Acid Stripping of Surface IgE Antibodies Bound to FcεRI Is Unsuitable for the Functional Assays That Require Long-Term Culture of Basophils and Entire Removal of Surface IgE

Abstract

Basophils are rare granulocytes and dysregulated functions of these cells are associated with several atopic and non-atopic allergic diseases of skin, respiratory system and gastrointestinal tract. Both cytokines and immunoglobulin E (IgE) are implicated in mediating the basophil activation and pathogenesis of these disorders. Several reports have shown that healthy individuals, and patients with allergic disorders display IgG autoantibodies to IgE and hence functional characterization of these anti-IgE IgG autoantibodies is critical. In general, anti-IgE IgG autoantibodies modulate basophil activation irrespective of allergen specificity by interacting with constant domains of IgE. Therefore, an ideal solution to prove the functions of such anti-IgE IgG autoantibodies would be to completely eliminate type I high affinity immunoglobulin E receptor (FcɛRI)-bound IgE from the surface of basophils and to demonstrate in an unequivocal manner the role of anti-IgE IgG autoantibodies. In line with previous reports, our data show that FcɛRI on peripheral blood basophils are almost saturated with IgE. Further, acetic acid buffer (pH 4) efficiently removes these FcɛRI-bound IgE. Although immediately following acetic acid-elution of IgE had no repercussion on the viability of basophils, following 24 h culture with interleukin-3 (IL-3), the viability and yield of basophils were drastically reduced in acid-treated cells and had repercussion on the induction of activation markers. Lactic acid treatment on the other hand though had no adverse effects on the viability of basophils and IL-3-induced activation, it removed only a small fraction of the cell surface bound IgE. Thus, our results show that acid buffers could be used for the elution of FcɛRI-bound IgE on the basophil surface for the biochemical characterization of IgE antibodies or for the immediate use of basophils to determine their sensitivity to undergo degranulation by specific allergens. However, these methods are not utile for the functional assays of basophils that require longer duration of culture and entire removal of surface IgE to validate the role of anti-IgE IgG autoantibodies that interact with FcɛRI-bound IgE irrespective of allergen specificity.

Basophils from Cancer Patients Respond to Immune Stimuli and Predict Clinical Outcome

“…Here, we identified a discrete population of CCR3 high SSC low basophils in the blood of patients with ovarian cancer of diverse histologies and treatment histories ( Table S3 ). Like basophils from healthy and atopic individuals [ 60 , 61 , 62 , 63 , 64 ], the basophils from ovarian cancer patients also expressed cell-surface FcεRI, which were partly occupied by endogenous IgE. Having confirmed this basophil phenotype, we showed that patient-derived basophils were susceptible to activation by IgE- and non-IgE-mediated stimuli irrespective of prior anti-cancer therapy.…”

Metal-Organic Framework Encapsulated Whole-Cell Vaccines Enhance Humoral Immunity against Bacterial Infection

“…To remove this surface-attached ZIF-encased bacteria, we included three rapid washes with a low pH buffer—a method developed originally to strip IgE antibodies from cell surfaces. 97-99 This completely dislodged the ZIF bound to the macrophage surface as confocal micrographs show no CFT@ZIF on the surface of the acid-washed macrophages ( Fig. S2C ).…”

“…To remove this surface-attached ZIF-encased bacteria, we included three rapid washes with a low pH buffer-a method developed originally to strip IgE antibodies from cell surfaces. [100][101][102] Another LDH assay was conducted to ensure that the low pH wash would not be toxic to the RAW 264.7 macrophages, which again showed the treatment did not kill the cells (Fig. S5E).…”

Metal–Organic Framework Encapsulated Whole-Cell Vaccines Enhance Humoral Immunity against Bacterial Infection

“…While this initially appeared to validate prior literature observation, upon closer inspection, we found a significant amount of the CFT@ZIF had adhered to the surface of the macrophages. To remove this surface-attached ZIF-encased bacteria, we included three rapid washes with a low pH buffera method developed originally to strip IgE antibodies from cell surfaces. − Another LDH assay was conducted to ensure that the low pH wash would not be toxic to the RAW 264.7 macrophages, which again showed the treatment did not kill the cells (Figure S5E). The low pH wash completely dislodged the ZIF bound to the macrophage surface as confocal micrographs show no CFT@ZIF on the surface of the acid-washed macrophages (Figure S5F).…”