**3. Results and Discussion**

### *3.1. Antibody Immobilization Efficiency*

The primary antibody (anti-TNF-α antibody) immobilization at the surface of electrospun nanofiber meshes (NFMs) was performed in a wide range of concentrations (0–12 μg/mL) to determine the maximum antibody immobilization capacity. The fluorescence of the unbound secondary antibody was measured and the values were plotted against the various primary antibody concentrations (Figure 1). As this is an indirect quantification method, higher fluorescence values of the unbound secondary antibody correspond to lower concentrations of anti-TNF-α primary antibody immobilization. Moreover, when the substrate is saturated, the fluorescence values of unbound secondary antibody reach a plateau, despite the increment on antibody concentration.

**Figure 1.** Box plot of anti-TNF-α antibody immobilization at concentrations ranging from 0 to 12 μg/mL. Data were analysed by nonparametric way of a Kruskal-Wallis test followed by Tukey's HSD test: (**a**) denotes significant differences compared to concentration 0, (**b**) denotes significant differences compared to concentration 1, (**c**) denotes significant differences compared to concentration 2, and (**d**) denotes significant differences compared to concentration 4.

The statistical analysis showed that the immobilized primary antibody, with concentrations above 4 μg/mL, displayed significantly lower values of fluorescence intensity than 0 μg/mL (*p* < 0.01). Concentrations of 6 μg/mL and 8 μg/mL exhibited significantly lower values of fluorescence intensity when compared to all lower concentrations (*p* < 0.001). Moreover, these two concentration values

did not display differences between themselves. Finally, when the antibody was immobilized at a concentration of 12 μg/mL, the fluorescence intensity of secondary antibody did not present significant differences when compared to the fluorescence intensity of NFMs with primary antibody immobilized at concentrations above 2 μg/mL (*p* < 0.01). Therefore, primary antibody immobilization at the surface of the nanofibrous substrate reached a maximum concentration of 6 μg/mL, since no statistically significant differences were found between the concentrations above. This value is within the range of concentrations systemically administrated in clinical practice using Infliximab (3–10 mg/kg or 3–10 μL/mL, considering water as body mass) [52].
