O phenylenediamine opd substrate

Recombinant ADAMTS13 (rADAMTS13) was donated from Shire (Benatzkygasse, Austria). Plasma-derived FXIa, α-thrombin, plasmin, and FXa were purchased from Haematologic Technologies (Essex Junction, VT, USA). α-FXIIa and kallikrein were from Enzyme Research Laboratories, Inc. (South, IN, USA). Rabbit polyclonal anti-ADAMTS13 antibody, specific for the MET domain, was from Abcam (Cambridge, MA, USA). Rabbit polyclonal anti-ADAMTS13 antibody, specific for the CUB1-2 domains, was from Santa Cruz Biotechnology (Dallas, TX, USA). Goat polyclonal anti-ADAMTS13 antibody, specific for the TSP4 domains, was from Bethyl Laboratories (Montgomery, TX, USA). Hirudin, O-phenylenediamine (OPD) substrate, and aprotinin were from Sigma-Aldrich (St. Louis, MO, USA). FRETS-VWF73 (VWF residues 1,596–1,668) was purchased from PeptaNova (Louisville, KY, USA).

The quantification of total IgG in children plasma from all groups was done by ELISA. At day 0, 7 and 30 after their inclusion in the study. Briefly, 96 microwell plates (reacti-bind 96 EIA Plate 100/PKG, Pierce) were coated with 5 μg/ml of purified sheep polyclonal anti-human IgG (Sigma-Aldrich, France) followed by overnight incubation at 4°C. After blockage with 1% gelatine and washing with 1% gelatin-PBS, serial dilutions of plasma samples were incubated overnight at 4°C. Bound IgG was detected using a peroxidase-conjugated polyclonal anti-human-IgG (The Binding Site, Birmingham UK). Binding was revealed using 0.5 mg/ml O-phenylenediamine (OPD) substrate (Sigma-Aldrich, France) and 0.03% H₂O₂ in 0.05 M phosphate-citrate buffer, pH5 and the product was quantified from the optical density (OD) at 450 nm. The mean of OD value was fitted into the sigmoidal standard curve using a specific ELISA programme running in Igor version 3.16 (Wavemetrics, Lake Oswego, OR).

Serum IgG antibody levels against NoV^{34 (link)} and RV^{55 (link)} were analyzed by ELISA as earlier described. Serum specimens were diluted two-fold starting at 1:100 and plated on NoV GI.3, GII.4 VLP or RV VP6 coated (1.0 µg/ml in phosphate-buffered saline, PBS) 96-well half-area microtiter plates (Corning Inc., Corning, NY) blocked with 5% skimmed milk in PBS. Serum dilutions were incubated on plates for 1 h at 37 °C. Bound antibodies were detected with goat anti-human IgG-HRP (Invitrogen, CA, USA) followed by o-phenylenediamine (OPD) substrate (Sigma-Aldrich, MO, USA) and H₂O₂. Optical density (OD) was measured at λ 490 nm using Victor2 1420 Multilabel Counter (Wallac, PerkinElmer) plate reader. Background signal from the blank wells (wells without serum) was subtracted from all of the OD readings on the plate. Each plate contained known NoV negative and positive control serum sample as an assay control. The cut-off value was determined as the mean OD reading of the negative control serum wells at a dilution 1:200 + 3 × standard error and at least 0.100 OD. End-point titer was expressed as a reciprocal of the final serum dilution giving an OD above the cut-off value. Seroconversion was defined as at least four-fold increase in the titer of successive sera.