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 cutoff 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 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.

Keywords: Cathepsin F; Coproantigen detection; Food-borne trematodiasis; Immunodiagnosis; Opisthorchiasis; Opisthorchis viverrini.

Fig. 1. Expression, purification and immunoreaction of recombinant cathepsin F from Opisthorchis viverrini

(A) Gel image of the gene encoding Ov-CF amplified from a cDNA library prepared from adult stage O. viverrini. M: 50 bp ladder. Lane 1: O. viverrini cathepsin F at 927 bp is shown by the white arrow. (B) SDS-PAGE analysis of the recombinant Ov-CF protease. M: molecular mass standards (masses shown on the left side); lane 1: lysate of E. coli with rOv-CF before induction; lane 2–4: lysate of E. coli with rOv-CF after induction for 1h, 2h and 3h respectively; lane 5: flow throw, Lane 6–9 rOv-CF fractions 1–4 after affinity purification and refolding. (C) Western blot analysis of a pool of positive human sera from individuals with parasitologically proven infection with O. viverrini. Pooled negative human sera was used as the control, (N and P) negative and positive human opisthorchiasis sera, respectively.

Fig. 2. Efficacy of rOv-CF as antigen in an immunodiagnostic test

(A) Receiver operating characteristic (ROC) curve of recombinant cathepsin F from Opisthorchis viverrini (rOv-CF) employed as the antigen in an indirect ELISA for parasitologically negative and positive opisthorchiasis individuals. The curve represents the suitable cut-off point at OD₄₅₀ 0.4003 and showed the area under the curve (AUC) was 0.7579 (95% confidence interval (0.692 to 0.823). (B) Serodiagnosis of O. viverrini infection among different infection intensity groups using the rOv-CF indirect ELISA. Data represent means ± 2 SD and are representative of 1 and 4 parasitologically negative and positive group respectively; these data show the positive correlation between EPG and rOv-CF indirect ELISA OD₄₅₀.

Fig. 3. Production, purification and specific immunoreaction of chicken IgY antibody raised against rOv-CF

(A) Extraction and purification of anti rOv-CF chicken IgY. M: Protein standards ladder. Lane 1: Position of heavy chain of purified anti rOv-CF IgY indicated with an arrow. (B) Immunoblot analysis of anti rOv-CF IgY. M: Protein standards. Lane 1: anti rOv-CF IgY recognized the purified rOv-CF antigen by revealing a single band of 40 kDa, indicated with an arrow.

Fig. 4. Efficacy of rOv-CF as an antigen detection immunodiagnostic test

Receiver operating characteristic curve of recombinant cathepsin F (rOv-CF) in IgY-based sandwich ELISA, calculated from OD at 450 nm values for feces from parasitologically negative and positive hamsters for opisthorchiasis. The curve represents the suitable cut-off point at OD 0.5601 and showed that the area under the curve (AUC) was 0.958 (95% confidence interval (0.872 to 0.992).