A Rapid Chemiluminescence Immunoassay for Plasmin-α2 antiplasmin Complex Utilizing Novel Monoclonal Antibodies

Abstract

This study aimed to develop novel, highly specific monoclonal antibodies for the rapid and precise detection of the plasmin-α2-antiplasmin complex (PIC), an early biomarker indicative of fibrinolytic activation. This approach addresses the limitations of existing methods, which are often timeconsuming and susceptible to interference from abundant free plasminogen and α2-antiplasmin in plasma, thereby improving the diagnosis and management of venous thromboembolism (VTE) and related disorders. We successfully generated self-assembled PIC complexes via co-expression technology to immunize mice and subsequently screened a pair of monoclonal antibodies (4C11 and 3E5) that specifically target novel epitopes of the PIC, exhibiting high affinity and specificity, with affinity constants of 1.0 × 10⁻¹² M and 8.9 × 10⁻¹⁰ M, respectively. Utilizing this antibody pair, we established a single-integrated chemiluminescence immunoassay capable of completing detection within 15 minutes. The developed assay demonstrated excellent analytical performance, characterized by a detection limit as low as 0.06 μg/mL, a broad linear range (0.08-40 μg/mL), and a total coefficient of variation below 10%. Multicenter clinical evaluations further confirmed strong concordance with the reference method (R² = 0.99), alongside enhanced sensitivity and specificity, as evidenced by an area under the curve (AUC) of 0.95 compared to 0.90. In summary, we have developed a rapid, high-performance PIC detection platform based on novel monoclonal antibodies. This platform is compatible with point-of-care testing (POCT) and represents a highly promising and reliable tool for the clinical assessment of hyperfibrinolysis.