2.4. Adsorption of PTH on Polysaccharide-Based Multilayer Films

A series of polysaccharide-based multilayer films were prepared to examine adsorption of PTH in vitro. Adsorption of PTH with an initial concentration of 1000 pg/mL on a series of multilayer films at the following time points were determined by using a PTH EIA kit (Sigma, Chicago, IL, USA): 10, 20, 30, 60, 120 and 180 min. Briefly, 25 µL of sample was collected at each time point and added to the 96-well microplate coated with secondary antibody, to which 50 µL Biotinylated PTH and 50 µL HRP-streptavidin solution was added. The sample was incubated for 2.5 h at room temperature with gentle shaking. Subsequently, the solution was discarded and washed 4 times with PBS solution. TMB regent (100 µL) was added to the solution, which was incubated for 30 min at room temperature in the dark with gentle shaking. Eventually, 50 µL of Stop Solution was directly added to each well, and measurements were taken at an absorbance of 450 nm in a multimode microplate reader (BioTek Instruments, Winooski, VT, USA).