Antigen-loss requires the simultaneous binding of antibodies with different epitope specificities. (A) C57BL/6 mice were injected with the indicated combinations of monoclonal antibodies. Mice were transfused with a mixture of mHEL RBCs labeled with DiI and C57BL/6 RBCs labeled with DiO. Six days after transfusion, peripheral blood was obtained and stained with polyclonal anti-HEL antiserum followed by fluorescently labeled anti–mouse IgG; anti-HEL staining was measured by flow cytometry. The combinations are arranged to maximize functional groupings of antibodies, and a complete table is presented that shows each condition twice but allows easier pattern analysis. Antigen loss is designated as “LOSS,” whereas no antigen loss is indicated by “ø.” The outcome of combining an antibody with itself, which is the same as injecting the antibody alone, was taken from the data with isolated antibodies in Figure 3. This experiment was reproduced in its entirety 2 times with identical results. Additional experiments tested smaller groups of antibody combinations with identical findings. (B) Each monoclonal antibody was directly conjugated to Alexa Fluor 647. mHEL RBCs were preincubated with the indicated unconjugated monoclonal antibodies followed by staining with the indicated conjugated antibodies. Blocking by unconjugated antibodies was measured by flow cytometry. Blocking in both directions is defined as epitope identity (I), failure to block as nonidentity (ø), and blocking in only one direction as partial identity (P). This experiment was reproduced 2 times with identical results.