Binding of purified chimeric human IgE and IgG1 anti-BLG antibodies clones 5D6.1, 7D5.1, 8C3.3, 8F7.1, 11B6.2 or 13A5.2 was done by using the BLG ELISA as described above. For detection, however, 1∶1,000 diluted HRP-conjugated goat anti-human IgE-specific antibodies (Sigma) and 1∶5,000 diluted HRP-conjugated goat anti-human IgG-specific antibodies (Jackson ImmunoResearch) were applied.
Bovine BLG (120 µg/480 µl non-reducing sample buffer) was loaded and electrophoresed in a one well pre-casted 4–12% BisTris gel/MOPS running buffer NuPage Novex system (Invitrogen). Then, bovine BLG was electro-blotted onto a polyvinylidene fluoride transfer membrane (Millipore). After blocking with PBS/0.05% Tween 20/1% BSA fraction V (Roche) for 1 hour at RT, this membrane was incubated with 1 µg/ml purified chimeric human IgE anti-BLG antibody clones 5D6.1, 7D5.1, 8C3.3, 8F7.1, 11B6.2 or 13A5.2 for 1 hour at RT. After washing in PBS/0.05% Tween 20, binding of these chimeric human IgE anti-BLG antibodies was determined with 1∶4,000 diluted HRP-conjugated goat anti-human IgE-specific antibodies (Sigma) for 1 hour at RT, followed by a ready-to-use solution of TMB substrate (Sigma) for colorimetric detection.
Bovine BLG (120 µg/480 µl non-reducing sample buffer) was loaded and electrophoresed in a one well pre-casted 4–12% BisTris gel/MOPS running buffer NuPage Novex system (Invitrogen). Then, bovine BLG was electro-blotted onto a polyvinylidene fluoride transfer membrane (Millipore). After blocking with PBS/0.05% Tween 20/1% BSA fraction V (Roche) for 1 hour at RT, this membrane was incubated with 1 µg/ml purified chimeric human IgE anti-BLG antibody clones 5D6.1, 7D5.1, 8C3.3, 8F7.1, 11B6.2 or 13A5.2 for 1 hour at RT. After washing in PBS/0.05% Tween 20, binding of these chimeric human IgE anti-BLG antibodies was determined with 1∶4,000 diluted HRP-conjugated goat anti-human IgE-specific antibodies (Sigma) for 1 hour at RT, followed by a ready-to-use solution of TMB substrate (Sigma) for colorimetric detection.