1

Mouse Anti- Ovalbumin Monoclonal IgE Antibodies for Studying Allergic Diseases
Catalog # 3006-3008

For Research Use Only - Not Human or Therapeutic UseINTRODUCTION
Type I hypersensitivity, which is characterized by an allergic reaction immediately following contact with innocuous antigens, is a typicalclinical feature of allergic diseases such as asthma, eczema, hay fever, or urticaria. This hypersensitivity mediated by the IgE antibodies iscalled “atopic reagin” and the clinical features of type I hypersensitivity are described as “atopy”. The high affinity receptor for IgE (FcεRI)
expressed on mast cells and basophils is another critical component in allergic responses. IgE bound to FcεRI drastically up-regulates FcεRI
expression on mast cells through stabilization and accumulation of FcεRI and contributes in further enhancing hypersensitivity responses toallergens (1). Specific allergens bound to IgE on cell surfaces will cross-link two FcεRIs and this linkage leads to the stimulation anddegranulation of mast cells (2-3). This is associated with the release of a variety of proinflammatory mediators and cytokines such as
histamine, proteolytic enzymes, heparin, and chemotactic factors, which cause the symptoms associated with type I hypersensitivity.
Mice are widely used experimental animals for studying pathogenesis and evaluating therapeutics in allergic disease models as a variety ofinbred strains, transgenic mice, and even gene knockout mice are available (2-6). In order to induce allergic diseases in animals, immunize
animals with an antigen adsorbed on aluminum hydroxide gel or expose animals to an aerosolized antigen repeatedly (7-9). In addition, IgE
antibodies against specific antigens can sensitize animals by passive transfer in vivo as well as sensitize mast cells in vitro (8-10). For
example, passive transfer of airway hyper-responsiveness by ovalbumin (OVA)-specific monoclonal IgE and IgG antibodies was reported byOshiba et al., although eosinophilic inflammatory response in the airway is modest despite high levels of serum IgE (8).

LIST OF IgE AND IgG MONOCLONAL ANTIBODIES AGAINST OVA
Chondrex, Inc. provides two IgE monoclonal antibodies specific to OVA, clone E-C1 and clone E-G6, in addition to one IgG1 monoclonal
antibody against OVA, clone L71 (see Type I Hypersensitivity Research Products) (13-15). In general, the cross-linkage of IgE molecules
bound to the receptor on mast cells by a polyvalent allergen is required to trigger degranulation of mast cells. Alternatively, IgG antibodies
specific to the allergen can trigger the activation of mast cells by cross-linking two allergens bound by IgE antibodies on the mast cell surfaces(10). E-C1 alone is capable of inducing degranulation of mast cells in vitro (Figure 1), and severe hypersensitivity in vivo (Figure 2). Therefore,
it is assumed that E-C1 might recognize repetitive epitopes of OVA. Furthermore, it has been reported that aggregated OVA, which carries
multiple epitopes, increases the formation of cross-linked IgE on mast cell surfaces (16). On the other hand, E-G5 is not capable of inducing
these hypersensitivity reactions by itself either in vitro or in vivo (Figures 1 and 2) and thus can be used as a control. Alternatively, Chondrex,
Inc also provide a mouse IgE monoclonal antibody, isotype control, clone 2A101A12 as a negative control antibody.
The IgG1 monoclonal antibody, L71, can be used for a variety of research purposes such as studying the interference between allergenspecific IgG and IgE antibodies as the pathogenic role of IgG in allergic diseases remains uncertain (11). For example, 1) IgG antibodies
might compete with IgE antibodies and block the immediate hypersensitivity reactions, 2) IgG antibodies might stimulate IgE-mediatedhypersensitivity reactions by cross-linking allergens bound to IgE on the surfaces of mast cells, or 3) IgG antibody-antigen immune complexes
might stimulate IgE mediated hypersensitivity reactions by forming multivalent antigens which might bind to IgE on the surfaces of mast cells.

© 2020 Chondrex, Inc. All Rights Reserved, 3006-3008 3.0
16928 Woodinville-Redmond Rd NE Suite B-101
Woodinville, WA 98072

Phone: 425.702.6365 or 888.246.6373
Fax: 425.882.3094

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support@chondrex.com

2TYPE I HYPERSENSITIVITY RESEARCH PRODUCTS

Catalog #Description

Antigen

In Vitro
Mast Cell
Activation

In Vitro
Hypersensitivity
Reaction

Quantity

Storage

3006

Mouse Monoclonal IgE
Antibody E-C1

OVA &Aggregated
OVA *Yes

Yes

1 mg/vial
Lyophilized with
Mouse Serum Albumin

-20°C

3007

Mouse Monoclonal IgE
Antibody E-G5

OVA Only **NoNo

1 mg/vial
Lyophilized with
Mouse Serum Albumin

-20°C

3008

Mouse Monoclonal
IgG1 Antibody L71

OVA &Aggregated
OVANoNo

1 mg/vial
Lyophilized with
Mouse Serum Albumin

-20°C

7129

Mouse Monoclonal IgE
Antibody 2A101A12(Isotype Control)

Not Determined(Not OVA)

NoNo

1 mg/ml in 0.05MPBS, 0.5 ml-20°C* Aggregated OVA can be prepared by mixing 2 ml of OVA solution (20 mg/ml in 0.05M PBS, pH 6.0) and 1 ml of 2.4% glutaraldehyde
diluted with 0.05M PBS, pH 6.0 at room temperature overnight. Add 1 ml of 1M glycine to neutralize excess amounts of glutaraldehyde
and react at room temperature for 30 minutes, then dialyze against 0.05M PBS, pH 7.2. In order to remove un-aggregated OVA, run
Sephacryl S-400 gel filtration chromatography (15).
** Does not react with glutaraldehyde-aggregated OVA.

EXPERIMENTAL PROTOCOLS
A. Handling the IgE Antibody SolutionIgE antibody is biologically active at very low levels, so extra caution is required for handling and diluting IgE antibodies. In general,
several 10 µg of protein will be adsorbed to 1 square cm of glass or plastic surfaces by non-specific hydrophobic binding. Therefore, if
1 ml of a 20 µg/ml IgE solution without blocking agents (such as PBS) is kept in a microcentrifuge tube, almost all the IgE antibodies
will be adsorbed to the tube and no detectable IgE antibodies will remain in solution. Do not dilute IgE solutions to less than 100 µg/ml
with PBS as it does not contain blocking agents. Add a suitable blocking agent to PBS, such as mouse serum albumin, bovine serum
albumin (BSA), or other suitable proteins. IgE Dilution Buffer (Cat # 3009) is preferred for diluting IgE for both in vitro and in vivo
experiments.
B. Assay for Degranulating Mast Cells In Vitro
1.

Culture mast cells such as rat basophilic leukemia cells (RBL-2H3) until the stationary phase. Re-suspend the cells at 2.5 x 105 cells/ml
in Eagle’s minimum essential medium containing 5% fetal calf serum (FCS) and transfer cells into 96-well (250 µl: 6.25 x 104 cell/well)or 24-well (1 ml: 2.5 x 105 cells/well) flat bottom cell culture plates, and continue to culture at 37°C overnight.

2.

Add IgE monoclonal antibodies diluted with PBS containing blocking agents or culture medium containing FCS to the wells (final
concentration: 0-10 µg/ml of medium) and incubate for 1 hour.

3.

NOTE: Instead of adding IgE into culture media after overnight cell culture, IgE can be added to culture media at the start of cell culture.
In this case, FcεRI expression could be significantly upregulated with the overnight culture. For example, it has been reported that FcεRI
expression will be upregulated 2-fold in RBL cell lines and up to 32-fold on bone marrow-derived mast cells within 24-48 hours (1).

© 2020 Chondrex, Inc. All Rights Reserved, 3006-3008 3.0
16928 Woodinville-Redmond Rd NE Suite B-101
Woodinville, WA 98072

Phone: 425.702.6365 or 888.246.6373
Fax: 425.882.3094

www.chondrex.com
support@chondrex.com

3

4.

Wash IgE-sensitized cells 3 times with Tyrode’s buffer (150 mmol/l NaCl, 2.5 mmol/l KCl, 12 mmol/l NaHCO3, 2 mmol/l MgCl2, 2 mmol/l
CaCl2, 1 mg/ml BSA, 1 mg/ml dextrose, pH 7.4).

5.

Add 200 µl (in 96-well plate) or 400 µl (in 24-well plate) of OVA solution diluted with Tyrode’s buffer (the final concentration of antigen:
0-5 µg/ml) and culture for 1 hour.

6.

Add 200 µl (in 96-well plate) or 400 µl (in 24-well plate) of a negative control antigen such as hen egg lysozyme (HEL) or BSA (5 µg/ml)
dissolved in Tyrode’s buffer into the negative control wells to determine spontaneous release of β-hexosaminase.

7.

Add 200 µl (in 96-well plate) or 400 µl (in 24-well plate) of 1% Triton X-100 solution dissolved in Tyrode’s solution into 100% control
wells to determine the total release of β-hexosaminase.

8.

Assay β-hexosaminase activity in the culture supernatant using p-nitro-phenyl-N-acetyl b-D-glucosaminide as a substrate (11).

Figure 1 - Degranulation of Mast Cells by Monoclonal IgE Antibody

6060

Hexosaminase Release (%)

Degranulation (%)

5040302010504030

20100
E-C1/OVA

E-C1/HEM

E-G5/OVA

L-71/OVA0

Anti-DNP

00.003

0.01

0.03

0.1

0.3

Anti-DNP

Monoclonal Antibody/ Antigen
IgE (mg/ml)a)Comparing the Three Monoclonal Antibodies: RBL Cells
cultured in a 24-well plate were sensitized by monoclonal
antibody, E-C1 (1 µg/ml), E-G5 (1 µg/ml) and L71 (50µg/ml), respectively, and then activated by OVA (5 µg/ml).
As a positive control, a combination of anti-DNP IgE
antibody (1:200 dilution in culture media) and DNP-HSA(0.05 µg/ml) was used. E-C1 alone is capable of activating
mast cells in antigen-specific manner, whereas E-G5 (IgE)
and L-71 (IgG1) are not capable under the same conditions.

b)

Dose Response Effect of E-C1 and E-G5: RBL Cells
cultured in a 24-well plate were sensitized with E-C1 and EG5 (0-0.3 µg/ml), respectively and activated by OVA (5µg/ml). As a positive control, cells were sensitized by antiDNP IgE antibody (1:200 dilution in culture media) andactivated by DNP-HSA (0.05 µg/ml).

Note: Aggregated OVA causes an equal degree ofdegranulation of E-C1-sentitixed mast cell at 1/5
concentration of OVA (data not shown).

C. Assay for Hypersensitivity Reactions In Vivo
Dilute IgE antibody solution (1 mg/ml) to 100 µg/ml with PBS containing a blocking agent such as mouse serum albumin. Inject 100 µl of thediluted IgE monoclonal antibody solution intravenously into mice (10 µg/mouse). Inject 25 µl of OVA (2 mg/ml in PBS) intradermally at thefoot pad (50 µg/foot pad) after 24 hours. Determine the swelling of the footpad by measuring the footpad thickness using a dial thickness
gauge every 1-2 hours for 24 hours.

© 2020 Chondrex, Inc. All Rights Reserved, 3006-3008 3.0
16928 Woodinville-Redmond Rd NE Suite B-101
Woodinville, WA 98072

Phone: 425.702.6365 or 888.246.6373
Fax: 425.882.3094

www.chondrex.com
support@chondrex.com

4

Figure 2 - Hypersensitivity Reaction in Foot Pad In Vivo
120
E-C1/OVA100

Foot Pad Swelling (10-2 mm)

Balb/c mice intravenously received 10 µg of monoclonal
antibody E-C1 or E-G5, then were challenged by OVA (50µg) or aggregated OVA (Agg-OVA, 50 µg) by intradermal
injection 24 hours later. Foot pad thickness wasdetermined by a Loop Handle Dial Thickness Gauge andshown as mm.

E-C1/Agg-OVA
E-G5/OVA80

PBS/OVAPBS/Aggreg OVA

60
40
20
0
00.5

12

345

24Time (hours)

REFERENCES
1.

S. Kubo, K. Matsuoka, C. Taya, F. Kitamura, T. Takai, H. Yonekawa, H. Karasuyama, Drastic up-regulation of FceRI on mast cells isinduced by IgE binding through stabilization and accumulation of FceRI on the cell surfaces. J Immunol 167, 3427-34 (2001).

2.

L. McKinley, J. Kim, G. Bolgos, J. Siddiqui, D. Remick, Reproducibility of a Novel Model of Murine Asthma-Like Pulmonary Inflammation.
Clin Exp Immunol 136, 224-31 (2004).

3.

J. Kim, A. Merry, J. Nemzek, G. Bolgos, J. Siddiqui, D. Remick, et al., Eotaxin Represents the Principal Eosinophil Chemoattractant ina Novel Murine Asthma Model Induced by House Dust Containing Cockroach Allergens. J Immunol 167, 2808-15 (2001).

4.

T. Morokata, J. Ishikawa, T. Yamada, Differential Susceptibility of C57BL/6 and DBA/2 Mice to Ovalbumin-Induced Pulmonary
Eosinophilia Regulated by Th1/Th2-type Cytokines. Immunol Lett 70, 127-34 (1999).

5.

S. Matsubara, G. Li, K. Takeda, J. Loader, P. Pine, et al., Inhibition of Spleen Tyrosine Kinase Prevents Mast Cell Activation and Airway
Hyperresponsiveness. Am J Respir Crit Care Med 173, 56-63 (2006).

6.

J. Kim, L. McKinley, J. Siddiqui, G. Bolgos, D. Remick, Prevention and Reversal of Pulmonary Inflammation and Airway
Hyperresponsiveness by Dexamethasone Treatment in a Murine Model of Asthma Induced by House Dust. Am J Physiol Lung Cell Mol
Physiol 287, L503-9 (2004).

7.

C. Taube, X. Wei, C. Swasey, A. Joetham, S. Zarini, T. Lively, et al., Mast cells, FceRI, and IL-13 are required for development of airway
hypersensitiveness after aerosolized allergen exposure in the absence of adjuvant. J Immuno 172, 6398-6406 (2004).

8.

Oshiba, E. Hamelmann, K. Takeda, K. Bradley, J. Loader, et al., Passive Transfer of Immediate Hypersensitivity and Airway
Hyperresponsiveness by Allergen-Specific Immunoglobulin (Ig) E and IgG1 in Mice. J Clin Invest 97, 1398-408 (1996).

9.

E. Hamelmann, K. Tadeda, A. Oshiba, E. Gelfand, Role of IgE in the Development of Allergic Airway Inflammation and Airway
Hyperresponsiveness--A Murine Model. Allergy 54, 297-305 (1999).

10. S. Kaul, K. Fötisch, F. Karamloo, S. Vieths, A. Hoffmann, Replacement of Murine Sera by Allergen-Specific Monoclonal IgE Antibodies:
A New Approach for the Characterisation of Allergen Extracts. Dev Biol (Basel) 111, 109-15 (2002).
11. P. Mehlhop, d. van, A. Goldberg, J. Brewer, V. Kurup, et al., Allergen-induced Bronchial Hyperreactivity and Eosinophilic Inflammation
Occur in the Absence of IgE in a Mouse Model of Asthma. Proc Natl Acad Sci U S A 94, 1344-9 (1997).
12. Hoffmann, A. Jamin, K. Foetisch, S. May, H. Aulepp, et al., Determination of the Allergenic Activity of Birch Pollen and Apple Prick TestSolutions by Measurement of Beta-Hexosaminidase Release From RBL-2H3 Cells. Comparison With Classical Methods in AllergenStandardization. Allergy 54, 446-54 (1999).
13. N. Mizutani, et al. Establishment and characterization of a murine model for allergic asthma using allergen-specific IgE monoclonal
antibody to study pathological roles of IgE. Immunol Lett 141(2):235-45 (2012).
14. N. Mizutani, T. Nabe, S. Yoshino, Interleukin-33 and alveolar macrophages contribute to the mechanisms underlying the exacerbationof IgE-mediated airway inflammation and remodelling in mice. Immunology 139, 205-18 (2013).
15. N. Mizutani, T. Nabe, S. Yoshino, IgE/antigen-mediated enhancement of IgE production is a mechanism underlying the exacerbation ofairway inflammation and remodelling in mice. Immunology 144, 107-15 (2015).
16. A. Collins, D. Thelian, M. Basil, Antigen Valency as a Determinant of the Responsiveness of IgE-sensitised Rat Basophil LeukemiaCells. Int Arch Allergy Immunol 107, 547-56 (1995).

© 2020 Chondrex, Inc. All Rights Reserved, 3006-3008 3.0
16928 Woodinville-Redmond Rd NE Suite B-101
Woodinville, WA 98072

Phone: 425.702.6365 or 888.246.6373
Fax: 425.882.3094

www.chondrex.com
support@chondrex.com