Anti human igm antibody

Blood was drawn from healthy adult donors in sodium-heparin anticoagulation tubes (BD Vacutainer) and isolated using Ficoll-Paque (GE Healthcare) as described previously (48 (link)). Before use, neutrophils were taken up in Hanks’ buffered salt solution (HBSS) without phenol red containing Ca²⁺ and Mg²⁺ plus 0.1% gelatin to the desired concentration. All experiments were performed with the same batch of pooled normal human serum (NHS) containing opsonizing antibodies and active complement obtained from GTI Diagnostics (catalogue no. PHS-N100). For IgM depletion, 2 ml of 20% NHS in PBS was incubated with 200 µl of PBS-washed Sepharose beads coupled to anti-human IgM antibody (Sigma). After 2 h of incubation while shaking at 4°C, Sepharose beads were removed by centrifugation, and sera were diluted to 10% with PBS and immediately stored at −80°C. Donor-specific NHS was obtained from healthy adult volunteers, using serum tubes (BD Vacutainer), centrifuged, and stored immediately at −80°C. Human nasal airway surface fluid was obtained from a healthy adult volunteer as described previously (49 (link)).

To remove IgM antibody from immune serum, the serum samples were mixed with agarose beads conjugated with anti-human IgM antibody (Sigma-Aldrich, St. Louis, MO, USA) and incubated for 2 hours at RT. Unconjugated agarose beads were used as a control. The mixtures were applied to Bio-Rad chromatography spin columns (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The columns were centrifuged at 2,000 r.p.m. for 1 minute at 4°C, and the eluted fraction was collected. The efficiency of depletion was monitored using the pneumococcal antibody ELISA. The optical density was measured at 405 nm to determine the concentration of anti-pneumococcal PS (anti-PnPS) IgM antibody in the 1:1,000 diluted absorbed serum (13 (link)).