The hybridoma-produced mouse IgE anti-DNP antibody clone SPE-7 is one of the most widely used molecules to study IgE biology and function. At the same time, SPE-7 has exhibited some unique characteristics such as the ability to trigger mast cell activation and survival independent of a cross-linking antigen,^{1, 2} the potential to bind DNP-unrelated antigens through conformational diversity,³ and an increased capability to bind mouse Fcγ receptors.⁴ Here, we compared IgE clone SPE-7 to other mouse IgE clones using enzyme-linked immunosorbent assays (ELISA), biolayer interferometry (BLI), liquid chromatography—mass spectrometry (LC–MS), and cell-based in vitro assays with mouse bone-marrow derived mast cells (BMMCs) and spleen-derived mouse B cells.

Surprisingly, we found that in contrast to other mouse IgE clones, SPE-7 was recognized by an anti-IgG detection antibody (Figure 1A–D, Figure S1). These IgG signals were lost by purification of SPE-7 using protein G columns. IgG present in SPE-7 could be captured with an anti-IgG2a/b antibody (but not with anti-IgG1) and detected with an anti-IgE antibody, suggesting the presence of IgG2a/b-IgE complexes in SPE-7 (Figure 1E,F). Similarly, SPE-7 ultracentrifugation reduced the contaminating IgG in SPE-7 (Figure S1).

The presence of IgG in SPE-7 was confirmed by LC–MS. We compared the cumulative distribution between samples using MaxLFQ and Top3 normalization protocols (suppl. Methods, Figure S1). Figure 1G shows an increased prevalence of IgG2a/b in IgE SPE-7 compared to IgE F127, and both IgG2a and IgG2b showed up in the top 8 up-regulated hits summarized in Figure 1H.

To test if IgE SPE-7 can functionally bind to IgG receptors, we displayed FcγRI (CD64) on BLI chips (Figure 2A) or on ELISA plates (Figure 2B). In both cases SPE-7, but no other IgE clone, showed binding activity to FcγRI. Using flow cytometry analysis, we next tested whether IgG influences the binding of IgE SPE-7 to the IgE receptor CD23 expressed on spleen-derived B cells. Indeed, Figure 2C shows that binding of IgE SPE-7 to CD23 was increased compared to other IgEs, or compared to IgG-depleted SPE-7.

The binding of SPE-7 to FcεRI-expressing BMMCs at 4°C was not significantly altered by IgG removal (Figure S2). When BMMCs were incubated with the different IgEs at 37°C, activation marker CD63 was slightly but consistently up-regulated by SPE-7 but not by IgG-depleted SPE-7 or control IgEs (Figure 2D). In survival tests over 3 days in absence of IL-3/SCF, IgE SPE-7 but not IgG-depleted IgE SPE-7 increased BMMC survival compared to medium controls (Figure 2E). Finally, in classical antigen-dependent degranulation assays, IgG depletion led to a striking increase in the ability of IgE SPE-7 to de-granulate mast cells (Figure 2F,G).

In conclusion, we unveil that purified monoclonal IgE SPE-7 can contain mouse IgG antibodies that form complexes with IgE and alter its functional profile. Our results are in line with previous studies that attributed the unique effects of SPE-7 to the presence of trimeric aggregates.¹ Our findings challenge the concept that IgE exerts antigen-independent effects on mast cell degranulation and survival. Moreover, the extent to which mouse IgE can bind to FcγRs needs to be carefully re-evaluated. The physiological relevance for IgG-IgE complexes is still given, as natural IgG anti-IgE autoantibodies and IgG-IgE complexes occur in mice and humans.⁵ Our findings are important to consider in the production of therapeutic IgE antibodies, which are gaining interest in cancer therapy.⁶ Contaminating IgGs may alter the functional and pharmacological profile of these therapeutic IgEs. Future studies will investigate how IgG makes its way into hybridoma-produced IgE clones.

AUTHOR CONTRIBUTIONS

P.E. conceptualized and supervised the study. M.S. and M.Z. purified IgE and performed ELISA. M.S. and P.E. performed cell-based assays. M.V. performed biolayer interferometry. A.C.U. and S.B.L. performed mass spectrometry. P.E. wrote the manuscript, A.C.U., M.S. and M.V. edited the manuscript.

FUNDING INFORMATION

This work was funded by a grant from Holcim Stiftung zur Förderung der wissenschaftlichen Fortbildung, and a grant from Novartis Foundation for Medical-Biological Research number 23B097 awarded to P.E.

CONFLICT OF INTEREST STATEMENT

The authors declare no conflict of interest in relation to this work.