Diagnosis of Fasciola gigantica infection using a monoclonal antibody-based sandwich ELISA for detection of circulating cathepsin B3 protease |
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| Affiliation: | 1. Division of Agricultural Science, Mahidol University, Kanchanaburi Campus, Saiyok, Kanchanaburi 71150, Thailand;2. Department of Anatomy, Faculty of Science, Mahidol University, Rama VI Rd., Bangkok 10400, Thailand;3. Department of Microbiology, Faculty of Science, Mahidol University, Rama VI Rd., Bangkok 10400, Thailand;4. Mahidol University, Nakhonsawan Campus, Nakhonsawan 60000, Thailand;1. Department of Parasitology, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand;2. Liver Fluke and Cholangiocarcinoma Research Center, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand;3. Department of Clinical Microscopy, Faculty of Associated Medical Sciences, Khon Kaen University, Khon Kaen, Thailand;4. Department of Public Health, Faculty of Science and Technology, Phetchabun Rajabhat University, Phetchabun, Thailand;5. Department of Pathology, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand;6. Section for Parasitology and Aquatic Diseases, Department of Veterinary Disease Biology, Faculty of Health and Medical Sciences, University of Copenhagen, Denmark;1. School of Veterinary Medicine, University College Dublin, Belfield D4, Dublin, Ireland;2. Veterinary Medicine Research and Development, Zoetis Inc, Mercuriusstraat 20, B-1930 Zaventem, Belgium;3. Veterinary Medicine Research and Development, Zoetis Inc, 333 Portage St, Kalamazoo, MI 49009, USA;4. School of Biological Sciences, Medical Biology Centre, Queen’s University Belfast, Belfast BT9 7BL, United Kingdom;1. Laboratorio de Parasitología, Facultad de Farmacia, Universidad de Santiago de Compostela, Santiago de Compostela, Spain;2. Laboratorio de Parasitología, Centro de Investigaciones Agrarias de Mabegondo, INGACAL, Abegondo, A Coruña, Spain;1. Department of Pathobiology, Faculty of Science, Mahidol University, Rama 6 Road, Ratchathewi, Bangkok 10400, Thailand;2. Faculty of Allied Health Sciences, Burapha University, Long-Hard Bangsaen Road, Saen Sook Sub-district, Mueang District, Chonburi 20131, Thailand;3. Faculty of Veterinary Medicine, Mahanakorn University of Technology, Cheum-Sampan Road, Nong Chok, Bangkok 10530, Thailand;4. Department of Anatomy, Faculty of Science, Mahidol University, Rama 6 Road, Ratchathewi, Bangkok 10400, Thailand;5. Faculty of Physical Therapy, Mahidol University, Putthamonthon Sai 4 Road, Salaya, Putthamonthon, Nakhon Pathom 73170, Thailand;6. Laboratory of Veterinary Parasitology, Faculty of Agriculture, Iwate University, Ueda 3-18-8, Morioka 020-8550, Japan |
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| Abstract: | A reliable monoclonal antibody (MoAb)-based sandwich enzyme-linked immunosorbent assay (sandwich ELISA) was developed for the detection of circulating cathepsin B3 protease (CatB3) in the sera from mice experimentally infected with Fasciola gigantica and cattle naturally infected with the same parasite. The MoAb 2F9 and biotinylated rabbit polyclonal anti-recombinant CatB3 antibody were selected due to their high reactivities and specificities to F. gigantica CatB3 antigen based on indirect ELISA and immunoblotting. The lower detection limit of the sandwich ELISA assay was 10, 100 and 400 pg/ml, when applied for the detection of rCatB3 antigen and CatB3 in whole body (WB) of newly excysted juveniles (NEJ) and metacercariae (Met) of F. gigantica, respectively. This sandwich ELISA assay could detect F. gigantica infection from day 1 to 35 post infection and revealed that circulating level of CatB3 peaked at day 1 post infection. In contrast, the antibody detection by indirect ELISA could only demonstrate the antibody level from 35 days post infection. The reliability of the assay method was evaluated using serum samples from mice infected with F. gigantica or Schistosoma mansoni, and hamsters infected with Opisthorchis viverrini, as well as normal mice and hamsters. In addition, sera from cattle infected with Paramphistomum cervi, Strongylid, Trichuris sp. and Strongyloides sp., as well as sera from normal cattle were also assessed. In experimental mice, the diagnostic sensitivity, specificity, positive predictive value, negative predictive value, false positive rate, false negative rate and accuracy of ELISA were 95%, 100%, 100%, 97.9%, 0%, 5.3% and 98.5%, while in natural cattle they were 96.7%, 100%, 100%, 98.5%, 0%, 3.4% and 98.9%, respectively. Hence, this assay method showed high efficient and precision for early diagnosis of fasciolosis by F. gigantica. |
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| Keywords: | Trematoda Immunodiagnosis Sandwich ELISA Cathepsin B3 Recombinant protein Monoclonal antibody Seroantigen Cross-reaction |
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