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. Author manuscript; available in PMC: 2017 Jul 1.
Published in final edited form as: Transfusion. 2016 Apr 4;56(7):1763–1774. doi: 10.1111/trf.13584

Fig. 3.

Fig. 3

Blocking ADAMTS13 inhibition with rabbit anti-idiotypic IgG. (A) ScFv 1-416 inhibited ADAMTS13 in the absence of rabbit IgG and in the presence of pre-immune rabbit IgG, but not in the presence of post-immune anti-scFv 1-416 rabbit IgG (right-hand set of bars; *, P=0.006; **, P=0.008; N.S., not significant). No scFv or irrelevant human anti-Rh(D) scFv E1M2 had any effect on ADAMTS13 activity under any rabbit IgG conditions (left-hand and middle sets of bars). Residual ADAMTS13 activity with scFv 1-416 and no rabbit IgG or pre-immune IgG (white and gray bars in right-hand set of bars) is higher than in Fig. 1 because the amount of scFv was 2.5-fold lower here to increase the sensitivity of rabbit post-immune IgG blocking. (B) In the presence of scFv 1-428, ADAMTS13 activity was inhibited when mixed with no rabbit or pre-immune rabbit IgG, but was “rescued” in presence of post-immune rabbit IgG (4th set of bars) as for scFv 1-416 in (A) (*, P=0.002; **, P=0.0006). Results were unchanged if performed in the presence of an 8-fold excess of human VH3-33-encoded anti-Rh(D) scFv E1M2 (5th set of bars) or human anti-keratinocyte VH1-69-encoded PX4-3 (6th set of bars). First 3 sets of bars are controls showing show that scFv’s E1M2 and PX4-3 had no effect on ADAMTS13 activity themselves. In this experiment, the amount of rabbit IgG was titered down to the point of just being able to block 1-428 in order to increase its sensitivity to any effects of E1M2 or PX4-3. (C) Gray bars show reduction of ADAMTS13 activity in normal plasma when mixed with heat-treated (56°C for 30 min to destroy any residual patient plasma ADAMTS13) TTP4-TTP7 plasmas and pre-immune rabbit IgG. Black bars show various degrees of “rescue” of ADAMTS13 activity if TTP4-TTP7 plasmas were pre-incubated with post-immune rabbit anti-idiotypic IgG raised against one of 4 inhibitory scFv’s as indicated. All differences significant (P range, 0.005 – 0.041) except for those marked “N.S.” (P range, 0.059 – 0.098). ADAMTS13 activities are averages of 2 measurements except for TTP7/1-431 which were measured once (*).