Cytofluorometric assessment for the presence of anti-hMOR IgG antibody in 10 healthy women and 12 healthy men.
The presence of anti-hMOR IgG in 22 healthy humans was examined by cytofluorometry after affinity purification of serum IgG on the hMOR/CHO cell clone and depletion of nonspecific antibodies on untransfected CHO cells. Recombinant hMOR/CHO cells and untransfected CHO-K1 cells (106) were incubated with serial dilutions of IgG samples for 45 minutes at 4°C. Bound antibodies were revealed with biotin-labeled, goat anti-human IgG F(ab′)2–specific antibodies and PE-labeled streptavidin. Panel A depicts the binding (filled histogram) to recombinant hMOR/CHO cells (upper panel) and to untransfected CHO-K1 cells (lower panel) of unpurified IgG (i), affinity-purified anti-laminin IgG (ii), affinity-purified anti–CHO-K1 IgG (iii), and affinity-purified anti-hMOR IgG (iv) from a large pool of human IgG at a concentration of 20 μg/mL. A representative binding activity of affinity-purified anti-hMOR IgG from a woman (v) and a man (vi) is shown. The background (open histogram) was cells stained with labeled goat anti-human IgG F(ab′)2–specific antibodies and PE-labeled streptavidin alone. In panel B, data are expressed as mean fluorescence intensity (MFI) obtained from IgG staining with hMOR/CHO cells minus MFI obtained from IgG staining with untransfected CHO-K1 cells. Backgrounds with untransfected and hMOR-transfected CHO-K1 cell clones were similar. The histogram shows, for each donor IgG sample (░), the value obtained with a dilution at which CHO-K1 cell staining was similar to the background. Controls were the specific antibody-binding activity of unpurified IgG (▪), affinity-purified anti-laminin IgG (▤), affinity-purified anti-CHO IgG (■), and affinity-purified anti-hMOR IgG (▨) prepared from a pool of human IgG.
