Goat anti human igg fcγ fragment

To quantify serum or supernatant human antibody, diluted samples were added to plates (Corning Incorporated) coated with goat anti-human IgG Fcγ fragment (Jackson) or goat anti-human IgM (Southern Biotech), washed, and blocked with 2% Bovine Serum Albumin (BSA, Fisher Scientific). Bound human Ig was detected using biotin-conjugated mouse anti-human IgG (BD Pharmingen) or biotin-conjugated mouse anti-human IgM (BD Pharmingen) secondary antibodies, followed by streptavidin-horseradish peroxidase (Thermo Scientific). Colorimetric change by 3,3′,5,5′-Tetramethylbenzidine substrate solution (Thermo Scientific) was stopped by 2M sulfuric acid (Sigma), and optical densities determined by spectrophotometer 450nm absorbance. Human serum with known IgM and IgG concentrations (Bethyl) established a standard.

BALB/c mice were divided into groups of three, and mice in each group were administered intraperitoneally with 100 µg of the indicated antibody. Blood was drawn from all animals at days 1, 2, 4, and 7 post antibody administration and serum was isolated and analyzed for levels of antibody in individual mice. For those animals in which antibody was detected at day 7, an additional blood collection at day 10 was performed and analyzed for antibody levels in serum. CoStar 96-Well EIA/RIA plates (Corning) were coated with 100 ng per well of goat anti-human IgG Fc-γ fragment (Jackson ImmunoResearch) overnight at 4°C. Plates were washed three times with PBS + Tween and blocked with PBS containing 5% milk and 0.5% BSA for 2 hours at room temperature. Mouse serum from the treated animals, and purified antibody in PBS for the standard curves, were added to the wells in 1:2 serial dilutions in PBS containing 2% milk and 0.2% BSA and incubated for 2 hours. After washing, peroxidase-conjugated goat anti-human IgG (Jackson ImmunoResearch) was incubated for 1 hour at room temperature. Samples were detected by TMB Liquid Substrate System (Sigma) and spectrophotometric readings were performed at 450 nm.