Evaluation of liver fluke recombinant cathepsin B-1 protease as a serodiagnostic antigen for human opisthorchiasis

Abstract

A cathepsin B-like cysteine protease belonging to family C1 is abundantly expressed in the transcriptome and proteome of the carcinogenic liver fluke of humans, Opisthorchis viverrini. This enzyme is present in excretory/secretory (ES) products released by parasites cultured in vitro. This study evaluated the performance of recombinant O. viverrini cathepsin B1 (rOv-CB-1) as an antigen for immunodiagnosis of opisthorchiasis. The full length Ov-CB-1 cDNA was cloned and recombinant protein was produced in catalytically active form in Pichia pastoris. The recombinant Ov-CB-1 (rOv-CB-1) was affinity purified via nickel-NTA chromatography and tested in enzyme-linked immunosorbent assays (ELISA) with human sera from an opisthorchiasis endemic area. Sera from egg-positive O. viverrini infections produced a strong IgG antibody response to rOv-CB-1 both in ELISA and immunoblot analysis. The sensitivity and specificity of the ELISA test was 67% and 81%, respectively. These findings support the feasibility of using recombinant Ov-CB-1 in ELISA for the serodiagnosis of human opisthorchiasis.

Immunodiagnosis of opisthorchiasis using parasite cathepsin F

“…With human sera, the indirect ELISA showed 62 % sensitivity and 84 % specificity, indicating the potential value of rOv- CF in immunodiagnosis of the infection. The diagnostic sensitivity and specificity of rOv- CF indirect ELISA were comparable to the performance of the r Cs CB protease as antigen in serodiagnosis of clonorchiasis (Chen et al 2011), Sm31 for S. mansoni (Klinkert et al 1991) or indeed recombinant cathepsin B of O. viverrini (termed Ov-CB-1 ) for serodiagnosis of opisthorchiasis (Sripa et al 2012). …”

“…The strips were incubated for 2 h in goat anti-human IgG conjugated with horseradish peroxidase (Thermo Scientific, USA) at a dilution of 1:1000. Subsequently, the strips were washed with PBST three times and a final wash with PBS after which immunoreactivity was visualized with the substrate o -diaminobenzidine (DAB) (Thermo Scientific, USA) (Sripa et al 2012). …”

“…The sensitivity, specificity and positive and negative predictive values analyzed using the formalin-ethyl acetate concentration technique (WHO, 1995) as the gold standard method. The statistical significance between the different groups was performed with one-way ANOVA and two sample t -test (Sripa et al 2012). Correlation between OD 450 and the ordinal infection level variable was calculated using Spearman’s correlation coefficient.…”

“…Earlier reports indicate that lysates of O. viverrini or excretory secretory products from these flukes are useful as antigens for serodiagnosis (e.g., Wongratanacheewin et al 1988; Akai et al 1995). However, defined or purified Opisthorchis antigens may provide more sensitivity and specificity (Wongratanacheewin et al 2003) and several purified parasite proteins have been tested with variable results including in our own laboratory (Laha et al 2008; Sripa et al 2012). …”

Opisthorchis viverrini: Analysis of the sperm-specific rhophilin associated tail protein 1-like

“…In Thailand, O. viverrini has attracted intense scientific interest due to the connection of opisthorchiasis and cholangiocarcinoma, a bile duct cancer with a uniquely high prevalence rate in this country (Sripa et al, 2007). Among others, released antigens, tegumental surface antigens and egg antigens of the parasite have been analyzed for their application as diagnostic markers, targets for intervention or their role in tumorigenesis (Ruangsittichai et al, 2006;Pinlaor et al, 2009;Smout et al, 2009;Eursitthichai et al, 2010;Piratae et al, 2012;Sripa et al, 2012) and yet, as this research will demonstrate, antigens of the parasite sperm could also be involved in the host-parasite interaction and might warrant further attention.…”