Fast-tracking rapid cyanotoxin detection: A novel immunoassay for emerging dihydroanatoxins

Dihydroanatoxin-a and dihydrohomoanatoxin-a are emerging cyanotoxins contaminating freshwater ecosystems, and they have recently been associated with numerous animal fatalities. While immunochemical methods have been developed and commercialized for the rapid analysis of the most relevant cyanotoxins, no highly performant immunoreagents have yet been obtained for these members of the anatoxin family. In the present study, the synthesis of two dihydroanatoxin haptens is described. On the one hand, hapten DAa was prepared with an azide functional group to enable protein conjugation via click chemistry. On the other hand, hapten DAc was synthesized with a carboxyl group to facilitate coupling via the active ester reaction. When the immunogenicity of the corresponding bioconjugates was evaluated, hapten DAc proved to be a more potent immunogen, capable of generating high-affinity antibodies with IC₅₀ values in the low nanomolar range. Furthermore, the resulting binders were able to recognize both dihydroanatoxin-a and dihydrohomoanatoxin-a with similarly high affinity, while showing low binding to anatoxin-a and homoanatoxin-a. These novel immunoreagents were then employed to develop the first reported enzyme-linked immunosorbent assay for the analysis of dihydroanatoxins. The optimized assay achieved a limit of detection for dihydroanatoxin-a as low as 0.04 ng mL^-1. No relevant matrix effects were observed when testing four distinct environmental water samples, and the immunoassay demonstrated excellent recovery rates and coefficients of variation across a concentration range of 2 and 4000 ng mL^-1 of dihydroanatoxin-a. Thus, this immunoassay offers a powerful strategy for the rapid analysis of dihydroanatoxins and it fills the gap for monitoring the most relevant cyanotoxins.