Immunodiagnostic monoclonal antibody-based sandwich ELISA of fasciolosis by detection ofFasciola giganticacirculating fatty acid binding protein

Abstract

Up to now, parasitological diagnosis of fasciolosis is often unreliable and possesses low sensitivity. Hence, the detection of circulating parasite antigens is thought to be a better alternative for diagnosis of fasciolosis, as it reflects the real parasite burden. In the present study, a monoclonal antibody (MoAb) against recombinant Fasciola gigantica fatty acid binding protein (rFgFABP) has been produced. As well, a reliable sandwich enzyme-linked immunosorbent assay (sandwich ELISA) has been developed for the detection of circulating FABP in the sera of mice experimentally and cattle naturally infected with F. gigantica. MoAb 3A3 and biotinylated rabbit anti-recombinant FABP antibody were selected due to their high reactivities and specificities. The lower detection limit of sandwich ELISA was 5 pg mL-1, and no cross-reaction with other parasite antigens was observed. This assay could detect F. gigantica infection from day 1 post infection. In experimental mice, the sensitivity, specificity and accuracy of this assay were 93·3, 100 and 98·2%, while in natural cattle they were 96·7, 100 and 99·1%. Hence, this sandwich ELISA method showed high efficiencies and precisions for diagnosis of fasciolosis by F. gigantica.

The in vitro anthelmintic activity of the ethanol leaf extracts of Terminalia catappa L. on Fasciola gigantica

“…As per the method described by Anuracpreeda et al (2016 b ). Adult F. gigantica were removed from the liver and bile duct of infected cattle and water buffaloes killed for consumption at local abattoirs in Pathumthani Province, Thailand.…”

Immunodiagnosis of paramphistomosis using monoclonal antibody-based sandwich ELISA for detection ofParamphistomum gracilecirculating 16 kDa antigen

“…Other trematodes, cestodes and nematode parasites were obtained for the cross-reactivity study as shown in Table 1. The whole body (WB) antigen of all parasites and excretory–secretory (ES) antigen of P. gracile were prepared as per the method described by Anuracpreeda et al (2006, 2013 a , b , 2016 b , c , d , e ; Panyarachun et al 2010). The 16 kDa antigen of P. gracile (16 kDaAgPg) was obtained as per the method described earlier by Anuracpreeda et al .…”

“…In the cross-reactivity study, proteins (10 µ g) in WB from P. gracile as well as WB from other trematode, cestode and nematode parasites were separated using 12·5% SDS–PAGE, and transferred onto nitrocellulose (NC) membranes for immunoblotting by the selected MoAb 1D10 according to the method of Anuracpreeda et al (2016 a ). Polyclonal anti-16 kDaAgPg for the detection of antigen captured by the immobilized MoAb was produced by immunizing New Zealand White rabbits with 16 kDa antigen of P. gracile as per the method of Anuracpreeda et al (2016 c ). The IgM fraction of MoAb and IgG fraction of PoAb were purified by affinity chromatography, and the purified IgG of PoAb was subsequently biotinylated as previously described (Anuracpreeda et al .…”

“…The IgM fraction of MoAb and IgG fraction of PoAb were purified by affinity chromatography, and the purified IgG of PoAb was subsequently biotinylated as previously described (Anuracpreeda et al . 2016 c ).…”

“…The sandwich ELISA was performed as per the method described previously by Anuracpreeda et al (2016 b , c ). For each step, 50 µ L well −1 was added unless otherwise mentioned.…”