Magellantm

Plasma and SF C5a/C5a desArg were measured by a sandwich ELISA from BD Bioscience. In brief, microtiter plates were precoated with monoclonal antibody specific for human C5a/C5a desArg. A standard was supplied with the kit containing lyophilized human serum containing a specific amount of C5a desArg. A calibrator curve was prepared by spiking C5a desArg standard into standard diluent (animal serum), ranging from 0.078 ng/ml to 5 ng/ml. In addition, three quality control samples (QCs) were prepared covering Low (0.25 ng/ml), Medium (2 ng/ml) and High (4 ng/ml) level of the standard curve. Any C5a/C5a desArg in the samples was bound to the plate, and was detected by a mixture of biotinylated anti-human C5a antibody and streptavidin-HRP followed by colorimetric detection using Tetramethylbenzidine. The plate was read at 450 nm and reference at 620 nm on a Tecan Sunrise reader controlled by Magellan^TM, Tecan Trading AG, Switzerland. The instrument response was exported to WatsonLIMS^TM software, ThermoFishes Scientific, where plasma concentrations were quantified.
SFs from RA patients were sent to Myriad RBM for analysis on their Human DiscoveryMAP® 175+ v1.0, a 45-biomarker Multi-Analyte Profile (MAP) designed to discern inflammatory biomarker patterns.

Pre-cultures were obtained by inoculating a loopful of each sample into 7 mL YEPD medium and grown 24 h at 37 °C (120 rpm). Biomass concentration was determined using a Thoma-Zeiss counting chamber and then diluted in YEPD and RPMI-1640 media to the final concentrations from 5 × 10⁷ to 1.19 × 10¹ cells mL⁻¹. All dilutions were obtained directly in 96-well plates in a final volume of 100 µL.
CMC2046, CMC1949 and CMC1839 (C. albicans, C. parapsilosis and C. tropicalis, respectively) strains were used to validate the protocol at four different cell densities (6.25 × 10⁶, 1.95 × 10⁵, 1.22 × 10⁴ and 1.53 × 10³ cells mL⁻¹, respectively). The 96-well plates were incubated in standardized conditions (24 h, 37 °C) using the multi-well plate reader Tecan infinite F-200. OD_405nm values were collected every 5 min, using the internal software of the instrument (Magellan^TM, Tecan Trading AG, Männedorf, Switzerland). All analyses were conducted in triplicate.