Development of an in-house enzymelinked immunosorbent assay (ELISA) for serodiagnosis of human fascioliasis

• Main Author: Sudirman, Nur Hafizah
• Format: Thesis
• Language: English
• Published: 2020
• Subjects: R Medicine
• Online Access: http://eprints.usm.my/47992/1/29.%20Thesis_Final%20Copy_THESIS_NUR%20HAFIZAH%20BINTI%20SUDIRMAN_P-SKM0014_19-24%20pages.pdf; http://eprints.usm.my/47992/

Fascioliasis is waterborne and foodborne parasitic disease caused by Fasciola spp. which infect animals and humans. Current gold standard of human diagnosis relies on coprological detection of fasciolid eggs which lacks sensitivity and only applicable in chronic infections. Serodiagnosis can be used to improve diagnostic capacity of fascioliasis for both clinical and research purposes. The aim of this study was to develop and optimize an in-house indirect ELISA based on F. gigantica crude antigen for serodiagnosis of human fascioliasis in local population. In the study, a crude soluble antigen (CSA) was prepared from adult flukes and was used in the optimization of in-house ELISA. Ninety archived human sera from a previous cross-sectional study (60 seropositive, 30 seronegative) were screened using the developed ELISA. The cutoff value of the assay was determined by plotting the receiver-operating characteristic (ROC) curve analysis and the percentage agreements were calculated in comparison to a commercially available ELISA kit (Diagnostics Automation/Cortez Diagnostics, USA). The coating antigen concentration, serum dilution and HRP-conjugated secondary antibody dilution were optimized at 20 μg/mL, 1:100, and 1:6000 respectively. The assay exhibited near perfect agreements with the commercial ELISA kit at a cut-off value of 0.65 (kappa value=0.910) in which the positive percent agreement and negative percent agreement were 100% and 96.7 % respectively. In conclusion, the developed in-house ELISA is comparable to that of commercial ELISA in this study and is potentially applicable for the serodiagnosis of human fascioliasis in local community.