Abstract: Objective To develop a novel animal model of erythrocyte alloimmunity via an antigen engineering strategy, thus providing a foundational platform for investigating the immune response triggered by erythrocyte transfusion. Methods The model antigen ovalbumin (OVA) was specifically modified onto the surface of C57BL/6J mouse erythrocytes using ethyl carbodiimide hydrochloride (EDC) coupling technology to prepare antigen-engineered erythrocytes (RBC-OVA). Mice of the same strain were continuously transfused with RBC-OVA, and the adhesion and clearance processes of erythrocytes in the liver and spleen were dynamically tracked using two-photon in vivo imaging technology. Results The OVA antigen modification efficiency reached 99.5%, with no significant impact on erythrocyte morphology, hemolysis rate, or cell membrane surface markers (CD47, PS). After four transfusions, the anti-OVA IgG antibody concentration in the serum of the model mice significantly increased, and the antibody specifically bound to RBC-OVA. Upon re-infusion of OVA-modified red blood cells, the peripheral blood recovery rate was significantly lower than that of the control group, with rapid clearance observed in the liver and spleen. Conclusion This model effectively mimics the clinical red blood cell alloimmune response and provides a robust experimental platform for investigating the mechanisms underlying antibody-mediated transfusion failure and for developing targeted intervention strategies.

Key words: RBC alloimmunization, Animal model, Antigen engineering, EDC conjugation