Sigmafast p nitrophenyl phosphate

Enzyme-linked immunosorbent assay (ELISA) was performed as described previously [19] (link). NUNC Maxisorp plates were coated overnight with antigen at 5 µg/ml in coating buffer. Plates were then blocked with 1% BSA before serum was added in PBS-0.05% Tween-20 and diluted stepwise. Bound antibodies were detected using alkaline-phosphatase conjugated, goat anti-mouse secondary antibodies (Southern Biotech) and Sigma-Fast p-nitrophenylphosphate (Sigma Aldrich). The absorbance at ODλ₄₀₅ nm was determined using an Emax microplate spectrophotometer (Molecular Devices, Germany). Relative reciprocal titres were calculated by measuring the dilution at which the serum reached a defined ODλ₄₀₅ nm.

Maxisorp 96-well plates (Nunc, Sigma-Aldrich, St. Louis, MO, USA) were coated overnight with 2 µg/mL of abrin in 50 mM pH-9.6 Carbonate-Bicarbonate Buffer (Sigma, C3041), then washed and blocked with PBST buffer (0.05% Tween 20, 2% BSA in PBS) for one hour. Antisera were then added to the plates for a one-hour incubation. The plates were then washed with PBST and incubated with the detecting antibody, AP conjugated goat anti-rabbit (Jackson, 111-055-003) followed by detection with SIGMAFAST p-Nitrophenyl phosphate (Sigma, N1891).

Maxisorp 96-well microtiter plates were coated overnight with 5µg/ mL Streptavidin (Sigma, S0677), then washed and blocked as described above. Biotin labeled 15aa length peptides (Table S1) (5 µg/mL in PBST) were then added for 20 minutes. The plates were washed, anti-holotoxin_abrin/anti-A_abrinB_ricin/anti-A_ricinB_abrin antisera were diluted in PBST and added to the plates for one-hour incubation. The plates were washed and incubated with AP conjugated anti-rabbit antibodies (Jackson, 111-055-003) for 15 minutes followed by detection with SIGMAFAST p-Nitrophenyl phosphate (Sigma, N1891).

MC38 cells overexpressing negative control (NC), ADAM12 wildtype (A12) or catalytically inactive ADAM12 (A12^E359Q) were seeded at a density of 60 × 10³ cells/well in 12-well plates, incubated overnight, and transiently transfected with AP-EGF plasmid (kindly provided by Dr. S. Higashiyama, Ehime University Graduate School of Medicine, Ehime, Japan), using Lipofectamine 3000 and following the supplier’s protocol. After 24 h incubation, cells were washed and 450 µl of DMEM without FBS was added to each well and incubated for 1 h. Collected media were spun down at 400 g for 5 minutes and supernatants transferred to new tubes to ensure no cell carryover. Cells growing in monolayers in 12-well plates were lysed using RIPA buffer supplemented with 1× HALT Protease and Phosphatase inhibitor cocktail (Thermo Scientific). Abundance of alkaline phosphatase in conditioned medium or cell lysates was quantified by adding a 2 mg/ml solution of SIGMAFAST p-Nitrophenyl phosphate (Sigma-Aldrich), incubating the samples for 1 h at 37 °C, and measuring absorbance at 405 nm. Relative EGF shedding was quantified using the formula S = C/(L + C) where S—relative shed EGF, C—quantity of AP in conditioned medium, and L—quantity of AP in the cell lysate.

Ninety-six well Maxisorp plates (Nunc, Thermo Fisher Scientific) were coated with 1 µg/ml of GMMA preparations expressing antigen or 1 µg/ml of recombinant protein in PBS overnight (O/N) at 4 °C. The next day, plates were washed 3 times with T-PBS (0.05% Tween-20 in PBS, pH 7.4) and blocked with 100 µl 2% BSA (Sigma Aldrich) for 1 hour at 37 °C. Each incubation step was followed by triple T-PBS wash. Serum samples were initially diluted 1:200 in 2% BSA in T-PBS, transferred to coated-blocked plates and serially 2-fold diluted followed by a 2-hours incubation at 37 °C. Then 100 µl/well of alkaline phosphatase-conjugated goat anti-mouse IgG (H + L) (Southern Biotech) 1:2,000 diluted were added and incubated for 2 hours at 37 °C. Bound alkaline phosphatase was visualized by adding SIGMAFAST p-Nitrophenyl phosphate (Sigma Aldrich). After 30 minutes at room temperature, plates were analyzed at 405 nm in a microplate spectrophotometer. The endpoint titer of a sample is defined as the reciprocal of the highest dilution that has a reading above the cut-off using the formula described by Frey and collaborators^{53 (link)}.

ELISA plates were coated with 5 μg/ml of sFliC (24 h at 4°C) and blocked with 1% BSA overnight at 4°C. Serum was diluted 1:100 in PBS−0.05% Tween, and was further diluted stepwise. Plates were incubated for 1 h at 37°C. Bound antibodies were detected using alkaline phosphatase conjugated, goat anti-mouse IgG, IgG1, and IgG2c Abs (Southern Biotech). Alkaline phosphatase activity was detected using Sigma-Fast p-nitrophenylphosphate (Sigma Aldrich). Relative reciprocal titers were calculated by measuring the dilution at which the serum reached a defined OD₄₀₅.