Detection of membrane-bound TT30 on PNH RBCs from an eculizumab-treated PNH patient. RBCs from a PNH patients on eculizumab were incubated 1 hour in aNHS with or without TT30 (± the anti-CR2 blocking mAb 1048); TT30 was detected by using either an anti-fH mAb (panels B,D,F,H) or an anti-CR2 mAb (C,E,G,I), both FITC-conjugated. The biotinylated anti-C3d mAb A702 was used to detect C3d (with APC-streptavidin); an APC-conjugated anti-CD59 was used to identify PNH RBCs. (A) Gating strategy on PNH RBCs. RBCs from an eculizumab-treated patient were used for this experiment; CD59 APC (y-axis) versus side scatter (SSC). PNH RBCs were identified by CD59 expression, and gated accordingly for further analysis of TT30 binding. (B-C) TT30 binding on fresh RBCs. Fresh PNH RBCs have a substantial C3d deposition, but do not show any cross-binding with the anti-fH or anti-CR2 mAb (B-C, respectively). C3d biotinylated-Streptavidin PE (y-axis; mAb A702) versus either fH or CR2-FITC (x-axis; B-C, respectively); analysis on CD59− RBCs (gate as shown in panel A). (D-E) TT30 binding after exposure in aNHS + TT30. After in vitro exposure to aNHS + TT30 3μM, TT30 can be detected on PNH (CD59−) RBCs by both the anti-fH (D) and the anti-CR2 (E). The saber-like pattern indicates the colocalization of C3d and TT30 on PNH RBC surface, with the most pronounced bound-TT30 detected on RBCs with the most abundant C3d deposition. C3d biotinylated-Streptavidin PE (y-axis; mAb A702) versus either fH or CR2-FITC (x-axis; D-E, respectively); analysis on CD59− RBCs (gate as shown in panel A). (F-G) TT30 binding after blocking by the anti-CR2 mAb 1048. Preincubation of TT30 with a 3-fold excess of the anti-CR2 mAb 1048 (TT30 1μM and mAb 1048 3μM) abrogates TT30 binding on PNH RBCs. C3d biotinylated-Streptavidin PE (y-axis; mAb A702) versus either fH or CR2-FITC (x-axis; F-G, respectively); analysis on CD59− RBCs (gate as shown in panel A).