Infinite f50 plus

The enzymatic activity of SgVnDPPIV was assayed with substrate Ala-Pro-p-nitroanilide (Sigma-Aldrich (Shanghai) Trading Co., Ltd., Shanghai, China) following the method described by Tereshchenkova et al. [3 (link)]. The substrate was initially dissolved in dimethylformamide, and its final reaction concentration was 0.25 mM. SgVnDPPIV (1 µg) was combined with 2.5 µL substrate. The freshly prepared PBS (phosphate-buffered saline) (0.1 M, PH 8.0) was added to the final volume of 200 µL. The mixture was incubated at 40 °C for 30 min. Its absorbance was measured periodically using an Infinite F50 Plus and Infinite F50 Robotic microplate reader (TECAN, Männedorf, Switzerland) at 405 nm for 60 min. Venom of S. guani and gut extract from the T. molitor larvae were used as the positive controls. The effects of the inhibitors, including vildagliptin, sitagliptin, diprotin A, and diprotin B (Sigma-Aldrich (Shanghai) Trading Co., Ltd., Shanghai, China), on the enzymatic activity of SgVnDPPIV were tested. The inhibitor concentration was 0.1 mM in the incubate assay with the SgVnDPPIV before its reaction with the substrate, as described above. All assays were performed in three independent biological replicates. The enzymatic activity was expressed in units (U).

TNF-α concentrations were measured in sera collected from healthy controls and patients with COVID-19. The human TNF- α uncoated ELISA kit (Invitrogen, Bender Med Systems GmBH, Vienna, Austria) was used to measure TNF-α levels within a detection range of 0.0128–5000 pg/mL. Standard curves were generated by making serial dilutions of a known concentration of TNF-α. The absorbance of sera from patients with COVID-19 and healthy controls were measured using an ELISA microplate reader (Infinite F50 Plus, Tecan, Männedorf Switzerland). The absolute concentration of TNF- α was calibrated using standard curves as suggested by the manufacturer.

CEE-cELISA was performed as previously described [11 (link)]. Briefly, 96-well plates (Thermo Fisher Scientific) were coated with SPEEDA^® rabies vaccine (Biovalys) overnight at 4 °C. The next day, the plates were washed with 0.05% Tween-20 in PBS 5 times prior to the addition of the blocking agent 3% BSA for 1 h at 37 °C. The blocking agent was removed before the addition of serially diluted serum samples at dilutions of 1:100, 1:200 and 1:400 together with diluted mouse mAb for competitive binding. The plates were incubated for 1 h at 37 °C, followed by washing and the addition of HRP-conjugated goat antimouse IgG as the secondary antibody. The plates were incubated at 37 °C for 1 h before removal and washing. To develop the samples, 3,3′,5,5′-tetramethylbenzidine (TMB) was added to the plates, followed by the addition of 1 N H₂SO₄ stop solution. The optimal density (OD) was measured at 450 nm using an Infinite F50 Plus (Tecan). The cutoff for adequate antibody levels was ≥ 0.7 EU/mL.