At the present, the identification of planktonic species in coastal water mainly relies on light microscopy
observations. This kind of analyses is performed by highly trained personnel, requires lab equipment and long
processing time. High-throughput and easy-to-perform methods are instead highly needed for routine costal and
ballast water monitoring.
Immuno-reagents are widely employed in the medical field for routine diagnostics, where they provide the necessary
sensitivity and specificity, as for example for cancer subtype characterization. Reagents of similar grade are so far not
widely available for both diagnostics and basic research of microalgae. We describe the first successful isolation of a
single-domain antibody (nanobody or VHH) from a pre-immune library, its engineering into application-ready
reagents, and its inexpensive production as recombinant fusion protein.
Alexandrium minutum was chosen as a model organism to test the feasibility of the procedure. The procedure foresees
the panning of a pre-immune phage library of VHHs that was used for in vitro selection against directly the target cells.
Monoclonal nanobodies specific for A. minutum cells were identified and optimized for recombinant production as
fusion with fluorescent proteins in bacterial hosts. Such fluorescently-tagged VHHs were validated by
immunofluorescence and cytofluorimetry for their selectivity by testing unicellular algal species that can be found in
the same environment of A. minutum. Two nanobodies were found to be highly specific for the target cells, were able
to bind also cysts of A. minutum and they gave no cross-reaction, even for a not-toxic strain of the closely related A.
tamutum.
Different tags can be then fused to the selected nanobodies and used instead of the fluorescent proteins to obtain a
reagent immediately applicable to further techniques, such as cell Enzyme Linked Immuno Sorbent Assay (ELISA) or
biosensor surface functionalization. The newly produced reagents can be applied for direct whole-cell detection in
seawater, bypassing the need of cell processing required for DNA or RNA diagnostics, and can be used for both alive
and fixed cells, guaranteeing the possibility to check old samples and to perform confirmatory morphological studies
