Duoset elisa development system

Mouse embryonic fibroblasts (MEF) were obtained from a wild-type pregnant mouse at 14 day post-coitum as previously described (30 (link)). Cells were plated at a density of 10⁵/ml and expanded in 6-well plates in RPMI 1640 medium supplemented with 2 mM L-glutamine, 10% FCS, and B-mercaptoethanol. MEF (5x10⁵ cells/well) were plated in a 24-well plate at passage 5-6 in RPMI 1640 medium supplemented with 2 mM L-glutamine, 1% FCS and stimulated with heat-killed K. pneumoniae (30 min at 70°C) at ratios of 5:1, 1:1 and 1:5 (bacteria:cells). LPS (10ng/ml) and TNFα (10ng/ml) were used as controls of PTX3 induction. Murine PTX3 concentration in supernatants was analyzed by ELISA (R&D DuoSet ELISA Development System, USA).

2 × 10⁵ monocytes per sample were pretreated for 2 h with RSV (10–40 μM) and then stimulated for 24 h with 100 ng/ml LPS from Salmonella typhimurim (Calbiochem, Merck KGaA, Darmstadt, Germany) with supernatant collection performed at different time points before being stored at -20 °C. For the TRUS patients, 3 × 10⁵ monocytes were plated per well and either left untreated or treated with 20 μM or 40 μM RSV for 24 h before collecting the supernatants. Measurement of IL-6, TNFα, IL-1β, and MCP-1 concentrations was performed with OptEIA™ Kit (BD Biosciences, San Jose, CA, USA). DuoSet ELISA Development System (R&D System, USA) was used to measure MIP-1α. HMGB1 levels were measured using Shino-Test’s HMGB1 ELISA kit II (Shino-Test Corporation, Japan). Cytokines and chemokines in the mouse model were measured using similar methods.

All plasma samples were assayed in duplicate blindly. Standard ELISA experiments were performed according to the manufacturer’s instructions (DuoSet^® ELISA Development System, R&D Systems, Minneapolis, MN). Briefly, plasma samples were diluted 1:1000 for sCD14, 1:80 for sCD163, 1:40 for Fractalkine, 1:10 for uPAR and Pentraxin, 1:2 for sTREM-1 and MIG and undiluted for Neopterin. Neopetrin level was tested in a competition ELISA test from IBL International R (Hamburg, Germany). The competition was evaluated between a peroxidase-conjugated and non-conjugated antigen for a fixed number of coated anti-Neopterin antibody binding sites. Unbound antigen was removed by washing, and the optical density (OD) was measured after substrate reaction. When the obtained OD values were out of the standard reference range, dilutions were modified accordingly.
In all experiments, analyte concentrations were calculated according to standard curves obtained by the assessment of specific recombinant human proteins elaborated by the manufacturers and determined in each ELISA plate, which systematically included negative control sera. The final results were expressed as ng/ml, with the exception of Fractalkine concentrations, which were expressed as pg/ml.

A20 B cell lines were loaded, or not, with different concentrations of OVAp (aa 323–339) overnight. Cells were then conjugated with purified mouse CD4⁺ T cells as described above and incubated for 20h. After the 20h incubation period, supernatant was recovered and stored at -20°C. IFN-γ production was quantified in the supernatants by sandwich ELISA using the commercial kit DuoSet® ELISA Development System (R&D systems), according to manufacturer’s instructions. 96-well plates were analyzed on an Infinite® M200 (Tecan Group, Ltd). T cells cultured for two to four days in the presence of 3μg/mL of anti-CD3 antibody were used as positive control of T cell activation. B cells cultured for 48h in the presence of 2.5μg/mL of anti-CD40 antibody and 5μg/mL of the F(ab’)2/F(ab) portion of an anti-mouse IgG antibody were used as positive control for B cell activation.

The concentrations of gastrointestinal lavage (GAL) cytokines and chemokines were measured by DuoSet^® ELISA Development system (R&D Systems, Minneapolis, MN, United States) according to the manufacturer’s instruction. Absorbance was measured at 450 nm using iMark^TM Microplate Reader (Bio-Rad, Hercules, CA, United States). Samples were measured in triplicate and statistical analyses were performed using GraphPad Prism version 6.0.