Rapid and sensitive detection of HIV-1 p24 antigen by immunomagnetic separation coupled with catalytic fluorescent immunoassay

In this study, a system of magnetic beads (MBs) coupled with catalytic fluorescent immunoassay for rapid and sensitive determination of HIV-1 capsid antigen p24 was developed. p24 was captured by antibody immobilized MBs, and the detection antibody was linked to horseradish peroxidase (HRP) through biotin–streptavidin recognition, catalyzing the oxidation of o-phenylenediamine (OPD) and hydrogen peroxide to produce a fluorescent product. This is the first reported utilization of the fluorescence of OPD oxidation product catalyzed by HRP for immunoassay. Optimization of conditions afforded a low detection limit of 0.5 pg/mL (3σ) for p24 with a linear range of 1.4–90.0 pg/mL. The assay exhibited good reproducibility with a relative standard deviation (RSD) of 4.4 %, 4.7 %, and 5.0 % for detecting 1.4 pg/mL, 22.5 pg/mL, and 45.0 pg/mL p24, respectively. The assay can be completed in less than 90 min. Moreover, the proposed method was successfully applied to detect p24 in spiked serum. This method overcomes the interference of MBs to the fluorescence signal and demonstrated higher sensitivity for detection of p24 than conventional ELISA kits. The system could be applied for detecting other antigens with high sensitivity, rapidity, specificity, and simple operation.