*4.7. Innate Humoral Parameters* 4.7.1. Antiprotease Activity

The antiprotease activity was determined as described by Ellis et al. [57], with some modifications. Briefly, 10 μL of plasma were incubated with the same volume of trypsin solution (5 mg mL−<sup>1</sup> in NaHCO3, 5 mg mL<sup>−</sup>1, pH 8.3) for 10 min at 22 ◦C. After incubation, 100 μL of phosphate buffer (NaH2PO4, 13.9 mg mL−1, pH 7.0) and 125 μL of azocasein solution (20 mg mL−<sup>1</sup> in NaHCO3, 5 mg mL−1, pH 8.3) were added and incubated for 1 h at 22 ◦C. Finally, 250 μL of trichloroacetic acid were added to the reaction mixture and incubated for 30 min at 22 ◦C. The mixture was centrifuged at 10,000× *g* for 5 min at room temperature. Afterwards, 100 μL of the supernatant was transferred to a 96 wellplate and mixed with 100 μL of NaOH (40 mg mL−1). The OD was read at 450 nm in a Synergy HT microplate reader (Biotek, Winooski, VT, USA). Phosphate buffer instead of plasma and trypsin served as blank, whereas the reference sample was phosphate buffer instead of plasma. The sample inhibition percentage of trypsin activity was calculated as follows: 100 – ((sample absorbance/Reference absorbance) × 100). All analyses were conducted in duplicates.
