Covalently Oriented AuNP–Antibody Bioconjugates Enable Early and Sensitive Detection of <i>Opisthorchis viverrini</i> Urinary Antigen

<i>Opisthorchis viverrini</i> (OV) infection remains a major public health concern in Southeast Asia due to its strong association with cholangiocarcinoma. Early and accurate detection of OV infection is crucial for timely intervention and reduction of cancer risk. While enzyme-linked immunosorbent assay (ELISA)-based urine antigen detection methods have shown high sensitivity, their dependence on laboratory infrastructure limits their field utility. This study aims to develop and compare two gold nanoparticle (AuNP)-based lateral flow immunoassay (LFIA) formats utilizing distinct antibody conjugation strategies for rapid, noninvasive detection of OV excretory-secretory (OV-ES) antigens in urine. Two types of AuNP-antibody bioconjugates were prepared: (i) physical adsorption of monoclonal anti-OV antibodies on citrate-capped AuNPs (AuNP-Citr-mAb-OV) and (ii) covalent conjugation with orientation control on amino-terminated AuNPs (AuNP-TEG-NH-mAb-OV). These were incorporated as signal reporters in LFIAs, which were evaluated for sensitivity, specificity, detection limit, cross-reactivity, and diagnostic performance against urinary ELISA results by using clinical urine samples. LFIA based on covalently oriented AuNP-TEG-NH-mAb-OV (LFIA-TEG-NH-mAb-OV) demonstrated significantly lower limits of detection (1.54 vs 5.94 ng/mL), higher sensitivity (98.81 vs 96.83%), and improved specificity (81.94 vs 62.37%) compared to the LFIA based on passive adsorption format (LFIA-AuNP-Citr-mAb-OV). Moreover, LFIA-TEG-NH-mAb-OV showed better agreement with the ELISA reference (κ = 0.805) and lower cross-reactivity with other helminth infections. This is the first study to report the use of covalently oriented antibody-AuNP conjugates for early urinary detection of OV, offering a field-deployable, accurate, and scalable solution for the diagnosis and control of opisthorchiasis in endemic regions.