Identification of Commercially-Available Antibodies that Block Ligand-Binding by BMPR2
Medicine and Health Sciences
Osteoporosis, a disease of low bone mineral density, affects 10 million Americans and is a significant health problem and a considerable socioeconomic burden. Current treatments for osteoporosis have significant limitations, necessitating identifying new treatment strategies via building a better understanding of the endogenous mechanisms regulating bone mass. We recently demonstrated that removal of the BMP type 2 receptor, BMPR2, in skeletal progenitor cells of Bmpr2-cKO mice leads to reduced age-related bone loss due to a sustained elevation in bone formation rate. The molecular mechanism underlying this phenotype is being pursued in other work. In the present study, we sought to advance the translational potential of our genetic model by identifying antibodies that neutralize the ligand-binding function of the BMPR2 extracellular domain (BMPR2- ECD). We first established a modified, cell-free immunoprecipitation assay wherein the ligand BMP2 is pulled-down by BMPR2-ECD conjugated to Protein G beads; the unbound BMP2, which is found in the supernatant, is subsequently quantified by ELISA. This yielded a standard assay wherein approximately 2 ug BMPR2-ECD leads to 70% reduction in BMP2 signal. Next, we examined the neutralizing ability of 3F6, which is a mouse monoclonal antibody raised against the ligand-binding region of BMPR2, and found a dose-dependent inhibition of BMPR2-ECD ligand-binding. Given that the commercial availability of this antibody is as an ascites preparation, specificity of this assay was confirmed by demonstrating that ligand-binding activity of BMPR2-ECD is unchanged in the presence of non-specific, negative control ascites. Using these results as a guide, we then evaluated 1F12, which is another mouse monoclonal antibody raised against the ligand-binding region of BMPR2, and found that this antibody is also capable of neutralizing the ligand-binding function of BMPR2-ECD. These results provide proof-of-concept data for future studies evaluating inhibition of BMPR2 function in vivo as a means to reduce age-related bone loss.
Copyright all authors
Schoerning, Laura OMSII; Gorrell, Ruthann E.; Newby, Jordan B.; Hum, Julia M.; and Lowery, Jonathan W. Ph.D., "Identification of Commercially-Available Antibodies that Block Ligand-Binding by BMPR2" (2016). MU-COM Research Day. 18.
This document is currently not available here.