Immunodiagnosis of opisthorchiasis using parasite cathepsin F

Abstract

Opisthorchis viverrini, a food-borne trematode parasite endemic in the lower Mekong countries, is conventionally diagnosed by stool examination. However, parasitological stool-based diagnosis can be unreliable in light infections. The goal of this study was to develop the immunodiagnosis of opisthorchiasis using cathepsin F cysteine protease of O. viverrini in both indirect and sandwich ELISA assays. A recombinant O. viverrini cathepsin F (rOv-CF) of 40 kDa was expressed in E. coli strain BL21 (DE3), affinity purified, and deployed in ELISA assays. Human sera from 272 cases were investigated by indirect rOv-CF based-ELISA. Positive antibody response to rOv-CF was found in 137 out of 272 cases (50.37%) using a cut off OD (0.400) determined by ROC analysis. In comparison to parasitological stool examined for fluke eggs, the gold standard, the rOv-CF indirect ELISA showed a sensitivity and specificity of 62.1% and 84.05%, respectively. Serum antibody levels correlated well with egg counts per gram feces (EPG) (P < 0.001). In addition chicken IgY antibody was raised against rOv-CF was tested in a sandwich ELISA for detection of coproantigen in the feces of experimentally infected hamsters. The sandwich ELISA using this chicken IgY in combination with rabbit antibody to O. viverrini somatic antigens showed sensitivity and specificity of 93.3% and 78.57%, respectively. Together these findings indicated the potential of rOv-CF for diagnosis of opisthorchiasis, including for uses with chicken IgY for detection of coproantigens of O. viverrini.

Chicken IgY-based coproantigen capture ELISA for diagnosis of human opisthorchiasis

“…There are reports describing the coproantigen detection in trematodes such as F. hepatica [32] and C. sinensis [33]. We have reported the establishment of coproantigen detection method using recombinant Ov -CF in hamsters infected with O. viverrini [22]. Here we applied the cathepsin F capture ELISA for coproantigen diagnosis of human opisthorchiasis.…”

“…The coding region of secretory O. viverrini cathepsin F was expressed in Escherichia coli, Bl-21 strain (DE3) as previously [22]. Briefly, the cDNA encoding the protease was amplified from an library of O. viverrini cDNAs [24] using the forward primer 5`- G CA TAT G AG AAC TAC CCC ATT CGAGCC TG and the reverse primer 5`- G CA TAT G CT ATT TGA CAA CGG CTG TAG TAA CTG C; Nde I restriction enzyme sites are underlined.…”

“…Egg laying Gallus domesticus chickens were immunized as described [22]. Briefly 0.5 mg of purified r Ov -CF was mixed with equal amount of complete adjuvant followed by 4 boosts with 3 weeks intervals.…”

“…A coproantigen sandwich ELISA for detection of O. viverrini cathepsin F was carried out as described in experimentally hamsters [22]. Briefly wells of 96-well Maxisorp ELISA plates (Nunc, Denmark) were coated with 5 µg of CF-IgY in 100 µl of carbonate buffer, pH 9.6.…”

“…Interestingly, IgY does not activate the mammalian complement system, and does not bind to mammalian rheumatoid factors, protein A or G or cross-react with mammalian immunoglobulin which make it a better choice for both diagnostic and therapeutic applications [21]. With these advantages of chicken IgY and the abundance of O. viverrini cathepsin F, we have successfully developed IgY-based capture ELISA in a hamster model of opisthorchiasis [22]. In this study, we applied this coproantigen detection technique to the diagnosis of human opisthorchiasis from an endemic region of northeastern Thailand.…”