Recombinant human gm csf

Buffy coats from healthy donors who consented to their use for scientific research were obtained from local blood donations. The study received approval from the Hôpital Erasme Ethics Committee (Route de Lennik 808, B-1070 Brussels). Peripheral blood mononuclear cells (PBMCs) were purified by density gradient centrifugation (Lymphoprep—Stemcell Technologies). CD14⁺ monocytes were purified from PBMCs by positive magnetic separation with anti-CD14 microbeads (Automacs—Miltenyi Biotec) and cultured in 75-cm² flasks with 20 ml of medium (RPMI containing 10% FCS, penicillin/streptomycin, non-essential amino acids, and glutamine) supplemented with recombinant human GM-CSF (800 U/ml—Gentaur) and IL-4 (200 U/ml—Wittycell). After 3 days, 16,000 U GM-CSF and 4,000 U IL-4 were added to the medium. At day 6–7, non-adherent cells corresponding to DCs (>90% CD14-negative HLA-DR-positive CD11c-positive) were recovered, washed, and used for subsequent experiments.

Human mAbs and total IgG from PAP sera were purified by protein A or protein G chromatography (GE Healthcare). Total IgG from healthy donors were purified using the HiTrap Protein A HP columns (GE Healthcare) and concentrated using Amicon Ultra filter units (100 K, Millipore). Total GM-CSF antibodies were affinity-purified from PAP sera using magnetic beads (Invitrogen) conjugated with human GM-CSF. Total IgGs were quantified using ELISA plates coated with anti-human IgG (SouthernBiotech) using Certified Reference Material 470 (ERMs-DA470, Sigma-Aldrich) as standard. Binding to GM-CSF was tested by ELISA using 384-well SpectraPlates (PerkinElmer) for primary screenings or 96-well MaxiSorp plates (Nunc) for any following test. Briefly, ELISA plates were coated with 1 μg ml⁻¹ of recombinant human GM-CSF (Gentaur), blocked with 1% BSA and incubated with titrated antibodies, followed by AP-conjugated anti-human IgG secondary antibodies (SouthernBiotech). Plates were then washed, substrate (p-NPP, Sigma) was added and plates were read at 405 nm. EC50 (ng ml⁻¹) was calculated for every sample by nonlinear regression analysis using the GraphPad Prism 5 software.