A trispecific triabody targeting A, B, and Rh(D) antigens for antigen blocking to expand red blood cell transfusion compatibility: An in vitro assessment

The need for compatible blood remains a persistent challenge to timely and safe transfusion support. Despite this, the stability and functional behavior of antigen-blocking strategies under storage and physiologically relevant conditions remain poorly understood, particularly regarding their stability during prolonged exposure to changing blood environments. This study evaluates a trispecific triabody designed to simultaneously target A, B, and Rh(D) antigens on red blood cells to block antigen recognition and prevent incompatibility-related hemagglutination. Triabody-coated RBCs were prepared through repeated incubation cycles and assessed for stability in incompatible plasma under storage (4 °C) and physiologically relevant temperatures (37 °C). Results showed that the triabody bound efficiently and reached saturation across tested blood groups. During storage at 4 °C, detachment remained minimal during early storage but increased after prolonged incubation, coinciding with declining pH and increased free hemoglobin. At 37 °C, triabody detached progressively, with anti-B dissociation occurring earlier than anti-A and anti-Rh(D), while differential stability was also observed during prolonged storage at 4 °C. Detachment showed a closer temporal association with pH decline than with ionic strength or free hemoglobin changes and was followed by hemagglutination in incompatible plasma. Triabody-coated RBCs maintained antigen blocking during early exposure but showed differential stability during prolonged incubation, supporting their potential to improve transfusion compatibility while highlighting the need for further in vitro optimization of triabody binding stability before in vivo validation.