Enhancing Red Blood Cell Compatibility: Mimicking O-Negative RBC Compatibility Using a Trispecific Triabody as a Blocking Fragment for Blood Group Antigens

Background 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 (RBCs) are universally compatible and essential for emergency transfusions; however, their scarcity, particularly in low-resource regions, poses significant challenges. To help overcome this challenge, a compact trispecific triabody was designed to block A, B, and Rh(D) antigens on RBCs, thereby conferring universal compatibility similar to O-negative RBCs. Results In this study, two combinations based on the placement of the anti-Rh(D) variable chain at the C- or N-terminus were generated, and fusion proteins from the first combination produced the closed (C1) and open (O1) triabodies. Intramolecular cooperative binding affinities of the selected triabody-C1 were predicted computationally using blood group antigens A, B, and Rh(D), with no significant changes observed in binding free energies. The triabody-C1 was expressed in Escherichia coli BL21(DE3) using a two-plasmid system and purified through a three-step process, yielding two fractions, AE3-B1 and AE3-B2. The hemagglutination potential of the triabody was evaluated both visually and microscopically through immunocytochemistry. Visually, no hemagglutination was observed, while microscopically, mixed-field hemagglutination occurred when AB+ and AB − RBCs were incubated with the triabody in fraction AE3-B1, but not when A+ or B+ RBCs were tested. No hemagglutination of AB+ RBCs was detected with the triabody in fraction AE3-B2. ELISA-based cooperative binding assays using free antigens showed that the triabody’s monomers functioned independently, with no changes in binding parameters K _D or B _max . In contrast, assays with RBC-bound antigens revealed increased K _D and decreased B _max across successive binding events, particularly when Rh(D) antigens were engaged first. Hemagglutination assays confirmed that triabody-coated RBCs exhibited a complete absence of hemagglutination with anti-A, anti-B, and anti-Rh(D) IgM antibodies, confirming effective antigen blocking. Conclusions The trispecific triabody effectively blocks A, B, and Rh(D) antigens, rendering RBCs with O-negative like universal compatibility and offering a promising strategy to expand the supply of universally transfusable blood, particularly in emergency and resource-limited settings.