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. 2016 Aug 1;11(8):e0160388. doi: 10.1371/journal.pone.0160388

Fig 5. Characterization of the ex vivo inhibitory effect of anti-mMDTCS mAbs 13B4 and 14H7.

Fig 5

Adamts13+/+ mice (n = 4, per condition) were injected with 2.50 mg/kg of mAb 13B4, 14H7 or 20A10 or with a combination of mAbs 13B4 and 14H7 (1.25 mg/kg each) on day 0 (black arrow). The optimal injection dose of mAb was determined in separate experiments (data not shown). Blood was retrieved 7 days before (‘day -7’) and 1, 3, 5, 7 and 14 days post injection. (A) The influence of the different mAbs on the proteolytic activity of mADAMTS13 was determined using the FRETS-VWF73 assay. Activities were calculated based on the slope of the proteolysis reactions (S1 Fig). (B) Plasma mAb levels (μg/mL) were determined using ELISA. Plates were coated with recombinant mADAMST13, blocked and plasma of the respective mice was added. Bound mAbs were detected using GAM-HRP. (C) The amount of mADAMTS13 (%) in plasma was determined using ELISA. Plasma mADAMTS13 was captured using the anti-mT2-CUB2 mAb 9F2. After blocking, the respective plasma samples were added. Finally, bound mADAMTS13 was detected using the polyclonal anti-mADAMTS13 rabbit IgG and GAR-HRP. (D) Platelet counts were measured of the respective mice samples. Error bars represent the SD (n = 4, per condition). (E) The plasma mVWF multimer pattern was determined for a new cohort of treated mice (n = 5, per condition) 7 days before (‘day -7’) and 1 and 3 days post injection of mAb(s) 20A10 or the combination of mAbs 13B4 and 14H7). Representative multimer patterns are given. Low, middle and high molecular weight (respectively LMW [1–5 bands], MMW [6–10 bands] and HMW [>10 bands]) multimers and UL-VWF multimers (brace) are indicated. (F) The percentage HMW multimers was calculated using the ImageJ 1.48v software.