ELISA plate-bound rCyp c 1 (5 μg/mL) was incubated with serial dilutions of the mCyp c 1–specific rabbit antiserum (diluted 1:100-1:819,200). Rabbit IgM was detected with a HRP-labeled anti-rabbit IgM from goat (1:2000 dilution; Rockland Immunochemicals, Limerick, Pa), rabbit IgA with a HRP-labeled anti-rabbit IgA from goat (1:2000 dilution; Thermo Fisher Scientific, Waltham, Mass), and rabbit IgG with a HRP-labeled anti-rabbit IgG antibody from donkey (dilution 1:2000; GE Healthcare). For the epitope mapping of rabbit anti-mCyp c 1 antibodies, ELISA plates were coated with rCyp c 1 or Cyp c 1 peptides (5 μg/mL) and exposed to the 1:50,000 diluted mCyp c 1–specific rabbit antiserum. For epitope mapping of IgG1 and IgG2a in mouse sera, a 1:250 serum dilution for IgG1 and a 1:50 dilution for IgG2a were used. IgG1 and IgG2a were detected as described above.
For IgE inhibition experiments, ELISA plate-bound rCyp c 1 (1 μg/mL) was preincubated with serial dilutions of the mCyp c 1–specific rabbit antiserum (1:20-1:100,000) or, for control purposes, with the preimmune serum (1:20). After washing, 1:10 diluted patients’ sera were added. Human IgE was detected with a HRP-labeled goat anti-human IgE antibody (1:2500; KPL). All measurements were performed as duplicates. The OD values shown are mean values with a deviation of <5%.
For IgE inhibition experiments, ELISA plate-bound rCyp c 1 (1 μg/mL) was preincubated with serial dilutions of the mCyp c 1–specific rabbit antiserum (1:20-1:100,000) or, for control purposes, with the preimmune serum (1:20). After washing, 1:10 diluted patients’ sera were added. Human IgE was detected with a HRP-labeled goat anti-human IgE antibody (1:2500; KPL). All measurements were performed as duplicates. The OD values shown are mean values with a deviation of <5%.