Figure 3.
Identification of the epitopes of VWF-activating nanobodies. (A) Representative BLI sensorgrams of immobilized AIM-A1, A1-CAIM, NAIM-A1, or tAIM-A1 binding to a serial dilution of purified nanobody 6C4 (blue), 6C11 (red), or 6D12 (green). Nanobody concentration ranges are 2 μM to 2 nM for 6C4 binding to AIM-A1, 250 nM to 2 nM for 6C11 and 6D12 binding to AIM-A1, and 500 nM to 1 nM for all others. The fitted affinity, when obtained, is shown in each panel. For AIM-A1, association was performed for 180 seconds and dissociation for 360 seconds. For A1-CAIM, NAIM-A1, and tAIM-A1, association was performed for 80 seconds and dissociation for 200 seconds. The fitting trace for each concentration is shown in black. (B) Western blots of nonreduced (NR) or reduced (R) AIM-A1 with noted nanobody or antibody 6G1. To accommodate the fast off-rate of 6C4, a biotinylated rabbit anti-VHH antibody (bio-RaVHH) and streptavidin-IRDye680 were used to detect blotting of AIM-A1 by 6C4. After blotting with 6C11 and 6D12, a rabbit anti-VHH antibody (RaVHH) and fluorescent goat anti-rabbit antibody (GaR) were used to visualize the blots. 6G1 binds to a linear epitope in the CAIM, and it was detected using a fluorescent goat anti-mouse secondary antibody (GaM). (C) Dot blots of biotinylated NAIM peptides (10 ng per spot) and bio-AIM-A1 (1 μg per spot). Each membrane strip was blotted with noted nanobody separately and detected with RaVHH and fluorescent GaR. To verify proper adsorption of biotinylated peptides and bio-AIM-A1 protein to the strip, the same strip was blotted with streptavidin-IRDye680. (D) ELISA binding isotherms of 6D12 to immobilized glycosylated AIM-A1 purified from mammalian cells (orange circles) and nonglycosylated AIM-A1 purified from E coli (black squares). Data are shown as mean ± SD (n = 3). Responses were fit to a hyperbola and the KD (10.7 ± 3.2 nM) was derived from half-maximal binding. (E) BLI binding traces of 6D12, in a dilution series, to immobilized NAIM-Fc, 1253-1266-Fc, and neuraminidase-treated NAIM-Fc (asialo-NAIM-Fc). Global kinetic fitting at ratio 1:1 is shown in red, and kinetic KD is listed on top of the sensorgrams.

Identification of the epitopes of VWF-activating nanobodies. (A) Representative BLI sensorgrams of immobilized AIM-A1, A1-CAIM, NAIM-A1, or tAIM-A1 binding to a serial dilution of purified nanobody 6C4 (blue), 6C11 (red), or 6D12 (green). Nanobody concentration ranges are 2 μM to 2 nM for 6C4 binding to AIM-A1, 250 nM to 2 nM for 6C11 and 6D12 binding to AIM-A1, and 500 nM to 1 nM for all others. The fitted affinity, when obtained, is shown in each panel. For AIM-A1, association was performed for 180 seconds and dissociation for 360 seconds. For A1-CAIM, NAIM-A1, and tAIM-A1, association was performed for 80 seconds and dissociation for 200 seconds. The fitting trace for each concentration is shown in black. (B) Western blots of nonreduced (NR) or reduced (R) AIM-A1 with noted nanobody or antibody 6G1. To accommodate the fast off-rate of 6C4, a biotinylated rabbit anti-VHH antibody (bio-RaVHH) and streptavidin-IRDye680 were used to detect blotting of AIM-A1 by 6C4. After blotting with 6C11 and 6D12, a rabbit anti-VHH antibody (RaVHH) and fluorescent goat anti-rabbit antibody (GaR) were used to visualize the blots. 6G1 binds to a linear epitope in the CAIM, and it was detected using a fluorescent goat anti-mouse secondary antibody (GaM). (C) Dot blots of biotinylated NAIM peptides (10 ng per spot) and bio-AIM-A1 (1 μg per spot). Each membrane strip was blotted with noted nanobody separately and detected with RaVHH and fluorescent GaR. To verify proper adsorption of biotinylated peptides and bio-AIM-A1 protein to the strip, the same strip was blotted with streptavidin-IRDye680. (D) ELISA binding isotherms of 6D12 to immobilized glycosylated AIM-A1 purified from mammalian cells (orange circles) and nonglycosylated AIM-A1 purified from E coli (black squares). Data are shown as mean ± SD (n = 3). Responses were fit to a hyperbola and the KD (10.7 ± 3.2 nM) was derived from half-maximal binding. (E) BLI binding traces of 6D12, in a dilution series, to immobilized NAIM-Fc, 1253-1266-Fc, and neuraminidase-treated NAIM-Fc (asialo-NAIM-Fc). Global kinetic fitting at ratio 1:1 is shown in red, and kinetic KD is listed on top of the sensorgrams.

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