A Novel Method for Total IgE Purification from Human Serum

Analysis of IgE is of importance in unraveling immunologic questions from allergic and autoimmune diseases. In relation to this, purification of total IgE from human serum of allergic patients and healthy controls is of importance for allergy research using in vitro models of allergic diseases. The percentage of IgE in the total pool of Igs in serum or plasma is relatively low compared with other isotypes, especially IgG. When using serum in immunoassays, the specific effect of IgE may be overruled or overcrowded due to competitive binding of epitopes by IgG or other Ig subclasses. In addition, for in vitro sensitization of cultured human mast cells, pooled serum from allergic patients cannot be used due to degranulation of these cells (I. Kortekaas Krohn, unpublished observations). Purified total IgE from allergic individuals would enable the analysis of IgE-mediated effects in allergic inflammation on the cellular and molecular levels in controlled experimental settings, including sensitized cultured human mast cells (1). Commercial kits for purification of IgE are not available, and methods for the isolation of total IgE have not been described. Until now, purification of Ab subclasses was based on high-affinity chromatography, which has the major advantage of high-affinity isolation of IgE (2–5). However, there are major disadvantages of this method, especially the limited access to high-affinity chromatography, the specific technical knowledge required, and the time-consuming nature of the procedure. Therefore, experimental research on IgE in in vitro and in vivo allergy models is hampered by technical barriers.

Because until today, no straightforward protocol for the isolation of high-purity total serum IgE was available, we developed a rapid and effective method for the purification of total IgE from human serum by combining the use of protein G–coupled spin columns for depletion of polyclonal IgG and subsequent IgE enrichment using anti-human IgE-coupled beads.

Human sera were obtained from 19 participants recruited at the Department of Dermatology at the Universitair Ziekenhuis Brussel. Inclusion was based on the presence of diseases often related with elevated IgE levels (atopic dermatitis, food allergy, allergic asthma). Sensitization (positive skin prick test) to one or more allergens was investigated with a panel of 14 common airborne and food allergens (including grass/tree pollen, house dust mites, fungi, cat, dog, milk, egg, soy, wheat, peanut, fish). To compare IgE Abs from patients with allergy versus those from healthy subjects, four nonsensitized patients were also included. None of the participants had malignant disease, ichthyosis vulgaris, or the presence of other clinically relevant conditions related to heart, lungs, liver, kidneys, gastrointestinal, metabolic, mental, or neurologic disorders at the time of inclusion. A washout period of 7 d was performed for subjects using medications. Reasons for exclusion were actively smoking or exposure to cigarette smoke, history of allergen-specific immunotherapy, pregnancy, or breastfeeding. The demographics of the subjects are depicted in Table I.

The study was approved by the local Ethics Committee (2018-346) and registered at the local Biobank of the Universitair Ziekenhuis Brussel. All subjects gave written informed consent. The study was conducted in accordance with the Good Clinical Practice guidelines and with the guidelines of the World Medical Association’s Declaration of Helsinki.

Peripheral blood was drawn and the blood samples were allowed to clot for 15–30 min. Next, the samples were centrifuged at 1400 × g for 10 min at 20°C, and serum aliquots were stored at −80°C until use.

Prior to the IgE purification, goat-anti human IgE Fc Abs (Thermo Fisher Scientific, no. 15700) were covalently coupled to epoxy-coated magnetic Dynabeads using an Ab coupling kit (Thermo Fisher Scientific, no. 14311D) according to the manufacturer’s instructions. The precoated beads were stored at 4°C until use and no longer than 14 d. The final bead concentration was 10 mg/ml Ab-coupled beads. Before use, the beads were washed once with PBS with 0.1% BSA for 5 min. The tube with the suspension of beads was placed in the magnetic field in front of the magnet (DynaMag-2, Thermo Fischer Scientific, no. 12321D) for 1 min. After removing the wash buffer, the beads were ready for immediate use or were resuspended in 100 µl of PBS containing 0.05% Tween 20 (PBST) (Sigma Aldrich, no. P1379) until use.

A volume of 50 µl of protein G–coated Dynabeads (Invitrogen, Thermo Fisher Scientific, no. 10003D) was transferred to a 1.5-ml microcentrifuge tube. The beads were washed 10 times with PBST to increase specificity and avoid unspecific proteins. The suspension was placed in front of the magnet for 1 min. After the last washing step, the buffer was removed and immediately used for the purification of IgG.

The protocol is summarized and visualized in (Fig. 1.

Undiluted human serum (500 μl) was added to a neutralizing Ab protein G spin column (Thermo Fisher Scientific, no. 89953). One column of immobilized protein G allows high-purity purification of 100–1000 µg of polyclonal IgG from human (or mammalian) serum. Total polyclonal IgG was purified according to the protocol of the manufacturer. This enriched IgG fraction can be stored if needed for further use. The flowthrough, containing serum without polyclonal IgG, was transferred to a clean 1.5-ml microcentrifuge tube.

Next, the flowthrough serum was incubated with 50 μl of the precoated Dynabeads coupled with goat anti-human IgE Fc Ab (Thermo Fisher Scientific, no. H15700) for 10 min at room temperature with rotation. The tube was placed in front of the magnet and the supernatant was removed. The beads were washed 10 times with 1 ml of PBST (0.05%). After the last washing step, 50 µl of elution buffer (200 mM glycine, pH 2.0, Merck Millipore, no. 1042010100) was added and incubated with rotation for 15 min at room temperature. Next, the tube was placed in front of the separation magnet and the suspension, containing the eluted Ab, was transferred to a clean 1.5-ml microcentrifuge tube. A new volume of 50 µl of elution buffer was added to the beads to capture the remaining IgE Abs and the tube was placed in front of the magnet. The suspension was also added to the tube with eluted IgE Abs and kept on ice. The pH of the eluate was adjusted by addition of 10 µl of 1 M Tris (pH 8.0). The beads were kept to determine the efficiency of the elution step.

To ensure high purity of IgE, the eluate was added to a new tube containing 50 µl of washed protein G–conjugated beads and incubated with rotation for 10 min at room temperature. The tube was placed in front of the magnet and the suspension, containing purified total IgE, was transferred to a clean tube.

The purity of total IgE and polyclonal IgG fractions as well as the elution process were evaluated using Western blot.

For validation, the following fractions were analyzed: purified total IgE, anti-human IgE Fc-coupled beads, purified polyclonal IgG, and protein G–coupled Dynabeads and positive controls (human IgE, Invitrogen, Thermo Fisher Scientific) diluted 1:10 in Tris and human IgG (Sigma-Aldrich) diluted 1:50 in Tris.

Loading buffer (2×) was added to the samples and heated on a thermo block at 96°C for 5 min. Then, the samples were loaded and separated on freshly prepared 12% SDS-PAGE at 110 V for 1.5 h at room temperature. Subsequently, proteins were transferred to nitrocellulose membranes (Thermo Fisher Scientific, no. 88018) at 100 V for 1 h at 4°C. The membrane was blocked with TBS with 0.1% Tween 20 (TBST) and 5% nonfat milk for 1 h at room temperature. For the detection of IgE, the membrane was incubated with an anti-human IgE Ab conjugated with HRP (Invitrogen, Thermo Fisher Scientific, no. A18793) diluted 1:2000 in TBST containing 5% BSA overnight at 4°C on a shaker. After the first visualization, the membrane was stripped with glycine stripping buffer to remove all anti-IgE Abs. The same membrane was stained with anti-human IgG-HRP Abs (Invitrogen, Thermo Fisher Scientific, no. A18817) diluted 1:10,000 in TBST with 5% nonfat milk for 1.5 h at 4°C on a shaker. For development of the blot, the membrane was incubated with Clarity Western ECL substrate (Bio-Rad, no. 1705062). Images were acquired with the Odyssey Fc imaging system (LI-COR Biosciences).

A human IgE uncoated ELISA kit (range, 3.9–500 ng/ml; Invitrogen, Thermo Fisher Scientific, no. 885061088) was used according to the manufacturer’s protocol. Paired measurements were performed to obtain concentrations of both total serum IgE and purified IgE fractions. All samples were measured in duplicates.

Protein concentration of purified polyclonal IgG was determined using the Pierce bicinchoninic acid protein assay (Thermo Fisher Scientific, no. 23227) following the manufacturer’s protocol for the 96-well microplate procedure. The detection range of the assay was 20–2000 μg/ml. All samples were measured in triplicates.

Peripheral blood–derived human mast cells were cultivated according to a previously described protocol (6). Mature mast cells were prepared at and provided by the Department of Dermatology and Allergy, Charité–Universitätsmedizin Berlin. Per condition, mast cells (5 × 10⁵) were sensitized with purified IgE (100 ng) from a single allergic patient or with a pool of purified IgE from four allergic patients in serum-free StemSpan culture medium (STEMCELL Technologies, no. 09650), supplemented with recombinant human stem cell factor (100 ng/ml, PeproTech, no. 300-07), recombinant human IL-6 (50 ng/ml, PeproTech, no. 200-06), and penicillin and streptomycin (200 μg/ml, Thermo Fisher Scientific) for 24 h at 37°C. Human isotype IgE (100 ng, Thermo Fisher Scientific, no. DIA HE1A-01) and/or culture medium were used for sensitization as positive and negative control conditions, respectively. On the next day, the mast cells were washed with PBS and activated with 1) rabbit anti-human IgE Ab (1 μg/ml, BIOKÉ, no. BET A80-109A), or 2) PMA (20 nM)/ionomycin (2 μM), or 3) a relevant allergen (based on positive skin prick test), or 4) culture medium (unstimulated control) for 30 min at 37°C. Cells were used for flow cytometry (expression of the activation marker CD63), and culture medium samples were collected and stored at −80°C until use for histamine release. Data are based on four individual experiments testing several samples (single donors or pooled IgE samples) and control conditions using three batches of mast cells from different donors.

The culture median was stained with Alexa Fluor 488–conjugated anti-human CD63 Ab (undiluted, BioLegend) and for viability using a Live/Dead fixable near-IR dead cell stain (1:50, Thermo Fisher Scientific, no. L10119) for 20 min at room temperature. Flow cytometry (BD LSRFortessa) was performed to detect increased CD63⁺ expression of the mast cells. Data were analyzed using FlowJo v9 software.

The histamine levels in the cell culture media released by activated mast cells were quantified using a histamine ELISA kit (Enzo, no. ENZ-KIT 1400-001; range, 0.098–25 ng/ml) according to the manufacturer’s protocol.

Serum samples of 19 participants (63% female; median, 24 y old; interquartile range, 23–33.5) were used for validation of the purification protocol. A skin prick test was performed with a panel of frequent aeroallergens and food allergens. Four participants had a negative skin prick test and 15 were positive for one or more allergens (Table I).

Western blot analysis revealed that IgE Abs of ∼70 kDa were observed in the positive control (human IgE) and in the purified total IgE fraction. This corresponds to the molecular mass of the H chain of IgE Abs. This was observed in all patient samples (Fig. 2, top row). For evaluation of the efficacy of the elution step, the Dynabeads used for IgE purification were analyzed. Only minor fractions of IgE remained on the beads, confirming an effective elution of the IgE Abs from the beads (Fig. 2, middle row).

After staining with anti-human IgG Ab, no residual IgG Abs were detected in the total IgE fraction, confirming the absence of IgG in the purified IgE fractions (which ensures a high purity of IgE Abs). Purified polyclonal IgG Abs corresponded with the molecular mass and the positive control of human IgG (Fig. 2, bottom row). This confirmed the depletion of polyclonal IgG from the serum samples.

Unused protein G–coupled Dynabeads stained negative for IgE and IgG. Furthermore, as expected, Dynabeads precoated with goat anti-human IgE Fc Abs (IgG isotype) stained positive for IgG, but not for IgE. Finally, the negative controls (TBS) with loading buffer were negative for the presence of IgE or IgG.

Measurements of total serum IgE and purified IgE samples were performed for each patient (Fig. 3A, 3B, Table I). The concentration of total serum IgE ranged from 8.9 to 5811.9 ng/ml. The purified IgE samples ranged from 82.9 to 2949.5 ng/ml, with three samples below detection limit (Table I). High levels of total serum IgE resulted in a high yield of purified IgE and vice versa. This confirms that total IgE is optimally purified from serum. Purified IgE levels also corresponded with the Western blot analysis (Fig. 2). Based on the present results, the minimum amount of total serum IgE prior to isolation is ∼370 ng/ml (=154 kU/l) for a detectable and useful amount of purified IgE, which results in ∼100 ng/ml. The median recovery percentage is 43.7% (interquartile range, 23.0–54.2%).

For determination of the purified polyclonal IgG concentrations, a bicinchoninic acid assay was performed (Fig. 3C). The concentrations ranged from 274.6 to 1049.8 μg/ml. As mentioned above, these high yields of polyclonal IgG were also confirmed by Western blot analysis (Fig. 2).

As a proof of concept, the functionality of the purified IgE was investigated using primary human mast cells. Mast cells were sensitized with purified IgE followed by activation with anti-IgE Ab or a relevant allergen (as determined by skin prick test). Strongly increased CD63⁺ expression was observed following activation with anti-IgE Ab, with a relevant allergen or with PMA/ionomycin (positive controls). Low levels of CD63⁺ expression were seen when incubated with culture medium (negative controls, Fig. 4). In line with the expression of CD63, the levels of histamine in culture medium samples were increased after stimulation with anti-IgE Ab (Fig. 5), although less pronounced after specific allergen activation (data not shown). Importantly, these results confirm the functionality of the purified IgE fractions and the possibility for sensitization of human mast cells with purified human IgE from allergic individuals.

The presented protocol provides a detailed description of a (to our knowledge) novel method for purification of total IgE from human serum. This protocol is time-efficient and relatively straightforward to perform with standard equipment present in life sciences laboratories. The present protocol used human serum samples and can be adapted for use of other mammalian species. As far as we are aware, no comparable and detailed protocol for purification of IgE from serum was published previously.

Recently, an abstract described the use of serum IgE isolation from betalactam allergic patients (7) in which ion-exchange chromatography was compared with conjugated Dynabeads with either polyclonal human anti-IgE Abs or with mAb omalizumab. The authors concluded that ion-exchange chromatography resulted in higher efficiency of the IgE purification compared with Dynabeads. However, the method using Dynabeads was more time-efficient. Unfortunately, no full-text protocol was published. Anti-IgE mAb conjugated to NHS Mag Sepharose has been used for purification of total IgE from 1 l of pooled serum from patients with chronic spontaneous urticaria or from healthy controls. After 24 h of incubation at 4°C, purified IgE was used to sensitize peripheral CD34⁺ stem cell–derived mast cells (6). Although this is an elegant protocol, the information on the purification is limited, and the process requires large amounts of serum and overnight incubation. Publications from other research groups between 1973 and 2004 were based on affinity chromatography, ion-change chromatography or DEAE cellulose chromatography (2–5, 8, 9). Although high purity and a high yield of purified IgE can be obtained, the technical procedure requires specific equipment, extensive knowledge, and is expensive and complex, which limits the use of this method.

The present protocol has some limitations. Relatively small volumes of serum (500 µl) were used for the development of this protocol, which results in relatively small amounts of IgE. However, the volume can be scaled up using the available protein G–coupled spin columns that allow larger volumes of serum and adjustment of the volume of the Dynabeads.

In this study, we present a (to our knowledge) novel protocol of a rapid and straightforward method for purification of IgE from human serum based on the use of a protein G spin columns followed by a positive selection using magnetic beads. The use of this technique may improve the use of in vitro and even in vivo models and contribute to the research within the field of allergy and clinical immunology.

We thank Hugo Vandenplas for support and technical assistance.

The authors have no financial conflicts of interest.