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Background

Allergy is an IgE-mediated immune reaction triggered by specific allergens followed by a series of clinical symptoms, such as itching, sneezing, and running nose. Serum concentrations of allergen-specific IgE and the ratio of specific IgE to total IgE are correlated with the appearance of allergic symptoms (1). Hence, results of serum IgE measurement are important for clinical allergy diagnosis and disease management (2). In the current serum IgE measurement protocol, human serum IgE is used as an IgE calibrator, and its preparation is reference to the World Health Organization international standard of human serum IgE (3). Since the IgE standards are prepared by mixing sera and plasma from blood donors, contamination of infectious pathogens, such as hepatitis B viruses, hepatitis C viruses, or human immunodeficiency viruses, is always a concern. The exhaustion of batch preparations is also an issue. Furthermore, due to the lack of allergen-specific human IgE monoclonal antibodies (mAbs) as calibrators, non-specific human IgE antibodies are employed to establish calibration curves. Subsequent detection methods differ from those used to detect allergen-specific IgE in specimens. The inconsistency between detection methods may influence the accuracy of IgE quantification from calibration curves. Also, results from different systems among manufactures may not be interchangeable if they exploit different detecting systems for the same calibrators. Therefore, IgE calibrators using allergen-specific human IgE mAbs or their equivalents, such as allergen-specific chimeric human IgE mAbs produced by transgenic animal and hybridoma technologies, can resolve these problems.

Proprietary gene knock-in mice

To generate human IgE knock-in mice, we constructed two gene-modified mouse embryonic stem cells (ES cells). In one ES cell genome, genes encoding mouse γ1 heavy chains were replaced with human ε heavy chains (Figure 1). In the other ES cell genome, genes encoding mouse κ light chains were replaced with human κ light chain (Figure 1). These two modified ES cells were transplanted into pseudo-pregnant female mice followed by breeding and cross-mating to generate the gene knock-in mice (HεκKI mouse) bearing both homozygous human ε and κ chain genes (Figure 1). These HεκKI mice produce complete chimeric human IgE antibody molecules, in which variable regions of IgE antibodies are mouse origin, and B cells expressing chimeric human IgE (4).

Figure 1

Human IgE knock-in mice - Antibody Supply Service

Preparation of chimeric human IgE mAbs

We employ conventional procedures of mouse hybridoma technologies to prepare chimeric human IgE hybridomas using HεκKI mice. Fusion proteins containing allergen components produced with recombinant DNA and mammalian protein expression techniques are used to immunize HεκKI mice. Blood titers of chimeric human IgE against allergen components are tested with enzyme-linked immunosorbent assays (ELISA). Fusion of mouse spleen cells and mouse myeloma cells (FO) followed by drug selections is carried out to produce hybridomas. After screening culture supernatants with ELISA and cloning positive single cells, IgE hybridomas producing allergen-specific chimeric human IgE mAbs are able to be prepared. Chimeric human IgE mAbs are purified from either hybridoma culture supernatants or peritoneal ascites of hybridoma-injected mice by affinity chromatography with KappaSelect resins.

Purity and Stability

The purity of chimeric human IgE mAbs purified with KappaSelect resins was examined by two antibody sources: culture supernatants of der p 1-specific IgE hybridomas (DP1-IgE) and peritoneal ascites of der p 2-specific IgE hybridoma-injected mice (DP2-IgE). Protein electrophoresis showed that both IgE mAbs exhibited high purity (>98%, Figure 2, Lane 2 and 5) and the sizes of ε heavy chains and κ light chains appeared at the corresponding molecular weights. We also tested the stability of IgE mAbs in two conditions: IgE mAbs were incubated at 37 °C for 48 hours or subjected to 5 freeze/thaw cycles between -20 °C and room temperature. Protein electrophoresis showed that heavy chains and light chains of IgE molecules were still intact after enduring these two tested conditions (Figure 2). This indicated that chimeric human IgE mAbs prepared by our system (1 mg/mL) were stable and resistant to temperature fluctuation.

Figure 2

Western Blot - IgE mAbs - Antibody Supply Service

Specificity

The specificity of chimeric human IgE mAbs was examined by incubating allergen-specific IgE mAbs in different blocking buffers containing various protein antigens, followed by reacting towards specific allergens using ELISA. A commercially available Der p 2-specific chimeric human IgE mAb, denoted as DP2-IgE (2B12), was also compared.5 Results showed that allergen-specific IgE mAbs (50 ng/mL) in the blocking solutions, bovine serum albumin (BSA), non-fat milk (NFM), or human serum (HS), were able to recognize their specific allergens without significantly losing their binding strength (Figure 3). The result also indicated that the specificity of mAbs prepared from HεκKI mice was comparable with that from normal mice.

Figure 3

ELISA - IgE mAbs - Antibody Supply Service

Cellular activity

The most important biological function of IgE is to mediate the degranulation of mast cells and basophiles releasing inflammatory substances. In this regard, the activity of chimeric human IgE mAbs was tested in vitro by sensitizing a rat basophilic leukemia (RBL) cell line SX-38 expressing human IgE receptors FcεRI with IgE mAbs followed by stimulation with specific antigens or cross-linking antibodies. The RBL SX-38 cells were incubated with 1 μg/mL human IgE or papain-specific chimeric human IgE mAbs (PAP-IgE). After washing unbound IgE away, specific antigens (Papain) or cross-linking antibodies (Goat anti-human IgE) were incorporated to trigger the degranulation of RBL SX-38 cells followed by measuring the activity of β-hexosaminidase in the medium. The results showed that PAP-IgE-sensitized RBL SX-38 cells were able to be stimulated in the presence of specific antigens or cross-linking antibodies (Figure 4). The percentage of β-hexosaminidase released in the PAP-IgE group was comparable with that in the human IgE group (Figure 4).

Figure 4

Degranulation - IgE mAbs - Antibody Supply Service

IgE calibrators

In current clinical protocol to measure allergen-specific serum IgE concentrations, IgE calibrators are used as reference standards to convert measured chemical signals into protein concentrations. The binding curve of IgE calibrators (a regression curve of measuring several known IgE concentrations) is established with the detection method similar to the sandwich ELISA (Figure 5, Method A). However, allergen-specific serum IgE is detected with a different method similar to the indirect ELISA (Figure 5, Method B). It is a cause for concern that using different methods to establish reference and measure samples may generate inaccurate results. In this regard, we compared the binding curves of chimeric human IgE mAbs generated from these two methods. The binding curves of human IgE and chimeric human IgE mAbs generated from the Method A in figure 5 were overlapped (Figure 6, Anti-human IgE mAbs). Conversely, binding curves were poorly matched among results of human IgE analyzed by the Method A and chimeric human IgE mAbs by the Method B (Figure 6, Specific allergens). Binding curves of the two chimeric human IgE mAbs specific to Der p 2 allergens were also different (Figure 6, Specific allergens). These results indicated that detection methods, allergen natures, and antibody clones (or affinity) were influential factors to determine the shape of antibody binding curves. If methods used to establish binding curves of IgE calibrators are in agreement with those used to detect allergen-specific serum IgE, the conversion of IgE concentrations from IgE calibrators will be more accurate and consistent. Allergen-specific chimeric human IgE mAbs may fulfill the requirements for better IgE calibrators.

Figure 5

Sandwich and Indirect ELISA - IgE mAbs - Antibody Supply Service

Figure 6

Binding Curves - Sandwich and Indirect ELISA - IgE mAbs - Antibody Supply Service

Advantages of chimeric human IgE mAbs

  • Easy to prepare IgE hybridomas
  • Allergen-specific
  • High affinity and purity
  • Stable in high concentrations
  • Capable of binding FcεRI receptors and mediating the activation of basophiles
  • Prepared in high concentrations and large quantities
  • Non-human specimen origin (comparing to human IgE)
  • Better IgE calibrators for measuring allergen-specific serum IgE/a>

References

1. Hamilton RG, et al., IgE antibody-specific activity in human allergic disease. Immunol. Res. 47:273-284 (2010)

2. Hamilton RG, et al., Human IgE antibody serology: A primer for the practicing North American allergist/immunologist. J. Allergy Clin. Immunol. 126:33-38 (2010)

3. Thorpe SJ, et al., The 3rd International Standard for serum IgE: international collaborative study to evaluate a candidate preparation. Clin. Chem. Lab. Med. 2014;52:1283-1289

4. Lu C-S, et al., Generating allergen-specific human IgEs for immunoassays by employing human ε gene knock-in mice. Allergy. 70:384-390 (2015)

5. DP2-IgE (2B12) is a Der p 2-specific chimeric human IgE mAb prepared by INDOOR Biotechnologies.

Article reproduced from AllerMAbs