Anti igm agarose

These studies were approved by the National Jewish Health Institutional Review Board, and informed consent was obtained from all specimen donors. The study was conducted in accordance with the Declaration of Helsinki. Whole blood was collected in EDTA K5 tubes from healthy donors and pwCF, and plasma was obtained following manufacturer’s guidelines. Whole human platelet-poor plasma (WP) samples were combined and aliquoted into three to five donor pools. Heat-inactivated human platelet-poor plasma (HP) was prepared by incubating WP at 56°C for 30 min or at 95°C for 30 min as indicated. To prepare opsonized M. avium, 50 μL of WP or HP was added to 100 μL of M. avium at OD₆₀₀ = 1.0. Complement C3-, C1q-, and factor B-depleted sera and control human serum (Complement Technology Inc, Tyler, TX) were also used to opsonize M. avium. Antibody-depleted plasma was prepared by incubating 300 μL of protein A- or protein G-agarose (Invitrogen, Waltham, MA) with 300 μL of WP at 37°C for 1 h with rotational mixing, followed by centrifuging at maximum speed and collecting the supernatant. IgM-depleted plasma was prepared by incubating 600 μL of anti-IgM-agarose (Sigma-Aldrich, St. Louis, MO) with 300 μL of WP and following the same procedure as the protein A and G plasma preparation. Antibody depletion was confirmed by Western blot.