Monoclonal antibody development against the Hepatitis C virus core protein enables rapid antigen detection.

Hepatitis C virus (HCV) remains a major global health burden, with limited access to affordable diagnostics impeding progress toward the World Health Organization target elimination by 2030. While HCV ribonucleic acid (RNA) testing is the gold standard, it requires costly infrastructure and technical expertise. HCV core antigen (HCVcAg) represents a reliable alternative viral analyte, but existing immunoassays remain laboratory-dependent and expensive. Here, we report the development of HCVcAg-specific monoclonal antibodies (mAbs) optimized for rapid, instrument-free antigen detection. BALB/c mice were immunized with HCVcAg, and antigen-specific plasma cells were isolated via single cell sorting on the Beacon optofluidic platform. Paired variable heavy/light regions were recovered by RT-nested PCR, cloned into human IgG1/kappa expression vectors, transiently expressed in HEK293 cells, and the mAbs were purified. Comprehensive molecular profiling identified mAbs with high avidity, diverse germline usage, and distinct epitope recognition. Strategic pairing yielded an optimal combination (M0004-M0007) capable of detecting contrived antigen concentrations as low as 0.3125 ng/ml. In a lateral flow format, this pair achieved superior diagnostic performance: 92.0% sensitivity, 100% specificity, 95.6% accuracy, 100% positive predictive value (PPV), 91.3% negative predictive value (NPV), Cohen's κ = 0.91, area under the curve (AUC) = 0.859, and non-significant McNemar χ². In low viremia samples, performance remained robust with 85.7% sensitivity, 100% specificity, 94.3% accuracy, 100% PPV, 91.3% NPV, κ = 0.88, 0.759 AUC, and non-significant McNemar χ². These findings establish a foundation for a clinically deployable HCVcAg rapid test with strong potential for point-of-care screening, decentralizing diagnosis, and treatment-response monitoring, particularly in resource-limited settings.