Screening of nanobody targeting alkaline phosphatase PhoX in Microcystis and detection of the PhoX in situ by fluorescence immunoassays

Cyanobacterial alkaline phosphatases (APases) play a key role in organophosphate utilization in freshwater. Tracking the distribution of APases can provide insights into the physiological response of phytoplankton to phosphorus nutrition. Extracellular APase PhoX, one of three prokaryotic APase families, is important for organophosphate utilization. Because the existing methods only give information on bulk APases activity, the specific contribution of the PhoX is little evaluated. To develop an immunoassay for PhoX detection, the phoX gene of Microcystis aeruginosa PCC7806 was expressed in Escherichia coli BL21 and the purified PhoX was used as a coated antigen for screening anti-PhoX nanobodies from an alpaca antibody library. After three rounds of panning, a nanobody 3H with the highest affinity was selected. Further tests showed 3H could specifically bind to Microcystis PhoX and be used for PhoX detection by immunoblotting analysis. Then we constructed two different fluorescent-labeled 3H, EGFP-3H (with an enhanced green fluorescent protein, EGFP) and FITC-3H (with fluorescein 5-isothiocyanate, FITC), as specific fluorescent dyes for PhoX. The fluorescence staining tests with laboratory strains and water bloom samples showed that both fluorescent-labeled nanobodies could visualize the distribution and evaluate relative expression levels of PhoX in Microcystis . The nanobody 3H could be a useful regent to develop fluorescence immunoassays for in situ analyses of Microcystis PhoX. Graphical Abstract