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.