KLH-conjugated synthetic RhD peptide significantly enhances in vitro antibody production, rivaling full-length protein.

The RhD antigen is a highly immunogenic blood group antigen, posing a significant risk of hemolytic transfusion reactions (HTRs) and hemolytic disease of the fetus and newborn (HDFN). Current methods for generating anti-RhD antibodies often rely on full-length RhD protein, which faces limitations due to restricted antigen availability, high production costs, and ethical concerns associated with in vivo immunization. Developing an efficient, ethical, and reproducible in vitro platform for antibody generation is crucial to overcome these challenges and ensure safer blood transfusion practices.

Researchers isolated human peripheral blood mononuclear cells (PBMCs) from O-negative blood units via Ficoll density gradient and depleted immunosuppressive cells using L-leucine methyl ester. PBMCs were stimulated with either full length RhD protein, synthetic RhD peptide, or peptide conjugated to Keyhole Limpet Hemocyanin (KLH). Conjugation efficiency was confirmed by ELISA and spectrophotometry (280/412 nm). Cultures were supplemented with IL-4, IFN-γ, and conditioned medium, then incubated at 37°C for one week. Antibody production in the culture supernatants was quantified using ELISA.

The synthetic RhD peptide demonstrated significant immunogenicity, particularly when conjugated. Peptide-KLH induced higher antibody levels at 84.87±13.6 ng/mL compared to free peptide at 52.4±9.8 ng/mL. While RhD protein alone elicited 108.7±10.4 ng/mL, a combined stimulation with peptide-KLH and RhD protein further enhanced antibody levels to 157.9±23.6 ng/mL. This synergistic effect highlights the potential for multi-antigenic approaches. A two-step immunization protocol significantly enhanced responses, yielding a P=0.006. The combination of IL-4 and IFN-γ was identified as providing the highest cytokine-driven enhancement of antibody production. > The combined stimulation of PBMCs with peptide-KLH and RhD protein achieved the highest antibody levels at 157.9±23.6 ng/mL, surpassing individual stimulations.