Analysis of wild-type and deglycosylated anti-RBC antibodies and their ability to induce RBC clearance and AMIS. Wild-type and deglycosylated antibodies were first evaluated for the efficiency of glycan removal. Glycans were released from IgG heavy chains by in-gel PNGase F digestion and then labeled with 2-aminoanthranilic acid and analyzed by UPLC. The symbols used for different glycan structures is based on Harvey et al.²²  (A) Heavy chains of all samples had detectable glycan structures in the wild-type form of the heavy chains (blue lines), but no detectable structures after deglycosylation (red lines). The loss of the heavy chain signal after the deglycosylation reaction with PNGase F indicates that the Fc glycan removal was successful. (B) The ability of anti-RBC antibodies (MIMA 29 and CBC-512) to induce RBC clearance was analyzed by determining the percentage of surviving PKH26⁺ HOD-RBCs in the circulation of mice. All mice except the naive treatment group received 10⁸ PKH26-labeled HOD-RBCs IV by tail vein injection. After 24 hours, mice were injected with no antibody (HOD) or 5 μg of each antibody assessed: wild-type MIMA 29 (MIMA 29), deglycosylated MIMA 29 (deMIMA 29), wild-type CBC-512 (CBC-512), or deglycosylated CBC-512 (deCBC-512). The percentage of remaining PKH26⁺ HOD-RBCs in the circulation was evaluated before (0 hour) and 2, 24, 48, and 72 hours after antibody injection. Mice were bled for serum 7 days after PKH26⁺ HOD-RBC transfusion and HEL-specific IgM (C) and IgG (D) antibody levels were evaluated by enzyme-linked immunosorbent assay. Data represent individual values from mice from ≥3 separate experiments. Data were expressed as mean ± standard error of the mean and analyzed by 1-way analysis of variance with Tukey’s multiple comparison test. ns, not significant. **P < .01; ***P < .001; ****P < .0001.