Enhancing red blood cell compatibility: in vitro hemagglutination prevention using a trispecific triabody as a blocking fragment for blood group antigens

Access to safe and timely blood transfusion is a cornerstone of modern healthcare but depends on a stable supply of voluntary donations and rigorous hemovigilance systems. O-negative red blood cells are universally compatible and essential for emergency transfusions; however, their scarcity, particularly in low-resource regions, poses significant challenges. To address this challenge, a compact trispecific triabody was designed to block A, B, and Rh(D) antigens on RBCs. In this study, two triabody configurations differing in the placement of the anti-Rh(D) variable domain were generated, producing closed (C1) and open (O1) formats. The selected triabody-C1 was expressed in Escherichia coli BL21(DE3) and purified into two fractions, AE3-B1 and AE3-B2. Hemagglutination assays demonstrated that AE3-B2 did not induce hemagglutination, whereas AE3-B1 showed mixed-field hemagglutination under standard conditions and complete hemagglutination under potentiator-enhanced hemagglutination conditions. ELISA-based binding assays indicated that the triabody’s monomers functioned independently with free antigens, while RBC-bound antigen assays revealed altered binding behavior upon sequential antigen engagement. Blood incompatibility related hemagglutination assays using monoclonal antibodies and incompatible O-negative blood plasma demonstrated complete prevention of hemagglutination by AE3-B2 triabody-coated RBCs, confirming effective antigen blocking. The trispecific triabody efficiently prevents hemagglutination by blocking A, B, and Rh(D) antigens on RBCs, enabling them to exhibit enhanced compatibility and hemagglutinating patterns similar to O-negative cells. These findings provide a promising strategy to increase the pool of compatible blood for transfusion, particularly in emergency and resource-limited settings, while emphasizing that future in vivo investigations are needed to confirm efficacy and safety.