Improved performance and quantitative detection of copro-antigens by a monoclonal antibody based ELISA to diagnose human opisthorchiasis

Copro-antigen detection has been advocated as a promising method for diagnosis of opisthorchiasis, particularly in people that harbored light infection or have had recent drug treatment. This study aimed to improve performance of a monoclonal antibody-based enzyme-linked immunosorbent assay (Mab-ELISA) for detection of Opisthorchis viverrini copro-antigen and assess the correlation between copro-antigen and intensity of infection. Four different treatment methods of 71 samples from the Lawa endemic area, Khon Kaen province were assessed for copro-antigen detection, namely (1) phosphate buffer saline (PBS), (2) heating (70 °C 30 min), (3) alkaline (pH 9.6 in carbonate buffer), and (4) trichloroacetic acid (TCA) treatment. Comparison of these protocols showed that the TCA method gave the best performance in discriminating O. viverrini positive and negative samples with high sensitivity (97.9%) and moderate specificity (54.2%) compared with other methods. Application of TCA-based Mab-ELISA method for antigen detection in fecal samples obtained from an endemic area of opisthorchiasis revealed that 86 of 141 samples (61.0%) were positive compared with 68 (48.2%) by PBS-based protocol, while the formalin ethyl-acetate concentration technique (FECT) yielded a positive proportion of 71.6%. Among 40 egg-negative samples confirmed by a gold standard parasitological method (FECT) from the same endemic area, 19 (47.5%) were positive by the TCA-based while only 6 (15%) were positive by PBS-based Mab-ELISA protocol. In addition, levels of antigen detection significantly correlated with intensity of infection (egg per gram feces). The results show that the improved Mab-ELISA method has high sensitivity and also quantifiable diagnosis of opisthorchiasis.