Binding of ab157289 to Osteoprotegerin. ab157289 binds to Osteoprotegerin:Fc. Method: Ligand binding assay: 96 well ELISA plates were coated O/N with 50 ng OPG-Fc per well. After a blocking step, the indicated concentrations of ab157289 were added for 1 hour. Bound ligand was revealed with an anti-FLAG antibody at 1 µg/ml for 30 minutes, followed by a rabbit anti-mouse IgG-HRP at 1/1000 dilution for 30 minutes. OPD was used as a substrate for the peroxidase. Absorbance was measured at 490 nm in an ELISA reader.

Mononuclear cells differentiate into Osteoclasts in the presence of M-CSF and ab157289. Method: Human CD14+ mononuclear cells isolated from adult peripheral blood were cultured in control medium (upper panel), in 25ng/ml M-CSF (middle panel), or in 25ng/ml M-CSF plus 50ng/ml ab157289 (lower panel). Osteoclasts were identified as Tartrate-Resistant Acid Phosphatase (TRAP)-positive multinucleated cells. Osteoclasts were detected exclusively in presence of ab157289 and in these culture conditions (M-CSF + ab157289), cells fused and generated multinucleated dark red TRAP-positive cells (arrows). Nuclei were stained with haematoxylin.