Human igg control antibody

Recombinant human TNF-α and human macrophage colony-stimulating factor (M-CSF) was purchased from Miltenyi Biotec (Bergisch Gladbach, Germany). Human oxidized low density lipoprotein (oxLDL), Dil(1,1´-dioctadecyl-3,3,3`,3`-tetramethylindocarbo-cyanine perchlorate)-labelled oxLDL and native LDL (nLDL) was from Kalen Biomedical LLC (Montgomery Village, MD). Antibodies for Western blot against ICAM-1, E-selectin were obtained from R&D Systems (Minneapolis, MN) and VCAM-1, Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Santa Cruz (Dallas, TX). An antibody for immunofluorescence against VE-cadherin was obtained from Santa Cruz. CD11b and CD11c antibodies labelled with allophycocyanin (APC) and PerCP/Cy5.5 respectively for flow cytometry were obtained from BioLegend (San Diego, SA). Appropriate secondary antibodies were from Jackson ImmunoResearch (West Grove, PA) Adalimumab (HUMIRA) was obtained from Abbott GmbH (Wiesbaden, Germany). Human IgG control antibody, 4',6-diamidino-2-phenylindole (DAPI) and lipopolysaccharide (LPS) were obtained from Sigma-Aldrich (Munich, Germany).

Total IgG secreted in B-cell culture supernatants was measured using a sandwich Enzyme-linked Immunosorbent Assay (ELISA). Briefly 96-well Maxisorp flat bottom plates (Nunc) were incubated overnight at 4°C with a goat anti-human IgG (AbDSerotec) diluted 1:1000 in coating buffer (Carbonate-Bicarbonate Buffer, pH 9.5). The following day, the coating buffer was removed, and plates were blocked with 2% non-fat milk solution in PBS, 0.05% Tween 20 (PBST) for 1 hour on an orbital shaker. Plates were then treated with either supernatants removed from B lymphocyte cultures or different concentrations of a human IgG control antibody (Sigma), from which the standard curve was derived. Plates were incubated for 2 hours at room temperature on an orbital shaker, and bound antibodies were detected with horseradish peroxidase-labelled goat anti-human IgG Fc-specific antibody diluted 1:1000 in PBST (Sigma) for 45 minutes at room temperature on an orbital shaker. After washing of unbound antibody, colour reaction was developed with o-Phenylenediamine dihydrochloride (OPD, Sigma) and OD was measured in an ELISA reader (BMG Labtech) at 492 nm (reference wavelength, 650 nm). Anti-folate receptor alpha IgG ELISA was performed as previously described. The lower detection levels of quantification were 3.125 and 0.625 ng/ml, respectively for 1:5 dilution and neat samples [15 (link), 16 (link)].