Fluorescent lectin assay to quantify particulate marine polysaccharides on 96-well filtration plates

Sven Uthicke, Lyndon Llewellyn, Florian Eder

Limnol. Oceanogr. Methods 7:449-458 (2009) | DOI: 10.4319/lom.2009.7.449

ABSTRACT: Our aim was to develop a method to more broadly quantify particulate polysaccharides (PPs) to complement Alcian blue quantification of transparent exopolymer particles (TEPs), which are a subset of the ocean¬ís pool of PPs. We used fluorescently labeled concanavalin A (FITC-conA), which is a lectin (a group of proteins that bind polysaccharides) and propose a method using 96-well filtration plate technology. ConA binds polysaccharides that contain the commonly occurring saccharides glucose and mannose. Using gum xanthan as a standard and field samples from the Great Barrier Reef (GBR), we demonstrated that marine particles (>0.4 µm) bound FITC-conA in a concentration-dependent manner. Particulate polysaccharides measured by FITC-conA and Alcian blue (TEP only) in samples from 11 locations in the GBR were significantly correlated (R2 = 0.61, P = 0.003). The lectin-based measurements were on average 80% higher, probably because Alcian blue stains acidic polysaccharides only, whereas lectins stain a broader polysaccharide spectrum. FITC-conA measurements of PPs in the wet season were on average 54% higher than in the dry season. PP concentrations correlated to concentrations of chlorophyll a, pheophytin, and particulate nitrogen, suggesting phytoplankton as an important source of PPs. Small volumes and few working steps enabled miniaturization of the FITC-conA protocol into a 96-well plate format, which provides for rapid sample throughput. In addition, there is extensive scope for further development using different lectins, naturally sourced standards, or filter size and capacity.