Eia kit

8-isoprostane expression in kidney was measured by the EIA kit (Oxford Biomedical Research, Rochester Hills, MI, USA).

Wildtype and CRF₂^−/− female mice (8 weeks of age) were sensitized by intraperitoneal injection (i.p.) with 20 µg of IgE monoclonal anti-dinitrophenyl in 100 µl PBS. The following day, mice were challenged with 1 mg of DNP in 100 µl PBS (i.p.). The mice were constantly observed and rectal body temperature recorded for 0, 15, 30, 60 and 120 minutes post DNP challenge (TH-5 Thermalert, Physitemp, Clifton, NJ). Mice were sacrificed at 0 min and 30 min post-DNP injection for serum collection via cardiac puncture and stored at −80⁰C until serum histamine level was quantified using a commercial EIA kit (Oxford Biomedical Research). Antalarmin experiments: PSA was performed as indicated above with the additional treatment group of antalarmin-treated CRF₂^−/− mice. Antalarmin (15 mg/kg) was injected (i.p.) in to male mice 15 minutes prior to DNP challenge. Mice were sacrificed 30 min post-DNP injection and plasma was collected via cardiac puncture for later histamine analysis.

Hematologic evaluation was performed using the hospital's clinical laboratory. We evaluated a panel of 27 cytokines, lipid mediators and soluble receptors to examine inflammation and immune activation using different immunoassays. The selection of variables was based on potential role in TB pathogenesis demonstrated by previous work from our group [6 (link)] and others [8 (link), 11 (link)]. We measured the biomarkers with commercial ELISA kits (for the soluble receptors CD40L, sIL-1R1, SIL-1R2, as well as MPO) (R&D Systems, Minneapolis, MN). The cytokines IL-1α, IL-Aβ, IL-1RA, IL-2, IL-5, IL-6, IL-8, IL10, IL-12p70, IL-17A, IL-18, IL-27, IFN-α, IFN-β, IFN-γ, TNF-α, TNF-β and resistin were measured using a Flowcytomix Multiplex Array kit (eBioscience, San Diego, CA) according to the manufacturer’s instructions. FlowCytomix is a bead-based ELISA like assay allowing for the results of several conventional ELISAs in a single assay [17 (link)]. This assay platform reduces sample volume requirements (<20µL of sample) and assay times, all performed using a standard flow cytometer. Levels of lipid mediators (PGE2, PGF2α, LXA4 and 15-EPI-LXA4) were assessed using EIA kits according to the manufacturer’s directions (Oxford Biomedical Research, Oxford, MI).