An ELISA-based method for measurement of food-specific IgE antibody in mouse serum: An alternative to the passive cutaneous anaphylaxis assay
ABSTRACT Passive cutaneous anaphylaxis (PCA) assay has been a gold standard method to measure allergen-specific IgE antibody (ASIgE Ab) levels in allergy mouse models. Many factors including stringent guidelines for laboratory animal use make PCA a difficult choice. Therefore, alternative methods are needed that can be readily applied for measurement of specific IgE antibody levels in mouse serum. Herein we describe a novel ELISA-based method that is more sensitive in comparison to PCA, IgE isotype-specific (because it has little cross-reactivity with IgG1 or IgG2a isotype) and highly reproducible (<10% inter- or intra-assay variation). Furthermore, we demonstrate the utility of this assay to measure specific IgE Ab against a variety of food extracts including chicken egg, peanut, almond, filbert/hazelnut and sweet potato. These findings are of particular interest to those who are seeking (i) to measure food-extract-specific IgE antibody in animal models and (ii) an alternative to the animal-based PCA method to measure mouse IgE antibodies.
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- "OVA-specific IgG1 and IgE antibody levels were determined as previously described using a modified enzyme-linked immunosorbent assay (ELISA) with OVA as coating antigen (Birmingham et al. 2003). Previous optimization of coating antigen concentration showed that high levels of IgG did not interfere with IgE determination and that IgG depletion using protein G was not necessary with this method. "
ABSTRACT: The mechanisms by which cannabinoid receptors CB(1) and CB(2) modulate immune function are not fully elucidated. Critical tools for the determination of the role of both receptors in the immune system are CB(1)/CB(2) double null mice (CB(1)/CB(2) null), and previous studies have shown that CB(1)/CB(2) null mice exhibit exaggerated responses to various immunological stimuli. The objective of these studies was to determine the magnitude to which CB(1)/CB(2) null mice responded to the respiratory allergen ovalbumin (OVA) as compared with wild-type C57BL/6 mice. The authors determined that in the absence of adjuvant, both wild-type and CB(1)/CB(2) null mice mounted a marked response to intranasally instilled OVA as assessed by inflammatory cell infiltrate in the bronchoalveolar lavage fluid (BALF), eosinophilia, induction of mucous cell metaplasia, and IgE production. Many of the endpoints measured in response to OVA were similar in wild-type versus CB(1)/CB(2) null mice, with exceptions being modest reductions in OVA-induced IgE and attenuation of BALF neutrophilia in CB(1)/CB(2) null mice as compared with wild-type mice. These results suggest that T-cell responses are not universally exaggerated in CB(1)/CB(2) null mice.Toxicologic Pathology 02/2010; 38(3):382-92. DOI:10.1177/0192623310362706 · 1.92 Impact Factor
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ABSTRACT: Enzymatic digestion of total protein along with liquid chromatography/tandem mass spectrometry (LC/MS/MS) was used to confirm the presence of a major peanut allergen in food. Several peptides obtained from the enzymatic digestion of the most abundant peanut allergen, Ara h 1, were identified as specific peptide biomarkers for peanut protein. Using ice cream as a model food matrix, a method was developed for the detection of the allergen peptide biomarkers. A key component of the method was the use of molecular mass cutoff filters to enrich the Ara h 1 in the protein extracts. By applying the method to ice cream samples containing various levels of peanut protein, levels as low as 10 mg/kg of Ara h 1 could routinely be detected. This method provides an unambiguous means of confirming the presence of the peanut allergen, Ara h 1, in foods and can easily be modified to detect other food allergens.Journal of Agricultural and Food Chemistry 06/2004; 52(10):2785-90. DOI:10.1021/jf035129h · 3.11 Impact Factor
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ABSTRACT: Allergen-specific IgE production is the central event in the pathogenesis of atopic disorders and increases in specific IgE serum antibodies are an indicator of immediate hypersensitivity responses in humans and in animal models of allergy. Consequently, accurate and user-friendly methods are needed to measure serum levels of allergen-specific IgE. This review examines historical and recent developments in in vivo and in vitro methods for the detection of allergen-specific IgE in humans and in animal models. Routinely, in vitro methods such as enzyme-linked immunosorbant assays or radioallergosorbant tests and in vivo methods such as the skin prick test (SPT) for humans and the passive cutaneous anaphylaxis assay (PCA) used in animals are utilized to detect allergen-specific IgE. While in vivo assays are usually more accurate than in vitro assays since they provide a functional readout of IgE activity, they are relatively costly and require considerable expertise. On the other hand in vitro assays are limited by the fact that the amount of allergen-specific serum IgG exceeds IgE antibody by several orders of magnitude, resulting in competition for allergen binding. Consequently, methods that use allergen as a direct capture step are limited by the availability of free allergen binding sites for IgE. In order to circumvent this problem, in vitro methods usually require prior depletion of IgG or use high amounts of allergen in order to facilitate availability of free binding sites for IgE detection. Clearly, these approaches are limited for small sample volumes and allergens that are in short supply. New methods such as protein microarray could potentially overcome this problem by providing high allergen concentrations in a relatively small reaction volume. Currently, in vitro methods are rarely used in isolation for prognosis but are used primarily to complement the information obtained from in vivo assays. With the emergence of new technologies it is conceivable that in vitro assays may in the future replace in vivo assays, however until then in vivo assays remain the gold standard of allergen-specific IgE detection.Journal of Immunotoxicology 08/2005; 1(3):189-99. DOI:10.1080/15476910490919140 · 1.91 Impact Factor