Chemical crosslinking of OVA and HEL to RBCs. OVA and HEL were crosslinked to leukoreduced RBCs, as described in “Study design.” HEL-RBCs and OVA-RBCs were stained with rabbit anti-HEL or rabbit anti-OVA, respectively (A-B). Cells were washed and incubated with goat anti-rabbit immunoglobulin conjugated to allophycocyanin. Staining of cells was analyzed by flow cytometry. OVA-RBCs were labeled with DiO, and unmodified RBCs were labeled with CM-DiI, as previously described.⁷  A mixture of labeled cells was transfused into C57BL/6 mice. Twenty-four hours after transfusion, peripheral blood was obtained, and the percentages of circulating RBCs were determined (C). The same blood specimen analyzed in panel C was stained with anti-OVA, as in panel B, and after appropriate gating, fluorescence was compared on transfused OVA-RBCs (solid line) compared with transfused unmodified RBCs (dashed line) (D). RBC ghosts were prepared by hypotonic lysis and were subjected to Western blot analysis with anti-OVA under reducing (E) or nonreducing (F) conditions. Standard curves of OVA were also generated using Western blots. To control for any changes in OVAimmunoreactivity as a result of the crosslinker, the OVA standard curve was generated using unreacted LC-SPDP-modified OVA.