Fig. 3

LILRB2 targeting antibody Ab29 showed potent blocking activity, high affinity, and specificity. a-b. Titration of blocking the activity of Ab29 against oAβ or PS-LILRB2 interactions. Plate-coated oAβ (a) or PS (b) was incubated with LILRB2-chimeric reporter cells under the presence of increasing concentrations of Ab29 or control IgG. The activation of LILRB2-chimeric reporter cells was observed as a percentage of GFP⁺ cells. Data are presented as mean ± SD (n = 3 independent experiments). c. Representative immunostaining images showing Ab29 blocks oAβ and PS from binding to LILRB2 expressed on the cell surface. Scale bar represents 5 μm. d. Ab29 blocks LILRB2 binding with oAβ as measured by BLI. LILRB2 was loaded onto protein A sensors via binding with sensor-captured Ab29. The LILRB2-loaded sensors were then incubated with biotinylated oAβ (1 μM) and the binding signals were amplified with streptavidin. The amount of oAβ bound onto the sensors was presented as wavelength shift in nanometers (nm). The red dotted vertical line marks the transit from incubation with biotinylated oAβ to signal amplification by streptavidin. e. Ab29 blocks LILRB2 binding with PS liposomes as measured by BLI. LILRB2 was loaded onto protein A sensors via binding with sensor-captured Ab29. The LILRB2-loaded sensors were incubated with PS liposomes (1 mM) (association stage) and then the sensors were dipped into kinetics buffer without PS allowing the bound PS to freely dissociate (dissociation stage). The amount of PS bound onto the sensors was presented as wavelength shift in nanometers (nm). The red dotted vertical line marks the transit from association stage to dissociation stage. f. Cross-reactivity of Ab29 against other LILRB and LILRA family receptors as measured by ELISA. Plate-coated LILRB and LILRA family receptors (shown in the x-axis) were incubated with Ab29 (4 nM). The amount of bound antibody was detected by anti-human F(ab)2 HRP, and the values are shown as OD₄₅₀. LILRB2-Fc was included as the positive control. Data are presented as mean ± SD (n = 4 independent experiments)