Crude Peanut Extract (CPE) and Mouse Anti-CPE IgE Monoclonal Antibody

Immediate hypersensitivity reactions to peanuts, an IgE-mediated food allergy, has been a major public health concern for many years, particularly in westernized countries where peanut allergies can persist into adulthood. For allergic patients, avoidance currently remains the only viable option (1). Peanut allergens contain eleven potentially major components: Ara h1, Ara h2, Ara h3, and Ara h6. Ara h2 and Ara h6, two highly related 2S albumins, especially contribute to the development of allergic reactions (2).

Mouse peanut allergy models are used to study the pathogenesis of the peanut allergy and to evaluate new treatments. The mouse models can be induced by administration of crude peanut extract (CPE) or each purified Ara allergen and evaluated for humoral immune responses such as serum anti-IgE and IgG antibodies against the allergen, T-cell mediated immune response associated cytokines levels, as well as body temperature and clinical signs of anaphylaxis. These factor changes observed in the disease models are useful for studying the efficacy of protective effects against the development of allergic reactions (3–9).

To evaluate humoral immune responses against CPE in mouse allergy models, Chondrex, Inc. provides CPE and anti-CPE IgE monoclonal antibodies for in-vitro and in-vivo studies. Additionally, Chondrex, Inc. manufactures Mouse Anti-CPE Antibody Assay Kits, Cytokine Detection ELISAs, and Chemokine Detection ELISAs for evaluating immune responses in mouse CPE type I hypersensitivity models. 

To learn more about CPE-Induced Type I Hypersensitivity models, please proceed to the Table of Contents below. If you have any questions about Chondrex, Inc.’s Mouse Allergic Disease Models, please contact us at support@chondrex.com.

NOTE: These CPE Type I Hypersensitivity models are highly reproducible due to CPE’s high antigenicity. However, different allergens, such as Ovalbumin (OVA), house dust mites (HDM), may also be involved in the pathogenesis of human allergic asthma. Chondrex, Inc, also provides OVA, anti-OVA IgE monoclonal antibodies, Mouse Anti-OVA Antibody Assay Kits, HDM extracts, and Mouse Anti-HDM Antibody Assay Kits for other mouse allergic disease studies.

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Table of Contents

  1. IgE, IgE receptors, and mast cells in mouse allergic models
  2. Characterization of anti-CPE IgE monoclonal antibodies
  3. Mast cell activation studies
  4. Animal studies
    1. Mouse footpad type I hypersensitivity studies
    2. Mouse anaphylaxis studies
  5. Summary of the biological activities in the four IgE monoclonal antibodies.

1. IgE, IgE receptors, and mast cells in mouse allergic models

Food allergies are immunologically characterized by dysregulated responses and the development of immediate hypersensitivity reactions to ingested food components. In allergic reactions, IgE antibodies against an allergen play critical roles. IgE antibodies can bind to two types of IgE receptors: the high-affinity Fc?RI and the low-affinity Fc?RII. Fc?RI is expressed primarily on mast cells, basophils, and dendritic cells. Crosslinking of Fc?RI by IgE antibodies and allergens (with IgG antibodies) can activate the receptors and initiate intracellular signaling pathways. The activated signaling induces both cell degranulation and release of preformed mediators, such as amines (histamine), proteoglycans, proteases, lysosomal enzymes ( b-hexosaminidase), newly formed lipid mediators, cytokines, and chemokines (GM-CSF, IL-1b, IL-8, IL-13, MCP-1)(10, 11) (Figure 1a and 1b). 

In murine models, the roles of IgE antibodies, Fc?RI, and mast cells are verified in the onset of immediate hypersensitivity reactions and anaphylaxis following acute food allergen challenge in sensitized animals (12). As a mast cell model, RBL-2H3 cells were established from rat basophilic leukemia cells and have comparable functions and phenotypes to mast cells. Thus, these cells are commonly used for in-vitro studies associated with allergens, IgE antibodies, FceRI receptors, and subsequent downstream events (13).

Figure 1. Two mechanisms of mast cell-bound IgE antibody cross-linking: a) cross-linkage by a single allergen and b) cross-linkage by a single IgG antibody bound to two allergen molecules. Mast cells are degranulated (activated) by cross-linking the two IgE receptors by which two adjacent IgE antibodies bound to the IgE receptors on mast cells capture a polyvalent allergen (Figure 1a). Alternatively, immune-complexes of allergens bound by IgG antibodies can also degranulate mast cells by bridging the allergens bound to IgE antibodies on mast cells (Figure 1b). Therefore, not only IgE antibodies, but also IgG antibodies against allergens play roles in the development of allergic reactions.
 

2. Characterization of anti-CPE IgE monoclonal antibodies

1) Immuno-blot analysis

The protein profile of crude peanut extract (CPE) was analyzed by 10% SDS-PAGE under reducing and non-reducing conditions. CPE revealed multiple protein bands including Ara h1, h2, h3, and h6 (Figure 2). Under non-reducing conditions, 6E10C12, 6B7B10, and 5E7B12 recognized a 68 kDa protein, while 2G11G7 bound to both 63 and 60 kDa proteins. This result suggests that their epitope is stable even after denaturing CPE at 100 degrees C under reducing conditions with SDS and linearization. However, under reducing conditions only 6B7B10 recognized both 68 and 63 kDa proteins (Figure 2). 

Figure 2. Immuno-blot analysis of anti-CPE IgE monoclonal antibodies against CPE
SDS-PAGE and immuno-blot analysis of CPE under reducing conditions (A) and non-reducing conditions (B). Coomassie brilliant blue R-250 staining; M: Molecular Marker and 1: CPE, Immuno-blot 2: 6E10C12, 3: 2G11G7, 4: 6B7B10, and 5: 5E7B12
 

2) ELISA

Four mouse IgE mAbs against CPE were evaluated in a sandwich ELISA employing anti-mouse IgE antibody-coated plates and biotinylated CPE as a tracer (Figure 3). The four IgE mAbs reacted with biotinylated CPE dose-dependently. 6E10C12, 2B11B7, and 6B7B10 showed similar reactivities between 1- 10 ng/ml while 5E7B12 had lower reactivities against CPE in the 10 -100 ng/ml range.

Figure 3. Dose-dependency of anti-CPE IgE monoclonal antibodies in a sandwich ELISA 
Mouse anti-CPE IgE monoclonal antibodies (mAbs): 6E10C12 (circle), 2G11G7 (triangle), 6B7B10 (square), and 5E7B12 (diamond), diluted with 0.1M Tris-buffered saline pH 7.5 containing 1% BSA were added to wells coated with 10 ?g anti-mouse IgE monoclonal antibody and incubated at room temperature for 2 hours. After washing the plates with 0.05M Phosphate buffered saline pH 7.4 containing 0.05% Tween 20, biotinylated CPE diluted with 0.1 M Tris-buffered saline pH 7.5 containing 2% casein was added and incubated at room temperature for 1 hour. After washing the plates with washing buffer, avidin-peroxidase diluted with casein buffer was added and incubated at room temperature for 30 minutes. After washing plates, the antibody binding was visualized with TMB at room temperature for 25 minutes, stopped, and read for OD values at 450 nm/630 nm. 

Then, the four mouse IgE mAbs were evaluated by indirect ELISA that consisted of CPE coated plates and anti-mouse IgE detection antibodies. In the indirect ELISA, 2G11G7 and 6B7B10 demonstrated higher, dose-dependent reactivities against CPE coated on the plate, with an antibody concentration range between 31.5 - 2000 ng/ml. In the same antibody concentration range, 6E10C12 showed lower reactivity and 5E7B12 failed to react with CPE coated on the plates.

Figure 4. Dose-dependency of mouse anti CPE IgE monoclonal antibodies in an indirect ELISA
Mouse anti-CPE IgE monoclonal antibodies (mAbs): 6E10C12 (circle), 2G11G7 (triangle), 6B7B10 (square), and 5E7B12 (diamond) diluted with 0.1M Tris-buffered saline pH 7.5 containing 1% BSA were added to wells coated with 1 ug of CPE and incubated at room temperature for 2 hours. After washing the plates with 0.05M Phosphate buffered saline pH 7.4 containing 0.05% Tween 20, biotinylated anti-mouse IgE antibodies diluted with 0.1 M Tris-buffered saline pH 7.5 containing 2% casein were added and incubated at room temperature for 1 hour. After washing the plates with washing buffer, avidin-peroxidase diluted with casein buffer was added and incubated at room temperature for 30 minutes. After washing plates, the antibody binding was visualized with TMB at room temperature for 25 minutes, stopped, and read for OD values at 450 nm/630 nm.
 

3. Mast cell activation studies

The biological activities of the four IgE mAbs were evaluated by beta-hexosaminidase release in degranulation assays using RBL-2H3 cells. The RBL-2H3 cells were sensitized with serial dilutions (0.128 - 2000 ng/ml) of the mouse IgE mAbs and then were challenged with 2 ?g/ml of CPE (Figure 5). A typical prozone effect was observed with a bell-shaped dose response curve. With 2G11G7 and 6E10C12, relatively low levels of beta-hexosaminidase release were recorded with high concentrations of IgE (400–2000 ng/ml), with the degranulation increasing to maximal levels between 3.2 and 80 ng/ml, ranging from approximately 20% to 60% release. At concentrations of 0.128 ng/ml, the beta-hexosaminidase release approached background levels. With 6B7B10, the highest beta-hexosaminidase release was lower, peaking at 15% between 3.2 and 80 ng/ml. Interestingly, 5E7B12 failed to degranulate RBL-2H3 cells. The prozone effect can be explained by the formation of monovalent complexes between multivalent antigens and IgE antibodies on their receptors at high concentrations. These complexes prevent the bridging of two IgE antibodies, leading to lower degranulation despite high antigen concentrations.

Figure 5. RBL-2H3 cell activation with CPE and mouse anti-CPE IgE monoclonal antibodies 
RBL-2H3 cells were cultured in 0.1 ml of DMEM containing 15% FBS at 10^6 cells/well in a 96-well plate at 37 degrees C for 3 hours and then treated with anti-CPE IgE monoclonal antibodies, 6E10C12 (circle), 2G11G7 (triangle), 6B7B10 (square), and 5E7B12 (diamond) at 37 degrees C for 16 hours. After washing cells with PBS two times, 2 ?g/ml of CPE in Tyrode’s buffer were added at 200 ul/well and incubated at 37 degrees C for 1 hour. 100 ?l of samples from each well were transferred to a 96-well plate and assayed for beta-hexosaminidase activity. The degranulation of RBL-2H3 cells were expressed as a ratio compared with 100% degranulation of the cells which received 1% Triton-X in Tyrode’s buffer.

The dose dependency of CPE concentration for the sensitization of RBL-2H3 cells was evaluated with three mouse IgE mAbs. At 100 ng/ml for both 2G11G7 and 6E10C12, CPE sensitization induced beta-hexosaminidase release dose-dependently from 0.5 to 8 ?g/ml. At 20 ng/ml for 6B7B10, the release was recorded between 2 and 8 ug/ml. In these assays, sensitization with a high concentration of CPE by itself also showed partial beta-hexosaminidase release (data not shown). RBL-2H3 cell activation by CPE alone may be caused by the inherent presence of LPS in CPE. RBL-2H3 cells express TLR2 and TLR4 receptors on their cell surface and even with washing by PBS, remaining serum components such as CD14 and MyD88 in the wells may work with the LPS in CPE to activate TLR4 receptors, resulting in some activation of the cells (14, 15).

Figure 6. Dose-dependency of CPE in RBL-2H3 cell degranulation with anti-CPE monoclonal antibodies
RBL-2H3 cells were cultured in 0.1 ml of DMEM containing 15% FBS at 10^6 cells/well in a 96-well plate at 37 degrees C for 3 hours and then treated with anti-CPE IgE monoclonal antibodies, 6E10C12 (circle), 2G11G7 (triangle), and 6B7B10 (square) at 37 degrees C for 16 hours. After washing cells with PBS two times, various concentrations of CPE in Tyrode’s buffer were added at 200 ul/well and incubated at 37 degrees C for 1 hour. 100 ?l of samples from each well were transferred to a 96-well plate and assayed for beta-hexosaminidase activity. The degranulation of RBL-2H3 cells were expressed as a ratio compared with 100% degranulation of the cells which received 1% Triton-X in Tyrode’s buffer.
 

4. Animal Studies

1) Footpad type I hypersensitivity studies 

Three IgE mAbs were evaluated for mouse type I hypersensitivity. 2B11G7 and 6B7B10 developed paw swelling, 1.10 +/- 0.42 mm and 1.25 +/- 0.78 mm, respectively, that peaked at 1-2 hours after the CPE injection at the footpad and resolved to base levels within 6 hours. However, 6E10B10 failed to induce paw swelling. The RBL-2H3 cells used previously were established from rat basophilic leukemia cells. Although mouse IgE antibodies can activate IgE receptors on rat cells, 6E10B10 may have a lower binding affinity to mouse IgE receptors on mouse mast cells. Therefore, the IgE mAb may not be capable of inducing type I hypersensitivity in-vivo (16). Further analysis is required regarding this discrepancy between cell-based assays and mouse studies for this antibody.

Figure 7. Footpad type I Hypersensitivity by anti-CPE IgE monoclonal antibodies
The footpad thickness of the mice after intravenous administration of 1 mg of monoclonal antibodies: 6E10C12 (circle), 2G11G7 (triangle), and 6B7B10 (square), followed by a 50 ?g CPE (solid line) or PBS (dashed line) intradermal injection at the footpad. 

The dose-dependency of paw swelling induced by 100, 300, and 1000 ug of mAb 2G11G7 was evaluated. The paw swelling of the type I hypersensitivity at 100, 300, and 1000 ug doses was 1.30 +/- 0.43 mm, 1.60 +/- 0.13 mm, and 2.07 +/- 0.10 mm, respectively.  The paw swelling lasted for 6 hours at all doses and resolved to base levels within 8 hours after CPE injection at the footpad.

Figure 8. Dose-dependency of footpad type I hypersensitivity by monoclonal antibody, 2G11G7
The footpad thickness of the mice after intravenous administration of each dose of monoclonal antibody, 2G11G7: 100 ?g (circle), 300 ?g (square), and 1000 ?g (triangle), followed by a 100 ?g CPE (solid line) or PBS (dashed line) intradermal administration at the footpad. 
 

2) Mouse Anaphylaxis Studies

With a CPE challenge, the mice who received 1 mg of 2B11G7 or 6B7B10 showed a drop in their body temperatures, 0.9 +/- 0.7 and 0.7 +/-0.7, respectively after 40 to 60 minutes. Meanwhile, 6E10C12 failed to induce a drop in body temperature. Body temperatures resolved after 90 minutes.

Figure 9. Anaphylactic reactions by anti-CPE IgE monoclonal antibodies
Mice who received 6E10C12 (circle), 2G11G7 (square), and 6B7B10 (triangle). Body temperatures after intravenous administration of 1 mg of CPE to anti-CPE IgE mAb administered mice to assess anaphylaxis was measured rectally from 10 to 90 minutes after injection. Mice had been pretreated 24 hours before challenge with their respective dose of 1 mg of anti-CPE IgE mAbs by intravenous administration. 

Mice were administered a different dose of 2G11G7 IgE mAb by IV injection, followed by intravenous challenge with 1 mg of CPE. After 10 minutes, mouse body temperatures showed drops of 1.2 - 2.7 degrees C. The degree of change for 1000 ?g, 100 ?g, and 300 ug was 2.7 +/-1.7 degrees C, 2.1 +/-1.5 degrees C, and 1.3 +/-1.6 degrees C, respectively. Body temperatures resolved to base levels within 30 minutes.

Figure 10. Dose dependency of anaphylactic reactions by mAb 2G11G7
Mice who received 100 ?g (circle), 300 ?g (square), and 1000 ?g (triangle). Body temperatures of anti-CPE IgE administered mice challenged with a 1 mg intravenous administration of CPE to assess anaphylaxis was measured rectally from 10 to 90 minutes. Mice had been pretreated 24 hours before challenge with their respective dose of anti-CPE IgE mAbs by intravenous administration.
 

5. Summary of the biological activities in four IgE monoclonal antibodies

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