Ultrasensitive and Specific Detection of Neutralization-Correlated Serum Antibodies Via Secondary Antibody−DNA Linker-Mediated Magnetic Bead−Based Proximity Extension Assay

Abstract Sensitive, accurate, and readily deployable detection of neutralizing antibody (NAb) titers is essential for evaluating vaccine efficacy and population-level immunity. Here, we report an Antibody-DNA Linker (AND Linker) comprising a fusion of Protein A/G, a G4S flexible peptide, and duck circovirus replication initiator protein (DCV). This design enables orthogonal, site-specific assembly: the N-terminal Protein A/G binds the IgG Fc region, while the C-terminal DCV covalently captures 5′ ssDNA at a defined 1:1 molar ratio. Unlike random chemical crosslinking, which suffers from heterogeneous attachment sites, variable stoichiometry, and poor reproducibility, the AND Linker ensures uniform functional orientation and minimal batch-to-batch variation. By optimizing ssDNA secondary structure, we further enhanced DCV recognition efficiency. Integrating this linker into a magnetic bead-based proximity extension assay (termed SADL-MPEA), we achieved femtomolar-level detection of serum antibodies highly correlated with neutralizing activity. For HPV16 and HPV18, SADL-MPEA demonstrated limits of detection (LOD) approximately three orders of magnitude lower than conventional PEA using random chemical conjugation. In clinical serum validation against the pseudovirus neutralization test (PBNT), SADL-MPEA showed strong concordance (Pearson’s r = 0.84−0.85), confirming its utility as a reliable surrogate for functional neutralization capacity. Diagnostic performance was exceptional, with sensitivities of 90.9% (HPV16) and 83.3% (HPV18), 100% specificity for both, and an area under the ROC curve of 0.99. Crucially, SADL-MPEA requires only antigen-coated magnetic beads to adapt to new targets, as the DNA-labeled secondary antibody remains universal. This platform offers a simple, high-throughput, and scalable solution for NAb-correlated antibody detection in research and clinical settings.