Antibody Depleted Human Serum & Complement

…362 Ng WHO X (1×10³ CFU in 40 µL HBSS) were added to wells containing diluted test sera 363 and incubated for 15 min at 37°C. Subsequently, 10 µL of either IgG/IgM-depleted normal human serum (NHS; Pel-Freeze Biologicals; Cat. # 34010-10, Lot 34943) or heat- inactivated NHS (HI-NHS; Cat. #34020-10, Lot 13844) was added as a 10% (v/v) complement source, and reactions were incubated for 1 h at 37°C. A single lot of…

…growth CHBA plate after a 4-h incubation. A volume of 10 µL of the bacterial suspension was added to each well of the microtiter plate. A volume of 10 µL of “active” antibody depleted human complement (Pel-Freeze, Inc., Brown Deer, WI) was added to all wells except the sample control wells for viable cfu and sample non-complement mediated lysis control, to which heat-inactivated antibody depleted human complement…

…allow opsonization, ~500 CFUs were incubated with various concentrations (1:20, 1:40, 1:80, 1:200, 1:400) of serum for 20 min, followed by the addition of 12% pooled human complement depleted of IgG/IgM (Pel Freez, cat. 34010, Lot 22344) and incubated for 60 min at 37 °C with 5% CO 2 . The highest dilution at which 50% killing was observed relative to no antibody control in 60 min was reported as the SBA titer.…

…strain lacking pora expression and expressing a hybridized porb (och) [18 ]. see table s3 for a complete description of pc reagents. 3.4 intrinsic sba growth assay igg/igm-depleted human complement (c′) pooled serum (pel-freez validation lot #03642) was screened for endogenous killing activity against ng strains f62, ms11, and fa1090 as previously described [19 ] with modifications. briefly, sialylated or non-sial…

…4-nitrophenyl phosphate disodium hexahydrate were purchased from Sigma-Aldrich (St. Louis, MO). Baby rabbit (3- to 4-week-old) and adult rabbit (8- to 12-week-old) complements were purchased from Pel-Freez Biologicals (Rogers, AR). Guinea pig and human complements were purchased from Rockland (Gilbertsville, PA) and Valley Biomedical (Winchester, VA), respectively. All complements were stored in aliquots at −80…