Peroxidase conjugated avidin

ELISA was used to quantitate the amount of a few important pro-osteogenic proteins in the DBM and BE-DBM. Equal amounts of both materials (10 mg) were placed in 96 well plates in quadruplicates. The material was blocked with 5% BSA for 1 h and incubated with the appropriate primary antibody overnight at 4°C. The samples were then washed four times in PBS and incubated with a biotinylated secondary antibody (1/250, Vector Laboratories) and then subsequently washed four times with PBS and incubated with peroxidase conjugated avidin (Vector Laboratories) for 1 h at room temperature. Finally, the samples were incubated with an ELISA substrate (Turbo TMB ELISA substrate, Thermo Scientific) for 5 min at room temperature. The reaction was stopped by the addition of 1 M sulfuric acid. The solution was then transferred to empty 96 well plate wells and absorbance at 490 nm was recorded. The following primary antibodies were used: rabbit polyclonal anti fibronectin antibody (1/500), mouse monoclonal anti BMP2 antibody (1/250), rabbit polyclonal anti VEGF antibody (1/250), mouse monoclonal anti phosphorylated serine, threonine, and tyrosine antibody (1/1000, abcam).