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