High sensitivity elisa

Animals were terminated by exsanguination. Lavage fluid neutrophils were evaluated by differential cytology on CytoSpin slides. Lavage protein was determined using a colorimetric assay (Bio-Rad, Hemel Hempstead, UK). CXCL1, Interleukin-6 (IL-6), and soluble Receptor for Advanced Glycation End-products (sRAGE) in lavage fluid, and plasma soluble TNF receptors were determined by ELISA (R&D systems, Abingdon, UK). Lavage fluid TNF was determined by high sensitivity ELISA (eBioscience, Altrincham, UK). Matrix metalloproteinase (MMP) activity was determined by fluorometric assay (abcam, Cambridge, UK). Other plasma biomarkers were determined by FlowCytomix assay (eBioscience).

Maternal antecubital venous blood samples were collected in serum tubes (BD Vacutainer) in early (12.6±2.8 weeks), mid (20.4±1.5 weeks) and late (30.3±1.3 weeks) pregnancy. Serum IL-6 concentration was determined using a commercial high sensitivity ELISA (eBioscience) with a sensitivity of 0.03 pg/ml. IL-6 concentration was moderately stable across pregnancy (ICC=0.45). Thus, IL-6 was averaged across pregnancy and base 2 logarithm transformed to normalize the distribution.

We studied human samples under protocols that were approved by the Stanford Institutional Review Board (IRB) and included subjects’ informed consent. Synovial fluids were obtained by needle aspiration of actively inflamed large or medium joints by a board certified rheumatologist at the Veteran’s Administration Hospital (Palo Alto, CA). Grossly bloody fluid was excluded from analysis. Synovial fluid was centrifuged at 1000 g for 10 min and supernatants removed and frozen at −80° C until use in experiments as described below. The diagnosis of gout was confirmed by identification of negatively birefringent intracellular needle shaped crystals on microscopic examination of synovial fluid under polarizing microscopy. The diagnosis of rheumatoid arthritis was made as defined by the 1987 revised criteria for rheumatoid arthritis (12 (link)).
Histamine levels were measured by competitive ELISA (Beckman coulter). IL-1β levels were measured using a high sensitivity ELISA (eBioscience; lower detection limit 0.16 pg/ml). Total tryptase levels were measured using an immunoCAP assay (CAP; Phadia Diagnostics, Uppsala, Sweden). Mature tryptase levels were measured by ELISA as described elsewhere (13 (link)). Both total and mature tryptase assays were performed in parallel at Virginia Commonwealth University (Richmond, VA, USA), by individuals not aware of the identity of individual specimens.

Serum IL-6 concentration was determined using a commercial high sensitivity ELISA (eBioscience) with a sensitivity of 0.03 pg/ml. The intra- and inter-assay coefficients of variability for IL-6 measurements were 10% and 14%, respectively. CRP concentrations were determined using a commercial Roche, COBAS Cardiac C-Reactive Protein (Latex) High-Sensitivity Test with a sensitivity of 0.15 mg/L, and intra- and inter-assay CVs of 1.6% and 8.4%, respectively. IL-6 and CRP were log₂ transformed to bring outliers closer to the mean and normalize the distribution.