A new enzyme-linked fluorescence assay (ELFA) for use with peroxidase-antibody conjugates: a comparison with ELISA for the quantitation of IgM antibodies to hepatitis B core antigen

This study describes a new enzyme-linked fluorescence assay (ELFA) using peroxidase-antibody conjugates and p-hydroxyphenylacetic acid (PHPA) as a fluorogenic substrate. The assay was developed to quantitate IgM antibodies against hepatitis B core antigen (HBcAg) and compared directly with the standard chromogenic ELISA method. The ELFA employs microtitration plates coated with anti-human IgM to capture patient antibodies, followed by addition of HBcAg antigen and peroxidase-labeled anti-HBcAg IgG. Fluorescence is generated when peroxidase oxidizes PHPA in the presence of hydrogen peroxide. Testing was performed on multiple sera including controls, HBsAg carriers, and acute hepatitis B patients. Results demonstrated excellent correlation between ELFA and ELISA methods, particularly in high-antibody samples (r=0.95, p<0.001). The fluorescent product proved stable and unaffected by light exposure. The ELFA showed comparable sensitivity to ELISA while offering potential advantages as a fluorescence-based immunoassay for future automation and optimization.

Key findings

• A new ELFA technique using p-hydroxyphenylacetic acid as substrate for peroxidase-conjugated antibodies was successfully developed and validated
• ELFA results showed strong correlation with standard ELISA for detecting IgM anti-HBcAg antibodies (r=0.95 in acute hepatitis B sera)
• The fluorescent product generated was highly stable and resistant to light degradation, remaining stable for at least 24 hours
• Both assays effectively differentiated between acute hepatitis B patients, HBsAg carriers, and control groups with statistical significance (p<0.001)
• ELFA demonstrates comparable sensitivity to ELISA and represents a useful addition to sensitive antibody detection methods with potential for technical improvement