Assessment of anti-hMOR F(ab′)2 specificity by cytofluorometry.
Affinity-purified anti-hMOR F(ab′)2 fragments were preincubated with either hMOR EL1 peptide, the hMOR EL3 peptide, or amino acid residues 70 to 86 of the guinea pig myelin basic protein (70-86 MBP) peptide for 1 hour at 37°C. Recombinant hMOR/CHO cells were incubated with 100 μL of the mixture for 45 minutes at 4°C. Bound antibodies were revealed by using biotin-labeled, goat anti-human IgG F(ab′)2–specific antibodies and PE-labeled streptavidin. Shown is the binding to the recombinant hMOR/CHO cell clone of 10 μg/mL affinity-purified anti-hMOR F(ab′)2fragments (thick black line) from either a large pool of samples from healthy donors (A), a sample from one healthy woman (B), or a sample from one healthy man (C) in the absence or presence of 20 μg/mL peptide. The binding activity to recombinant hMOR/CHO cell clones of 5 μg/mL affinity-purified anti–CHO-K1 F(ab′)2 fragments (thick black line) in the absence or presence of 20 μg/mL peptide is shown (D). The background (thin gray line) was the binding activity of anti-hMOR antibodies to untransfected CHO-K1 cells, which was superimposable to the staining of recombinant hMOR/CHO cells obtained by using either anti-laminin F(ab′)2 fragments or labeled goat anti-human IgG F(ab′)2–specific antibodies and PE-labeled streptavidin alone. One representative experiment of 3 is shown.
