Verification of Coupling of Amelogenin and Emdogain® to Plastic by Enzyme-linked Immunosorbent Assays (ELISA)

and Emdogain® to Plastic by Enzyme-linked Immunosorbent Assays (ELISA) To assess whether recombinant amelogenin and Emdogain® used in the cell attachment experiments indeed coupled to plate surfaces, we coated a two-fold serial concentration range (1.5 g–1.5 and 0 ng/well) of each protein in duplicate on Dyclone 2HB 96-microwell plates overnight at 4°C in MAM, imitating the conditions used in the cell attachment experiments. Subsequently, plates were rinsed thoroughly to remove unbound protein with PBS, 0.5% (v/v) Tween-20, pH 7.4, and non-specific binding sites were blocked with 2.5% (w/v) BSA in PBS, pH 7.4, for 1 hr at RT. To detect bound recombinant amelogenin, we used a polyclonal antibody, which is raised in rabbits and is specific for the recombinant human amelogenin used in the studies. The working concentration was 1/2400 in PBS, 0.05% (v/v) Tween-20, 0.25% (w/v) BSA, pH 7.4, and the incubation time was 1 hr at RT°C. We also used this primary antibody to detect Emdogain® after coating, because a major proportion of this porcine-derived product is amelogenin that is also recognized by the antibody. After further rinses, bound primary antibody was reacted with a goat anti-rabbit alkaline-phosphatase-conjugated antibody (BioRad) used at a concentration of 1/1000 for 1 hr at RT°C. The reaction was visualized with PNPP (Sigma) as substrate, and the color reaction was monitored and recorded on a Dynex microplate reader at 405 nm. Negative controls included wells with no protein-coated or no primary antibody.