Sector 6000

The biotinylated C3a (Clone 2991, Hycult HM2074BT-B) capture antibody was diluted in MSD Diluent 100 (catalog number: R50AA-2) and was added to MSD GOLD 96-Well Streptavidin SECTOR Plate (catalog number: L15SA-1). Following 1h incubation the plate were washed 3 times in PBS with 0.05% Tween 20 and blocked with blocking solution (2% BSA in PBS + 0.05% Tween) overnight at 4C. The next day, normal human plasma was diluted to 6% in alternative pathway buffer (7.5mM HEPES, 150mM NaCl, 7mM MgCl₂, 10mM EGTA) and activated with zymosan (1mg/ml). Small molecule Factor B inhibitor (CFBi) and factor B blocking antibody with appropriate negative (DMSO vehicle for CFBi and IgG1 isotype antibody) and positive control (EDTA) were added to wells of the 96-well U-bottom plate and incubated for 1.5 hours at 27C with shaking at 750rpm. The reaction was stopped with 5mM EDTA and 50l aliquot was transferred to the anti-C3a coated MSD plate. A C3a standard curve was constructed using purified human C3a (ComTech, A118) in concentrations ranging from 10nM to 0.00977nM. Following 1.5 hours of incubation at 27C with shaking the plates was washed in PBS/0.05% Tween 20 and the detection ruthenylated anti-C3a antibody (clone 474, made at GSK) was added. Following 3 washes, plates were developed with MSD Read buffer and electroluminescence was recorded on MSD Sector 6000 plate reader.

The concentration of total soluble TNFR1 in mouse samples containing DMS5540 was determined using a MSD assay. Briefly, 96-well standard bind MSD plates (MSD #L11XA-6) were coated overnight with a rat anti-mouse TNFR1 mAb (R+D Systems #MAB425). Plates were then washed and incubated with assay buffer (PBS containing 3% BSA (Sigma #A7030) and 0.1% Tween-20 (Fisher #BPE337)) followed by incubation with MSD Serum Cytokine Assay Diluent (MSD # R51BB-2). Mouse serum samples were added directly to the relevant wells alongside a standard of mouse sTNFR1 (R&D Systems #425-R1) of known concentrations. Plates were incubated at room temperature for 20 hours before washing. Any bound sTNFR1 was detected using a MSD sulfo-tagged (MSD #R91AN-1) anti-TNFR1 dAb (DMS5541), which recognises an epitope independent of DMS5540. After incubation, plates were washed and 2xMSD read buffer T with surfactant (MSD #R92TC-1) was added and plates were read immediately using the MSD SECTOR 6000.