Purification and Labeling of Complement Proteins

The recombinant proteins Efb, and Ecb, the leukocidin components Luk S37, and Luk S45, were expressed and purified as previously described [13 (link), 25 (link)]. The FH fragments FH1-4, FH5-7, and FH19-20 were expressed in Pichia pastoris [26 (link)–28 (link)] and FH and C3 were purified from plasma as described previously [15 (link)]. C3b was prepared from C3 using trypsin [29 (link)]. Soluble CR1 (sCR1) was obtained from CelldexTherapeutics (product code CDX-1135; Needham, MA), and human and bovine serum albumin (HSA and BSA, respectively) were purchased from Sigma-Aldrich (St. Louis, MO). Normal human serum (NHS) was obtained by pooling serum from at least five healthy consented laboratory workers and stored at -70°C until used (Ethical Committee decision 406/13/03/00/2015, Hospital district of Helsinki and Uusimaa). Labeling of sCR1 and C3b was performed with ¹²⁵I (Perkin Elmer, Boston, MA) using the Pierce Iodination Reagent (Thermo Scientific, Rockford, IL) resulting in specific activity of 4.6–8.0 x 10⁶ cpm/μg for sCR1 and 6.0–6.8 x 10⁶ cpm/μg for C3b. The antibodies used were rabbit anti-human C3c (Dako, Denmark), Alexa Fluor® 488-labeled goat anti-rabbit (Invitrogen, Eugene, OR), and FITC-labeled goat anti-human C3 (Protos Immunoresearch, Burlingame, CA).