Humanized Mediator Release Assay as a Read-Out for Allergen Potency

Wenger, Mario; Bethanis, Athanasios; Johnson, Litty; Aglas, Lorenz

doi:10.3791/62702

Cited by 3 publications

(2 citation statements)

References 8 publications

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“…The functional activity of the hIgE mAb was assessed by using humanized rat basophilic leukemia (huRBL) cells transfected with human FcɛRI for passive sensitization with hIgE mAb (RBL-2H3 cell line) ( 26 , 27 ). β -Hexosaminidase release was used as a marker of allergen-induced degranulation.…”

Section: Methodsmentioning

confidence: 99%

Unique allergen-specific human IgE monoclonal antibodies derived from patients with allergic disease

Smith,

Reid Black,

Bermingham

et al. 2023

Front. Allergy

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IntroductionAllergic reactions are mediated by human IgE antibodies that bind to and cross-link allergen molecules. The sites on allergens that are recognized by IgE antibodies have been difficult to investigate because of the paucity of IgE antibodies in a human serum. Here, we report the production of unique human IgE monoclonal antibodies to major inhaled allergens and food allergens that can be produced at scale in perpetuity.Materials and methodsThe IgE antibodies were derived from peripheral blood mononuclear cells of symptomatic allergic patients, mostly children aged 3–18 years, using hybridoma fusion technology. Total IgE and allergen-specific IgE was measured by ImmunoCAP. Their specificity was confirmed through ELISA and immunoblotting. Allergenic potency measurements were determined by ImmunoCAP inhibition. Biological activity was determined in vitro by comparing β-hexosaminidase release from a humanized rat basophilic cell line.ResultsHuman IgE monoclonal antibodies (n = 33) were derived from 17 allergic patients with symptoms of allergic rhinitis, asthma, atopic dermatitis, food allergy, eosinophilic esophagitis, or red meat allergy. The antibodies were specific for five inhaled allergens, nine food allergens, and alpha-gal and had high levels of IgE (53,450–1,702,500 kU/L) with ratios of specific IgE to total IgE ranging from <0.01 to 1.39. Sigmoidal allergen binding curves were obtained through ELISA, with low limits of detection (<1 kU/L). Allergen specificity was confirmed through immunoblotting. Pairs of IgE monoclonal antibodies to Ara h 6 were identified that cross-linked after allergen stimulation and induced release of significant levels of β-hexosaminidase (35%–80%) from a humanized rat basophilic cell line.ConclusionsHuman IgE monoclonal antibodies are unique antibody molecules with potential applications in allergy diagnosis, allergen standardization, and identification of allergenic epitopes for the development of allergy therapeutics. The IgE antibody probes will enable the unequivocal localization and validation of allergenic epitopes.

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Section: Methodsmentioning

confidence: 99%

Unique allergen-specific human IgE monoclonal antibodies derived from patients with allergic disease

Smith,

Reid Black,

Bermingham

et al. 2023

Front. Allergy

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“…The medical community does not widely accept these tests [200] since there is a lack of well-designed controlled trials confirming their efficacy [201]. One of these methods is the mediator release test (MRT), which measures the release of chemical mediators by white blood cells upon exposure to food antigens [202]. Another approach is ALCAT testing, which assesses immune cell reactions to a panel of food antigens [203].…”

Section: Other Food-specific Intolerancesmentioning

confidence: 99%

Myths and Facts about Food Intolerance: A Narrative Review

Zingone,

Bertin,

Maniero

et al. 2023

Nutrients

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Most adverse reactions to food are patient self-reported and not based on validated tests but nevertheless lead to dietary restrictions, with patients believing that these restrictions will improve their symptoms and quality of life. We aimed to clarify the myths and reality of common food intolerances, giving clinicians a guide on diagnosing and treating these cases. We performed a narrative review of the latest evidence on the widespread food intolerances reported by our patients, giving indications on the clinical presentations, possible tests, and dietary suggestions, and underlining the myths and reality. While lactose intolerance and hereditary fructose intolerance are based on well-defined mechanisms and have validated diagnostic tests, non-coeliac gluten sensitivity and fermentable oligosaccharide, disaccharide, monosaccharide, and polyol (FODMAP) intolerance are mainly based on patients’ reports. Others, like non-hereditary fructose, sorbitol, and histamine intolerance, still need more evidence and often cause unnecessary dietary restrictions. Finally, the main outcome of the present review is that the medical community should work to reduce the spread of unvalidated tests, the leading cause of the problematic management of our patients.

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Biological activity of human IgE monoclonal antibodies targeting Der p 2, Fel d 1, Ara h 2 in basophil mediator release assays

Pena-Castellanos,

Smith,

Pomés

et al. 2023

Front. Immunol.

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BackgroundHuman Immunoglobulin E monoclonal antibodies (hIgE mAb) are unique tools for investigating IgE responses. Here, the biological activity of hIgE mAb, derived from immortalized B cells harvested from the blood of allergic donors, targeting three allergens (Der p 2, Fel d 1 and Ara h 2) was investigated.MethodsThree Der p 2-, three Fel d 1- and five Ara h 2-specific hIgE mAb produced by human B cell hybridomas, were combined in pairs and used to passively sensitize humanized rat basophilic leukemia cells and compared with sensitization using serum pools. Sensitized cells were stimulated with corresponding allergens (recombinant or purified), allergen extracts or structural homologs, having 40-88% sequence similarity, and compared for mediator (β-hexosaminidase) release.ResultsOne, two and eight pairs of Der p 2-, Fel d 1- and Ara h 2-specific hIgE mAb, respectively, produced significant mediator release (>50%). A minimum hIgE mAb concentration of 15-30 kU/L and a minimum antigen concentration between 0.01-0.1 µg/mL were sufficient to induce a pronounced mediator release. Individual sensitization with one Ara h 2-specific hIgE mAb was able to induce crosslinking independently of a second specific hIgE mAb. Der p 2- and Ara h 2-specific mAb showed a high allergen specificity when compared to homologs. Mediator release from cells sensitized with hIgE mAb was comparable to serum sensitization.ConclusionThe biological activity of hIgE mAb reported here provides the foundation for novel methods of standardization and quality control of allergen products and for mechanistic studies of IgE-mediated allergic diseases, using hIgE mAb.

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Humanized Mediator Release Assay as a Read-Out for Allergen Potency

Cited by 3 publications

References 8 publications

Unique allergen-specific human IgE monoclonal antibodies derived from patients with allergic disease

Unique allergen-specific human IgE monoclonal antibodies derived from patients with allergic disease

Myths and Facts about Food Intolerance: A Narrative Review

Biological activity of human IgE monoclonal antibodies targeting Der p 2, Fel d 1, Ara h 2 in basophil mediator release assays

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