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HuCAL anti Human sclerostin antibody, clone AbD09097 (HCA230Z) used in a functional assay to inhibit internalization of sclerostin and sclerostin modulated LRP6 signaling.
Inhibition of Sclerostin function by antibodies.
(A) Sclerostin-GFP was pre-incubated with Fc or the different Fabs raised against Sclerostin for 30 minutes at room temperature, next the mixtures were added to 293-LRP6 cells for 1.5h at 4°C. After binding the cells were transferred to a 37°C incubator for 30 minutes, next the cells were fixed and confocal images were taken. (B) KS483 cells were affinity labeled with [125I]Sclerostin that was pre-incubated for 30 minutes at room temperature with controls (Fc and aGFP-IgG) or Sclerostin neutralizing antibodies (AbD9097 and aSCL-IgG), followed by chemical crosslinking. Cells were lysed and samples were separated by SDS-PAGE. The radioactive signal was visualized using a phosphorimager screen. (C) KS483 cells were transiently transfected with the BAT-luc reporter construct. The cells were stimulated with conditioned control media (pcDNA CM) or Wnt3a conditioned media (Wnt3a CM), together or without Sclerostin conditioned (SCL CM) media that was pre-incubated with either anti GFP-IgG2a (aGFP-IgG) or anti Sclerostin-IgG2a (aSCL-IgG) for 30 minutes at room temperature. (D) KS483 cells were stimulated with control conditioned media or Wnt3a conditioned media (Wnt3a CM), together or without Sclerostin conditioned media that was pre-incubated with either anti GFP-IgG2a (aGFP-IgG) or anti Sclerostin-IgG2a (aSCL-IgG) for 30 minutes at room temperature. RNA was isolated and AXIN2 mRNA levels were determined by qRT-PCR. (E) KS483 cells were stimulated with the indicated conditioned media, which were pre-incubated with the indicated Fab antibodies. Three days after stimulation cells were fixed and stained for alkaline phosphatase activity. Results were quantified using spectrometry (F).
From: van Dinther M, Zhang J, Weidauer SE, Boschert V, Muth E-M, Knappik A, et al. (2013)
Anti-Sclerostin Antibody Inhibits Internalization of Sclerostin and Sclerostin-Mediated Antagonism of Wnt/LRP6 Signaling.
PLoS ONE 8(4): e62295.