Development and Evaluation of an IgY-Based ELISA for the Detection of Hepatitis B Surface Antigen

ABSTRACT Hepatitis B virus (HBV) remains a major global health burden, affecting over 254 million people worldwide, with a disproportionate impact on resource-limited countries due to late testing and limited access to affordable diagnostics. To address this need, this study aimed to develop an enzyme-linked immunosorbent assay (ELISA) for detecting the HBV surface antigen (HBsAg) using immunoglobulin Y (IgY) from chicken egg yolk. Laying hens were immunized with two synthetic peptides representing immunodominant HBsAg epitopes. Antigen-specific IgY was extracted from egg yolks via water dilution and salt precipitation, yielding a highly pure (>97%) antibody preparation as confirmed by SDS-PAGE. Immunoreactivity was assessed by indirect ELISA, which identified IgY raised against the combination of both peptides (P1+P2) as the most reactive, with an endpoint titer of 0.312 µg/100 µL. This anti-P1+P2 IgY was subsequently used in a sandwich ELISA format. When tested against clinical samples, the assay successfully discriminated all ten HBsAg-positive from ten negative human serum samples, with absorbance values for positives (1.360 – 2.265) significantly (P