Quantification of the H-ficolin concentration was performed blinded to subject identity as previously described using normal human serum as standard except for a few changes39 (link). In brief, serum was thawed, diluted in assay buffer, and added to microtiter wells (FluoroNunc, Thermo Scientific, Waltham, MA, USA) coated with acetylated bovine serum albumin (B2518, Sigma-Aldrich, St. Louis, MO, USA), which is recognized by H-ficolin. Standards, samples, and controls were added automatically to plates using a Janus Varispan automated work-station (PerkinElmer, Waltham, MA, USA). In-house biotinylated anti-H-ficolin antibody, europium-labelled streptavidin (PerkinElmer) and enhancement solution (Ampliqon, Odense, Denmark) were added in successive steps with triple washing in between. The europium fluorescence intensity was detected with a Victor X5 fluorometer (PerkinElmer). Intra-assay and inter-assay coefficients of variation were below 10% and 16%, respectively. H-ficolin concentration was used as a predictor as a continuous variable as well as by comparing subjects grouped according to H-ficolin quartiles.
B2518
Manufactured by Merck Group
Sourced in United States
B2518 is a lab equipment product manufactured by Merck Group. It is designed for general laboratory applications. The core function of B2518 is to provide a reliable and consistent tool for researchers and scientists in their day-to-day laboratory activities.
Automatically generated - may contain errors
2 protocols using b2518
1
H-ficolin Quantification Assay Protocol
2
Controlled Release of bFGF from PEG Hydrogels
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Fifty microliters PEG hydrogel was formed with 2.5 µg bFGF for a final gel concentration of 50 ng/µl. This was then aliquoted into 10 µL droplets and allowed to set. Gels were placed in 500 µL 1% BSA (B2518; Sigma) PBS, pH 7.4 and incubated on a shaker at 37°C. Every 24 h, the gel in each sample was transferred to a fresh tube containing 500 µL BSA PBS, and the old sample was flash-frozen and stored at -20°C until the ELISA was performed. Samples were taken over 5 days (n = 4). A commercial ELISA system (DY233-05, DuoSet; R&D Systems, Minneapolis, MN) was used according the manufacturer's specifications. Samples were diluted 1:50 to ensure values were within the established standard curve (0-2 ng/mL generated using a serial dilution of stock solution provided by the manufacturer) Plates were read on a Bio-Rad microplate reader (170-6800; BioRad, Rosebank, Johannesburg, South Africa) using a 595 nm filter (170-6914; BioRad).
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