Enhanced serum IgE clearance by anti-IgE IgG compared to
anti-IgE(PNG) IgG
We next wanted to investigate the effects of anti-IgE and
anti-IgE(PNG)-IgG antibodies on the serum clearance of secondarily
administered IgE and their ability to prevent sensitization of effector
cells. Therefore, we isolated IgG antibodies from pooled sera by Protein
G chromatography and analyzed them by ELISA. As expected, these
antibodies reacted against IgE and Fel d 1 (Supp. Fig. 3A for IgE-Fel d
1, and 3D for IgE(PNG)-Fel d 1). Thus, to remove anti-Fel d 1-specific
antibodies, the IgG antibodies were separated using a Fel d 1-coupled
column. ELISA results of the eluate fraction show successful removal of
Fel d 1-specific IgG antibodies (Suppl. Fig. 3B and 3E for IgE and
IgE(PNG) specific IgG; Fig. 3C and 3F for Fel d 1 specific IgG). From
now on, the anti-IgE IgG isolated from mice vaccinated with IgE-ICs will
be called anti-IgE IgG, and the ones from mice immunized with
IgE(PNG)-ICs will be called anti-IgE(PNG) IgG.
The purified anti-IgE IgG or anti-IgE(PNG) IgG antibodies were injected
into mice 1h before IgE administration. Serum IgE was measured 30min,
2h, and 24h after IgE injection by ELISA. Additionally, Basophil surface
IgE was measured one week after IgE injection (see the outline in Fig
2A). Anti-IgE IgG significantly increased the clearance of passively
administered IgE in serum (Fig. 2B). In contrast, using anti-IgE(PNG)
IgG did not lead to a significant reduction in serum IgE levels (Fig.
2C). Flow cytometry evaluated IgE sensitization of basophils one week
after administration of IgE. The anti-IgE IgG group showed significantly
decreased IgE surface levels compared to the IgE-only control group. The
anti-IgE(PNG) IgG group also showed decreased IgE levels on basophils.
However, their levels were significantly higher than the IgE levels of
the anti-IgE IgG group (Figs. 2D, 2E). Avidity ELISA was performed to
analyze the avidity of the anti-IgE IgG antibodies. The results show
that approximately 13% of the anti-IgE IgG and 4% of the anti-IgE(PNG)
IgG have high avidity (Fig 2F). This difference could explain why
anti-IgE IgG are better able to clear IgE from serum than
anti-IgE(PNG)-IgG.
These experiments conclude that anti-IgE IgG antibodies induced by
glycosylated IgE are more efficient in downregulating passively
administered IgE.