Quantifying Plasma Complement Proteins

Plasma levels of human complement-related proteins were measured using MILLIPLEX MAP Kit Human Complement Magnetic Bead Panels 1 and 2 (HCMP1MAG-19K and HCMP2MAG-19K, Merck Millipore, USA) according to the instructions of the kit manufacturer as described previously (30 (link)). Protein levels in the plasma samples were quantified using 5PL logistic regression algorithms that are built into the Bio-Plex Manager 6 software, which was used for the quantification of all plasma samples with reference to the standards provided by the kit manufacturer using overnight incubation protocol. The samples were run on a Bio-Plex 200 (Bio-Rad, Hertfordshire, UK) instrument. The plasma samples were diluted 200 times for the measurement of complement panel-1 proteins (C2, C4b, C5, C5a, C9, factor D, mannose-binding lectin, and factor I) and 40,000 times for the measurements of complement panel 2 (C1q, C3, C3b/iC3b, C4, factor B, factor H, and properdin)-related proteins to attain the levels of the proteins within the reference range of the standard curve. The working range and assay precision for different complement-related proteins were reported previously (30 (link)).