Pvdf membrane based syringe filter unit

Directly after antigen loading, the OVA-encapsulated microspheres (~20 mg) were rinsed with ddH₂O, as described above, to remove any unencapsulated OVA. The microspheres were then immediately resuspended in 0.5 mL of PBS, pH 7.4, and incubated at 37 °C under constant agitation (240 rpm/min). At different incubation times (1, 3, 5, and 7 days, and then every 7 days until day 49), the mixture was centrifuged at 7000 rpm for 5 min, and the supernatant was collected through a low protein-binding Durapore (PVDF) membrane-based syringe filter unit (Millipore Corporation, USA). Fresh PBS release media (0.5 mL) was then added to the sample, and the microspheres were resuspended to continue the release study. The OVA content in the supernatants was analyzed by SE-HPLC.

After self-encapsulation of the protein, the microsphere samples were centrifuged at 7000 rpm for 5 min. The supernatant was passed through a low protein-binding Durapore (PVDF) membrane-based syringe filter unit (Millipore Corporation, USA) and the filtrate collected. The microspheres were then washed once with ddH₂O and the rinse filtered and collected. A modified Bradford assay was used to determine OVA concentration in the filtrates. Coomassie Plus reagent (Thermo Fisher Scientific, USA) was added to the appropriate volume of standard OVA solution or filtrate sample in a 96-well plate (Nunc, Thermo Scientific, USA). After 10 min, the absorbance was read at 595 nm using a Dynex II MRX microplate reader (Dynex Technology Inc., USA). The mass of OVA encapsulated by the microspheres was calculated by subtracting the mass of OVA in the filtrates from the mass of OVA in the initial loading solution. Percent w/w loading and encapsulation efficiency (EE) were quantified with the following formulas:
$$\%\mathrm{w}/\mathrm{w}\text{ loading}=\frac{\text{mass of OVA encapsulated by the microspheres}}{\text{mass of microspheres in loading sample}+\text{mass of OVA encapsulated}}\times 100\%\text{EE}=\frac{\text{mass of OVA encapsulated by the microspheres}}{\text{mass of OVA in initial loading solution}}\times 100$$