P nitrophenyl phosphate solution

The enzymatic activity of ALP was measured in osteoblasts at days 0, 2, 7 and 14, according to previously published methods [57 (link)], adapted to our experimental conditions. Briefly, the osteoblast cultures were rinsed twice with PBS (Sigma-Aldrich, 38297 Saint-Quentin Fallavier, France) prior to freezing at −20 °C. The cells were then lysed by freeze–thaw cycles and homogenized in diethanolamine/magnesium chloride hexahydrate buffer (pH 9.8; Sigma-Aldrich). The cell lysates (10 μL) were added to 200 μL of p-nitrophenyl phosphate solution (6 mg/mL) (Sigma-Aldrich, St. Louis, MO, USA). Absorbance was assessed at 405 nm, 30 °C, every 150 s for 30 min, using an ELX808 microplate reader (BioTek Instruments Inc, Winooski, VT, USA). Protein measurement was performed using a BioRad protein assay (BioRad, Munich, Germany). ALP activity was expressed as micromoles of p-nitrophenol per hour per milligram of protein [58 (link)].

TfRMAb-TNFR fusion protein binding to TNF-α (Additional file 2: Fig. S1B), and TfRMAb-TNFR and etanercept plasma concentrations were quantified by a sandwich ELISA. The human TNF-α (hTNF-α) (PeproTech, Rocky Hill, NJ, USA) was the capture agent and 2 µg/mL of hTNF-α was plated in 96-well plates, and incubated overnight at 4 ºC. The wells were blocked with TBS containing 1% bovine serum albumin (TBSB) (0.01 M Tris/0.15 M NaCl/1% BSA/pH7.4) for 30 min at room temperature. Plasma samples (diluted 1:10 in TBSB) were added to the wells and incubated for 1 h at room temperature followed by washing with TBST. Alkaline phosphatase-conjugated detector agents, goat anti-human IgG-Fc fragment antibody (Bethyl, TX, USA) that binds to the human Fc domain of etanercept and goat anti-mouse light chain (kappa) antibody (Bethyl Laboratories, Inc., TX, USA) that binds to the TfRMAb domain of the TfRMAb-TNFR, were added to the wells for 1 h at room temperature followed by washing with TBST. Wells were then incubated with P-nitrophenyl phosphate solution (Sigma Aldrich, St. Louis, MO, USA) for 15 min in the dark at room temperature and the reaction was stopped by adding 1.2 M NaOH. Blank corrected absorbance measured at 405 nm was used to calculate plasma concentrations using a standard curve.

Development and validation of an E.coli ClpB immunoassay has been described [18 (link)]. In brief, rabbit polyclonal anti-E.coli K12 ClpB antibodies (Delphi Genetics) were coated onto 96-well Maxisorp plates (Nunc, Rochester, NY). One hundred microliters of plasma samples or ClpB protein (Delphi Genetics) standard dilutions were incubated for 2 h at room temperature (RT). Mouse monoclonal anti-E.coli K12 ClpB antibodies (Delphi Genetics) and goat anti-mouse alkaline phosphatase conjugated IgG (Jackson ImmunoResearch Laboratories Inc., West Grove, USA) were used for ClpB detection and p-nitrophenyl phosphate solution (Sigma-Aldrich, St. Louis, USA) as a substrate. The reaction was stopped by 3 N NaOH and optical density was determined at 405 nm using a microplate reader Metertech 960 (Metertech Inc., Taipei, Taiwan). Concentration was measured by referring to the ClpB protein standard curve.

Direct enzyme-linked immunosorbent assays (ELISAs) were used to determine anti-norovirus serum IgG titers. Medium binding polystyrene plates were coated with 2 µg/mL norovirus VLPs. Unbound VLPs were removed by washing the plate 5 times in PBS-T (phosphate buffered saline + 0.025% Tween 20). The plates were blocked overnight at 4 °C with 5% Blotto in PBS-T. After 5 washes, the plates were incubated for 1 h at room temperature with duplicate serum samples diluted 1:50 in blocking solution. Unbound antibody was removed by washing 5 times with PBS-T. The secondary antibody (alkaline phosphatase-labeled rabbit α-human IgG, Sigma-Aldrich Co., St. Louis, MO, USA) was diluted 1:2500 in blocking solution and incubated for 30 min at room temperature. After a final 5 washes in PBS-T, 100 µL of p-nitrophenyl phosphate solution (Sigma-Aldrich Co.) was added to each well, and the plate was incubated at room temperature in the dark for 10 to 30 min. The optical density (OD) at 405 nm was determined using an ELx800 plate reader (BioTek Instruments, Inc. Winooski, VT, USA). For each norovirus VLP evaluated, positive control serum from confirmed cases was included on each plate, except for GIV.1, where positive serum from a previous seroprevalence study was used.

Antibody was raised in rabbit against SPA4 peptide, and was affinity purified (Thermo Fisher Scientific, MA). An ELISA method was developed using SPA4 peptide and affinity-purified antibody. Briefly, microwell strips were coated with 0.1 μg SPA4 peptide or BSA per well diluted in 0.1 M NaHCO₃, pH 9.6, overnight at 4°C and at room temperature for 1 h. Nonspecific sites were blocked using 1% BSA in Tris buffered saline containing 0.05% Tween 20 (TBST), for 1 h at 30°C. The microwells were then washed with TBST and incubated overnight with 1:10, 1:100, 1:1000 diluted mouse serum samples or 1:2000 diluted affinity purified SPA4 peptide-specific antibody (1.34 mg/ml, positive control) at 4°C. After washing with TBST, the microwells were then incubated with 1:2000 diluted alkaline phosphatase-conjugated anti-mouse IgG (whole molecule) or anti-rabbit IgG (whole molecule) antibody at 30°C for 1 h. Finally, the immune complexes were detected by using p-nitrophenyl phosphate solution (Sigma-Aldrich, MO). The reaction was stopped by the addition of 30 μl of 3 M NaOH, and the optical density (OD) was read at 405 nm spectrophotometrically. The ∆OD405 readings were obtained by subtracting the reading obtained for BSA-coated wells from those obtained from SPA4 peptide-coated wells for the mouse serum samples and positive control.