Único / Anticorpos Antibacterianos Tipo de estudo: Diagnostic_studies Idioma: En Ano de publicação: 2021 Tipo de documento: Article