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Nanobody-Dependent Detection of Microcystis aeruginosa by ELISA and Thermal Lens Spectrometry

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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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Authors and Affiliations

  1. Laboratory for Environmental and Life Sciences, University of Nova Gorica, Vipavska cesta 13, 5000, Nova Gorica, SI, Slovenia

    Oginni Gbenga Folorunsho, Sandra Folarin Oloketuyi, Elisa Mazzega, Hanna Budasheva, Dorota Korte, Mladen Franko & Ario de Marco

  2. Istituto Nazionale di Oceanografia e di Geofisica Sperimentale (OGS), Trieste, Italy

    Alfred Beran & Marina Cabrini

Authors
  1. Oginni Gbenga Folorunsho
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  2. Sandra Folarin Oloketuyi
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  3. Elisa Mazzega
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  4. Hanna Budasheva
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  5. Alfred Beran
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  6. Marina Cabrini
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  7. Dorota Korte
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  8. Mladen Franko
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  9. Ario de Marco
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Contributions

OGF, SFO, EM, and HB performed the experiments. AB and MC prepared the microalgae. DK and MF supervised the TLS experiments. AdM supervised the nanobody-related experiments. MF and AdM conceived the project. All the authors contributed to write the manuscript.

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Correspondence to Ario de Marco.

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Folorunsho, O.G., Oloketuyi, S.F., Mazzega, E. et al. Nanobody-Dependent Detection of Microcystis aeruginosa by ELISA and Thermal Lens Spectrometry. Appl Biochem Biotechnol 193, 2729–2741 (2021). https://doi.org/10.1007/s12010-021-03552-6

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  • DOI: https://doi.org/10.1007/s12010-021-03552-6

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