A continuous fluorescence assay for the determination of calcium-dependent secretory phospholipase A2 activity in serum

BackgroundCalcium-dependent secretory phospholipase A2-IIA (sPLA2-IIA) in the circulation is a marker of inflammation, associated with acute and chronic disease processes. We describe a quick, sensitive and reliable microplate continuous fluorescence assay for determining sPLA2 activity in serum.MethodsLiposomes composed of a fluorescent probe and varying amounts of l-α-phosphatidylglycerol (PG) and 1,2-dioleoyl-l-α-phosphatidylcholine (DOPC) were used as substrates to determine the optimal protocol for sPLA2 activity determination without interference from serum albumin and lipoproteins.ResultsHydrolysis of the labeled substrate by sPLA2-IIA, characterized by increase in fluorescence intensity (FI) and confirmed by end-product analysis, occurred in a time-, calcium-, and protein-dependent manner. Liposomes containing 100% PG were most suitable for measurement of sPLA2 activity without interference from serum components; LDL produced a Ca2+-independent increase in FI when liposomes containing DOPC were used. The assay determined that sPLA2 activity in serum spiked with sPLA2-IIA and illustrated that endogenous sPLA2 activity was markedly higher in sera from patients with sepsis than in healthy subjects. Intra-assay and inter-assay CVs were in the ranges of 1.6–8.8% and 3.0–11.5%, respectively.ConclusionsThe described method has potential for rapid and sensitive screening of sPLA2 activity in both clinical and research settings.