M999 competes with TRC105 for ENG binding and both antibodies block BMP9/ENG interaction.

<p>A) Sequence of protein layering in the Bio Layer Interferometry antibody competition assay: 1) a strepatavidin-coated biosensor is loaded to saturation with 2) biotinylated recombinant human Endoglin (10 μg/ml), then with 3) a negative control IgG1k or the M999 anti-Endoglin rat IgG2 antibodies (10 μg/ml) prior to the addition of 4) M999, TRC105, SN6, or SN6h (10 μg/ml). (B) Wavelength shifts (WS) triggered by the addition of the individual anti-ENG antibodies to ENG in the presence of a ctrl antibody (blue bars), or in the presence of the anti-ENG antibody M999 (red bars). Competition with M999, calculated as (WS^CTRL-WS^M999)/WS^CTRL*100 is indicated at the bottom of the graph. (C) Sequence of protein layering in the Bio Layer Interferometry BMP9 binding assay: 1) a strepatavidin-coated biosensor is loaded to saturation with 2) biotinylated recombinant human Endoglin (10 μg/ml), then with 3) a negative control IgG1k or the tested anti-ENG antibodies (M999, TRC105, SN6, SN6h) (10 μg/ml) prior to the addition of 4) BMP9 (10 μg/ml). (B) Wavelength shifts (WS) triggered by the addition of BMP9 in addition to the individual anti-ENG antibodies. BMP9 binding inhibition, calculated as [1-(WS^ENGAB/WS^CTRLAB)]*100, is indicated at the bottom of the graph. Results are the mean +/− standard deviation of technical triplicates.