Increased specificity of Fasciola hepatica excretory-secretory antigens combining negative selection on hydroxyapatite and salt precipitation

A single and rapid method to obtain an antigenic fraction of excretory-secretory antigens (ESAs) from Fasciola hepatica suitable for serodiagnosis of fascioliasis is reported. The procedure consists in the negative selection of F. hepatica ESAs by hydroxyapatite (HA) chromatography (HAC; fraction HAC-NR) followed by antigen precipitation with 50% ammonium sulphate (AS) and subsequent recovery by means of a Millex-GV or equivalent filter (Fi-SOLE fraction). Tested in indirect ELISA, the Fi-SOLE antigens detected natural infections by F. hepatica with 100% sensitivity and 98.9% specificity in sheep, and 97.7% sensitivity and 97.7% specificity in cattle, as determined by ROC analysis. The SDS-PAGE and proteomic nano-UHPLC-Tims-QTOF MS/MS analysis of fractions showed that the relative abundance of L-cathepsins and fragments thereof was 57% in fraction HAC-NR and 93.8% in fraction Fi-SOLE. The second most abundant proteins in fraction HAC-NR were fatty-acid binding proteins (11.9%). In contrast, free heme, and heme:MF6p/FhHDM-1 complexes remained strongly bond to the HA particles during HAC. Interestingly, phosphorylcholine (PC)-bearing antigens, which are a frequent source of cross-reactivity, were detected with an anti-PC mAb (BH8) in ESAs and fraction HAC-NR but were almost absent in fraction Fi-SOLE.