Ultrasensitive chemiluminescent detection of plasma Aβ1-40 based on soluble macromolecular signal amplification carrier.

The research presents the development of an ultrasensitive chemiluminescent immunoassay (CLIA) for detecting the Alzheimer's disease (AD) biomarker amyloid-β (Aβ) 1-40 in plasma, utilizing novel monoclonal antibodies and an innovative soluble macromolecular signal amplification carrier, Ficoll400-Streptavidin (Ficoll400-SA) conjugates. The study successfully generated two high-affinity monoclonal antibodies (KD ≈ 10-10 M), designated 2F10 and 12H6, which are epitope-specific and target distinct N-terminal and C-terminal regions of Aβ1-40. These antibodies demonstrated exceptional specificity, exhibiting minimal cross-reactivity (<0.5%) with closely related isoforms like Aβ1-42. The Ficoll400-SA carrier, formed by conjugating multiple streptavidin molecules to the Ficoll400 backbone and further crosslinking, enabled substantial signal amplification by facilitating rapid, homogeneous reactions and high-density enzyme loading capacity. Optimization of assay conditions demonstrated a 13-fold signal enhancement compared to conventional streptavidin-horseradish peroxidase (SA-HRP) systems, achieving an ultra-low detection limit of 0.28 pg mL-1 and a broad dynamic range (0.54-1000 pg mL-1). The assay exhibited excellent precision (CV < 8%), robust stability under accelerated and onboard conditions, and outstanding specificity with negligible cross-reactivity. Clinical validation against a commercial Aβ1-40 assay using human plasma samples showed a remarkable correlation (Spearman's ρ = 0.99) and no systematic bias, confirming the assay's accuracy and translational potential. Overall, this integrated approach offers a robust, cost-effective, and ultrasensitive platform for Aβ1-40 quantification, advancing blood-based AD diagnostics and demonstrating the versatility of soluble macromolecular carriers in immunoassay signal amplification.