Application of Immunoreaction Enhancer Solutions to an Enzyme-Linked Immunosorbent Assay for Antigen-Specific IgE in Mice Immunized with Recombinant Major Mite Allergens or Ovalbumin

Abstract

Background: Weak signals for allergen-specific IgE are a problem in murine models for the study of allergies. It has been reported that the removal of IgG from murine sera enhances signal intensity. Very recently, buffer solutions designed to enhance signals in immunoassays have been developed and made commercially available. Methods: Sera from mice immunized either with a recombinant form of one of the major mite allergens Der p 1, Der f 1 and Der f 2, or with ovalbumin adsorbed to alum were used for the assays. Total IgE was measured by a sandwich enzyme-linked immunosorbent assay (ELISA). Allergen-specific IgE was assayed using plates coated with the allergens after the removal of IgG from sera with protein G-coupled sepharose beads in wells of other plates or with the use of commercially available enhancer solutions without the removal of IgG. IgE binding was detected with horseradish peroxidase-conjugated anti-murine IgE monoclonal antibody as the secondary antibody. Results: Significant levels of total IgE were produced after the immunizations. The in-well pretreatment of diluted sera (1/10 dilution) with protein G-coupled beads enhanced the signals for allergen-specific IgE. The use of the enhancer solutions for dilution of the sera and secondary antibody and prolonged incubation remarkably enhanced the signals at a more extensive dilution of sera (1/200 or less) without the removal of IgG. Conclusions: An ELISA simply modified with the use of immunoreaction enhancer solutions has advantages in terms of signal intensity and ease of handling for the detection of allergen-specific murine IgE and would be useful for the study of allergies with murine models.

Cupressaceae Pollen Grains Modulate Dendritic Cell Response and Exhibit IgE-Inducing Adjuvant Activity In Vivo

“…Although administration of 20 g (or less) of grains/wk without OVA did not increase serum total IgE levels (Fig. 6, A and C, 10 g; and data not shown), it does not exclude the possibility that Ag-specific IgE was increased (39). An assay system for IgE specific to pollen-derived Ags with high sensitivity should be established in a future study.…”

“…OVA-specific Abs were detected on plates coated with 1 mg/ml OVA and blocked with BlockAce (Snow Bland) and developed with HRPconjugated Abs specific to the murine IgE and IgG subclasses as described previously (39,40) with some modifications as follows. Sera and detection Abs were diluted with solutions 1 and 2 of CanGetSignal (Toyobo), respectively.…”

IL-33–Mediated Innate Response and Adaptive Immune Cells Contribute to Maximum Responses of Protease Allergen–Induced Allergic Airway Inflammation

“…Serum total IgE and allergen-specific Abs were measured by a sandwich ELISA, as described previously (10,(32)(33)(34)(35) with minor modifications. Plates were coated with papain (30 mg/ml for the experiment of Fig.…”

House dust mite induces direct airway inflammation in vivo: implications for future disease therapy?

“…Der p 1 belongs to the papain-like cysteine protease family and exhibits cysteine protease activity [4]. This proteolytic activity has been suggested to be involved in the pathogenesis of allergies by: increasing the permeability of epithelial cells and allowing the passage of their own and other allergens across the epithelium [5][6][7]; cleaving and/or interacting with cell surface molecules and intrinsic protease inhibitors [8][9][10][11]; and modulating the function of a range of cells, such as basophils, mast cells, alveolar macrophages and airway epithelial cells [12][13][14][15]. The effect on the airway epithelium is especially interesting, as it is likely to be the first cell type to interact with the allergen.…”