Immunomagnetic Sample Preparation Targeting Host- and Viral-Derived Antigens for HIV-1 Isolation from Limited Plasma Volumes

Rapid and accurate HIV detection is crucial for early diagnosis and viral load (VL) monitoring in individuals receiving antiretroviral therapy (ART). To address the challenge of limited sample volume, we developed a novel immunomagnetic sample preparation method to isolate HIV-1 virions from 25 μL of plasma─about one-tenth of a small finger-prick sample. Immunomagnetic conjugates were produced by coupling antimouse IgG magnetic beads with mouse IgG antibodies targeting host- and virus-derived antigens on the HIV surface. In buffer spiked with inactivated HIV-1 grown in the HUT-78 cell line, these conjugates achieved capture efficiencies of 45-97%. Optimization of conjugate concentrations (0.71-5.71 mg/mL) improved capture efficiency by 19%, with 15 min identified as the optimal capture time. In spiked plasma (25 μL), immunoconjugates targeting different surface antigens captured inactivated HIV-1 across inputs of 148-53,145 RNA copies/25 μL (5,920-2,125,800 RNA copies/mL) with consistent efficiencies ranging from 41% to 100%. In clinical specimens from individuals living with HIV with VLs ranging from 100,000 to >1 million copies/mL, capture efficiencies varied from 5% to 60% depending on the conjugate used. Notably, combining CD44 and CD46 targeting conjugates, which recognize different surface markers, did not enhance virion capture, suggesting the coexpression of targeted antigens on HIV-1. Together, our results established immunomagnetic isolation as a promising approach for HIV-1 sample preparation, enabling more accessible, sensitive, and rapid VL quantification.