Mab21501

Changes in the C5a levels in the plasma of individual Tg2576 mice before (at the age of 8 months) and after the last immunization (at the age of 15 months) with AFF1-, AFF2-, and vehicle-containing vaccines was determined by a C5a sandwich ELISA. Briefly, 96-well Nunc-MaxiSorp plates were coated with the monoclonal rat anti-mouse C5a antibody (R&D, MAB21501). Plates were blocked with 1× PBS/1 % BSA. Subsequently, plasma obtained 1 day before the first immunization and 8–9 weeks after the last immunization were applied at a starting dilution of 1:10 and serially diluted 1:2 in 1× PBS/0.1 % BSA/0.1 % Tween 20 and incubated for 1 h at 37 °C. For the detection of bound C5a, the biotinylated polyclonal goat anti-mouse complement component C5a antibody (R&D, BAF2150) was applied for 1 h at 37 °C. Horseradish peroxidase coupled to streptavidin (Roche) was added (30 min, 37 °C) followed by the addition of the substrate ABTS (BioChemica, AppliChem) (30 min, RT). The OD at 405 nm was determined by a microwell plate reader (Sunrise, Tecan, Switzerland). The relative changes in OD values at a dilution of 1:40 of plasma samples obtained before (=100 %) and after the last immunization of each individual mouse were evaluated. Plasma obtained from untreated wt mice was used as a control. The group means ± SEM of n animals are presented.

BMCs were seeded on 96-well plates for osteoclast differentiation assay or osteo assay plates for resorption assay at a density of 1.0 × 10⁵ cells per well and cultured in alpha-modified Eagle’s minimum essential medium (α-MEM) with 15% FBS containing 20 ng/mL M-CSF (#315–02; Peprotech, Rocky Hill, NJ, USA) and 50 ng/mL murine soluble recombinant receptor activation of nuclear factor kappa-B ligand (sRANKL, #315–11; Peprotech). After 2 days, BMCs were cultured in the presence of 20 ng/mL M-CSF and 50 ng/mL sRANKL with 10% FBS and 5% mouse serum for 3 days. Sera were prepared from each group of mice. In regard to the neutralization of C5a, 0.5 μg/mL anti-mouse C5a antibody (#MAB21501; R&D) or rat IgG2 isotype control (#MAB006; R&D) was added to the culture medium. After 3 days, differentiated osteoclasts were identified by TRAP staining and TRAP-positive multinucleated (more than three nuclei) cell numbers were evaluated. Resorption areas were measured by ImageJ after removal of the cells with 1 M NH₄OH.