In vitro isolation of nanobodies for selective Alexandrium minutum recognition: A model for convenient development of dedicated immuno-reagents to study and diagnostic toxic unicellular algae.

Graphical abstract Highlights • A pre-immune library of nanobodies was used to recover in vitro binders for A. minutum. • Such nanobodies are highly specific and can be produced as functional fluobodies. • They enable rapid and precise detection of A. minutum in complex water samples. • The procedure is rapid (few weeks) and inexpensive, suitable for ballast water analysis. • The procedure can be applied to any unicellular alga. Abstract At the present, the identification of planktonic species in coastal water is still a time intensive process performed by highly trained personnel that relies either on qPCR or on light microscopy observation and in vitro culturing. Furthermore, the increasing danger represented by Harmful Algal Blooms (HABs) inside phytoplankton community and the recent implementation of the legislation on ballast water management to prevent the introduction of HABs and NIS (Non Indigenous Species) urge the development of faster and reliable diagnostic methods. Immuno-based approaches could fulfil this need provided that the costs for antibody selection and production will be reduced. In this work it is demonstrated for the first time the feasibility to recover nanobodies (VHHs) selective for native surface epitopes of Alexandrium minutum by direct whole cell bio-panning using a pre-immune phage display library. The recombinant nature of VHHs enabled their rapid engineering into eGFP fluorescent reagents (fluobodies) that were produced recombinantly in bacteria and are directly suitable for fluorescence microscopy and flow cytometry. Immune-detection identified also cysts and anti- Alexandrium fluobodies showed no cross-reactivity with indigenous not-toxic phytoplankton microalgae belonging to different geni. The fluobodies were able to bind selectively to the target cells in both fixed and fresh samples with minimal processing. [ABSTRACT FROM AUTHOR]