Evaluation of the immunogenicity of polymerized hemoglobin solutions in a rabbit model

Chemical modification of proteins carries the risk that neo-antigens are introduced. To investigate the potential immunogenicity of human glutaraldehyde-polymerized hemoglobin (polyHbX1), we analyzed the antibody responses of rabbits after hyperimmunization with complete Freund's adjuvant. In view of the species difference, we also tested rabbit hemoglobin that was modified in the same way as human polyHbX1. Thereafter, we studied the antibody response after weekly intravenous infusion of clinically relevant doses of polyHbX1 to evaluate whether an immune response is likely to occur when modified hemoglobin is used as blood substitute. The occurrence of an antibody response was tested by using an enzyme immunoassay (ELISA). To find out whether antibodies were directed against neo-epitopes on human polyHbX1 we used a competitive ELISA. The results showed that polymerized hemoglobin may weakly activate the immune system in special conditions, but is unlikely to do so when it is used as blood substitute.