Victor x5 fluorometer

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 changes^{39 (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.

Quantification of PolyP in egg homogenates was done using a general protocol based in the fluorimetric analysis of the characteristic emission of the DAPI-PolyP complex, as described before by [35 (link)]. Excitation wavelength was 420 nm and the emission wavelength was of 535 nm. PolyP₆₅ was used for a standard curve ranging from 90 to 540 ng of PolyP. Measurements were made using a 2030 Victor X5 fluorometer (Perkin Elmer).