hepatica has a cosmopolitan distribution, in temperate zones mainly, even though F. and serum examples (r = 0.730, p < 0.01 and r = 0.608; p < 0.01, respectively). Conclusions These data showed that the use of MoAb-based sandwich ELISA for the detection of F. gigantica coproantigens in stool specimens was superior to serum samples; it provides a highly efficient, noninvasive technique for the diagnosis of active F. gigantica infection. Keywords: Fasciola gigantica, Monoclonal antibodies, Sandwich ELISA, Coproantigen, Seroantigen Background Fasciola hepatica and F. gigantica are two trematode species which have an important impact on public health due to the infections they cause in humans and livestock. F. hepatica has a cosmopolitan distribution, mainly in temperate AZD9567 zones, while F. gigantica is found in tropical regions of Africa and Asia [1-3]. Although the majority of cases are attributed to F. hepatica, human infections with F. gigantica are also present in many countries [4-6]. In the Nile Delta of Egypt, beside the two species, a third intermediate form of Fasciola sp. has been identified [3] using molecular approaches [7]. Parasitological diagnosis of human fascioliasis is often unreliable and has low sensitivity, as parasite eggs are not found during the pre-patent period and shedding of parasitic eggs is intermittent Rabbit Polyclonal to ETS1 (phospho-Thr38) [8-10]. Moreover, Fasciola eggs may be found in the stools of uninfected persons who have eaten raw infected liver leading to false positive diagnosis [11]. Alternatively, detection of circulating Fasciola antigen in both serum and stool was found to be more sensitive and specific [12]. The majority of methods based on antigen detection are applied to F. hepatica infection, but only few are applied to F. gigantica infection [13-15]. This research was carried out to establish a highly efficient MoAb-based sandwich ELISA to diagnose active F. gigantica infection by detecting excretory/secretory antigens (ES Ags) in both serum and stool samples of infected patients for comparative purposes. Methods Study Population Patients admitted to Gastroenterology and Hepatology Department, AZD9567 Theodor Bilharz Research Institute (TBRI), who complained of abdominal pain, loss of body weight, dyspepsia, fever and diarrhea were subjected to parasitological stool examination on three consecutive days using merthiolate-iodine-formaldehyde concentration method [16]. The number of eggs per gram stool was determined by the modified Kato-thick smear technique [17]. Three groups were used; F. gigantica infected group where patients had the characteristic large operculated Fasciola eggs in their stool samples with no evidence of other parasitic infections (n = 50). Other parasites group (n = 60) included S. mansoni (n = 20), S. hematobium (n = 20) and Hymenolepis nana (n = 20). Control group (n = 30) were age- and sex-matched parasite-free healthy individuals. Stool Elute Preparation and Serum Samples Collection Aqueous elutes of a portion of AZD9567 each stool specimen were prepared by adding approximately 3 parts of 0.01 M phosphate-buffered saline (PBS), pH 7.2, containing 0.05% Tween 20 (PBS/T) to 1 1 part of stool in a centrifuge tube [18]. The mixture was homogenized and then centrifuged at 900 g for 5 min. The supernatant was aspirated and stored at -80C until use. Whole blood was collected from each subject and centrifuged at 760 g at 4C for 10 minutes and the obtained serum samples were stored at -80C until use. Fasciola Excretory/Secretory (ES) Antigens Livers of infected cattle were obtained from a local abattoir at Giza District, Egypt. Live intact F. gigantica adult worms were collected from the bile ducts and thoroughly washed at room temperature with 0.9% sodium chloride. The worms were individually incubated at 37C in 5 ml RPMI 1640 medium, pH 7.4, supplemented with 100 U of penicillin and 100 g of streptomycin per ml medium (Sigma Chemicals, St. Louis, USA). Following 24 h incubation, the medium was centrifuged at 1500 g for 10 min at 4C. The supernatants containing the ES Ags.