Development of ready-to-use microtiter plates for enhanced ELISA detection of Opisthorchis viverrini antigens in urine samples.

A monoclonal antibody against Opisthorchis viverrini antigen (OvAg) immobilization step for OvAg detection in urine samples by ELISA significantly influences the reduction in overall procedural time to conduct the assay. Herein, the monoclonal antibody against OvAg immobilization step was improved using a combining approach between a crosslinking agent, APTES, and a coupling agent EDC-NHS. Results showed that the combining approach method is effective when immobilization of monoclonal antibody is applied for approximately 30 min providing excellent potential for OvAg detection by ELISA. Moreover, it exhibited a good linear relationship with OvAg concentration in a range of 0.98 to 31.25 ng mL. The limit of detection was 2 ng mL which was a 15-fold improvement compared to that of conventional immobilization. The approach shows high diagnostic sensitivity, specificity, and accuracy, 91.36 %, 80.36 %, and 84.44 %, respectively, compared to the conventional immobilization method. The combining approach method for monoclonal antibody immobilization has been verified in field settings for OvAg detection in urine samples with excellent diagnostic performance. This approach effectively reduces procedural time and enhances sensitivity for detecting Opisthorchis viverrini in urine samples. Providing ready-to-use microtiter plates supports the detection, control, and elimination of human opisthorchiasis in resource-limited areas of the Mekong River Basin, Southeast Asia.