Sudirman, Nur Hafizah
(2020)
Development of an in-house enzymelinked
immunosorbent assay (ELISA)
for serodiagnosis of human
fascioliasis.
Masters thesis, Universiti Sains Malaysia.
Abstract
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.
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