Evaluation of the Luciferase Assay-Based In Vitro Elicitation Test for Serum IgE

Nakamura, Ryosuke; Ishiwatari, Ayano; Higuchi, Masakazu; Uchida, Yuka; Nakamura, Rika; Kawakami, Hiroshi; Urisu, Atsuo; Teshima, Reiko

doi:10.2332/allergolint.11-oa-0407

Cited by 23 publications

(20 citation statements)

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“…This suggests that basophils may offer a sensitive way of measuring the presence of parasite antigen-specific IgE in infected individuals, and, perhaps more importantly in the context of vaccination, to demonstrate the ability of allergens to induce basophil or mast cell activation, in contrast to measuring allergen binding by specific IgE alone. Therefore, we recently developed a new detection system for antigen-specific IgE based on the NFAT-dependent luciferase expression in a humanised rat basophilic leukaemia cell line (RS-ATL8) [22], [23]. When sensitised with egg white-allergic patient's serum, this cell line detected at least 1 fg/mL of egg white extract proteins as a luciferase expression [23].…”

Section: Introductionmentioning

confidence: 99%

Use of Humanised Rat Basophilic Leukaemia Cell Line RS-ATL8 for the Assessment of Allergenicity of Schistosoma mansoni Proteins

et al. 2014

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BackgroundParasite-specific IgE is thought to correlate with protection against Schistosoma mansoni infection or re-infection. Only a few molecular targets of the IgE response in S. mansoni infection have been characterised. A better insight into the basic mechanisms of anti-parasite immunity could be gained from a genome-wide characterisation of such S. mansoni allergens. This would have repercussions on our understanding of allergy and the development of safe and efficacious vaccinations against helminthic parasites.Methodology/Principal FindingsA complete medium- to high-throughput amenable workflow, including important quality controls, is described, which enables the rapid translation of S. mansoni proteins using wheat germ lysate and subsequent assessment of potential allergenicity with a humanised Rat Basophilic Leukemia (RBL) reporter cell line. Cell-free translation is completed within 90 minutes, generating sufficient amounts of parasitic protein for rapid screening of allergenicity without any need for purification. Antigenic integrity is demonstrated using Western Blotting. After overnight incubation with infected individuals' serum, the RS-ATL8 reporter cell line is challenged with the complete wheat germ translation mixture and Luciferase activity measured, reporting cellular activation by the suspected allergen. The suitability of this system for characterization of novel S. mansoni allergens is demonstrated using well characterised plant and parasitic allergens such as Par j 2, SmTAL-1 and the IgE binding factor IPSE/alpha-1, expressed in wheat germ lysates and/or E. coli. SmTAL-1, but not SmTAL2 (used as a negative control), was able to activate the basophil reporter cell line.Conclusion/SignificanceThis method offers an accessible way for assessment of potential allergenicity of anti-helminthic vaccine candidates and is suitable for medium- to high-throughput studies using infected individual sera. It is also suitable for the study of the basis of allergenicity of helminthic proteins.

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Section: Introductionmentioning

confidence: 99%

Use of Humanised Rat Basophilic Leukaemia Cell Line RS-ATL8 for the Assessment of Allergenicity of Schistosoma mansoni Proteins

et al. 2014

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“…[7][8][9][10] Briefly, the cells 4 cells/50 µL/well) were sensitized with 10 ng/mL anti-OVA IgE Abs and plated on a clear-bottom white 96-well plate (ViewPlate; Perkin Elmer, Inc.). After overnight incubation, the cells were washed three times with PBS and then stimulated with OVA diluted in MEM containing 10% FCS (50 µL/well) at 37°C under an atmosphere of 5.0% CO 2 for 3 h. After stimulation, 50 µL of luciferase substrate solution containing cell lysis reagent (ONE-Glo; Promega Corporation, Madison, WI, U.S.A.) was added to the cells, and chemiluminescence was measured using an EnVision multilabel plate reader (PerkinElmer, Inc.).…”

Section: Reagentsmentioning

confidence: 99%

“…To overcome this blocking effect, excessive amounts of antigens are immobilized in the solid phase of immunochemical allergy tests, such as the radioallergosorbent test and fluoroenzyme immunoassay ImmunoCAP ® (CAP test). 4,5) However, immobilization may affect the physicochemical nature of the antigens, resulting in discrepancies of the allergenicity of the molecule between in vivo responses and in vitro tests.We developed a new allergy test, called the IgE crosslinking-induced luciferase expression (EXiLE) test, [6][7][8][9][10] which is dependent on the activation of the humanized rat mast cell line RS-ATL8 sensitized with serum IgE of allergic patients. The EXiLE test detects the ability of IgE to crosslink multivalent antigens in the liquid phase, resulting in a more reliable diagnosis of egg white food allergy than the conventional CAP test.…”

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confidence: 99%

“…We developed a new allergy test, called the IgE crosslinking-induced luciferase expression (EXiLE) test, [6][7][8][9][10] which is dependent on the activation of the humanized rat mast cell line RS-ATL8 sensitized with serum IgE of allergic patients. The EXiLE test detects the ability of IgE to crosslink multivalent antigens in the liquid phase, resulting in a more reliable diagnosis of egg white food allergy than the conventional CAP test.…”

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confidence: 99%

“…The EXiLE test detects the ability of IgE to crosslink multivalent antigens in the liquid phase, resulting in a more reliable diagnosis of egg white food allergy than the conventional CAP test. 7) The EXiLE test can be used to determine the effects of the physicochemical nature of allergens on allergy test results. Here, we used two ovalbumin (OVA)-specific IgE monoclonal antibodies (Abs), E-C1 and E-G5, which can and cannot induce mast cell degranulation, respectively.…”

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Different Results of IgE Binding- and Crosslinking-Based Allergy Tests Caused by Allergen Immobilization

Okamoto‐Uchida¹,

Nakamura²,

Y³

et al. 2016

Biological & Pharmaceutical Bulletin

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The physicochemical nature of allergen molecules differ from the liquid phase to the solid phase. However, conventional allergy tests are based on the detection of immunoglobulin (Ig)E binding to immobilized allergens. We recently developed an in vitro allergy testing method using a luciferase-reporting humanized rat mast cell line to detect IgE crosslinking-induced luciferase expression (EXiLE test). The aim of the present study was to evaluate the effects of antigen immobilization on the results of different in vitro allergy tests using two anti-ovalbumin (OVA) antibodies (Abs), E-C1 and E-G5, with different properties in the OVAinduced allergic reaction. Both Abs showed clear binding to OVA with an enzyme-linked immunosorbent assay and by BIAcore analysis. However, only E-C1 potentiated EXiLE response for the liquid-phase OVA. On the other hand, OVA immobilized on solid-phase induced EXiLE responses in both E-C1 Ab-and E-G5 Ab-sensitized mast cells. Western blotting of OVA indicated that E-C1 Ab binds both to OVA monomers and dimers, unlike E-G5 Ab, which probably binds only to the OVA dimer. These results suggest that antigen immobilization enhanced IgE crosslinking ability through multimerization of allergen molecules in the solid phase, resulting in an increase in false positives in IgE binding-based conventional in vitro allergy tests. These findings shed light on the physicochemical nature of antigens as an important factor for the development and evaluation of in vitro allergy tests and suggest that mast cell activation-based allergy testing with liquid-phase allergens is a promising strategy to evaluate the physiological interactions of IgE and allergens.Key words immunoglobulin E (IgE); allergen; immobilization; in vitro allergy test; luciferase assay; IgE crosslinking-induced luciferase expression (EXiLE) test Immunoglobulin (Ig) E is the primary factor for mast cell activation and type I allergic reactions, such as histamine release and production of inflammatory factors. Although immunochemical allergen-specific IgE tests based on the binding of serum IgE to an allergen are used worldwide, the results cannot always be translated into a clear diagnosis, especially in the case of food allergies.1,2) The existence of allergen-specific IgE indicates that the individual has been previously sensitized to the allergen. However, IgE is not always capable of crosslinking high-affinity IgE receptors (FcεRI) with the soluble allergen because multiple epitopes of an allergen molecule are required.3) Additionally, there is a greater abundance of antigen-specific IgGs in sera than IgE, which may interfere with the binding of IgE to the antigen. To overcome this blocking effect, excessive amounts of antigens are immobilized in the solid phase of immunochemical allergy tests, such as the radioallergosorbent test and fluoroenzyme immunoassay ImmunoCAP ® (CAP test). 4,5) However, immobilization may affect the physicochemical nature of the antigens, resulting in discrepancies of the allergenicity of the molecule betw...

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Use of Humanized RS-ATL8 Reporter System for Detection of Allergen-Specific IgE Sensitization in Human Food Allergy

Ali

Nakamura

Falcone

2017

Methods in Molecular Biology

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Allergen-specific Immunoglobulin E (IgE) determination lies at the heart of diagnosis of sensitization to food and other allergens. In the past few years, reporter systems capable of detecting the presence of allergen-specific IgE have been developed by several labs. These rely on humanized rat basophil leukemia cell lines stably transfected with reporter genes such as firefly luciferase. In this chapter, we describe protocols for the use of the RS-ATL8 cell line (IgE cross-linking-induced luciferase expression; EXiLE) in 96-well and 384-well formats. We also describe optional treatment steps for enveloped virus and complement inactivation.

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Evaluation of the Luciferase Assay-Based In Vitro Elicitation Test for Serum IgE

Abstract: In contrast to in vivo tests, the EXiLE test appears to be a useful tool in diagnosing patients suspected of having IgE-dependent EW allergy without the risk of severe systemic reactions.

Cited by 23 publications

References 21 publications

Use of Humanised Rat Basophilic Leukaemia Cell Line RS-ATL8 for the Assessment of Allergenicity of Schistosoma mansoni Proteins

Use of Humanised Rat Basophilic Leukaemia Cell Line RS-ATL8 for the Assessment of Allergenicity of Schistosoma mansoni Proteins

Different Results of IgE Binding- and Crosslinking-Based Allergy Tests Caused by Allergen Immobilization

Use of Humanized RS-ATL8 Reporter System for Detection of Allergen-Specific IgE Sensitization in Human Food Allergy

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