Assessment of the Sensitizing Potential of Proteins in BALB/c Mice: Comparison of Three Protocols of Intraperitoneal Sensitization

Abstract

Most food allergy cases are associated with a limited group of allergens. This could be attributed to an increased ability of some foods to sensitize and trigger allergic reactions. However, there are no validated animal models to evaluate the sensitizing or allergenic potentials of proteins. Our aim was to evaluate three protocols of adjuvant-free intraperitoneal sensitization that differ in the time points for sample collection (days 14, 28 and 35 from beginning of the sensitization) and also in the number of immunizations (2, 5 and 3, respectively). Ovalbumin (OVA; 0.05 mg), cow milk proteins (CMP; 0.025, 0.05 and 0.25 mg), and potato acid phosphatase (PAP; low allergenic protein; 250.0 mg) were administered intraperitoneally (ip) to BALB/c mice (n = 4–6) and the protein-specific IgE and IgG antibody responses were evaluated using ELISA. Additional serum protein-specific IgE antibodies evaluations were carried out after IgG depletion. Anti-OVA IgE antibodies were detected in mice from all three protocols. The responses were higher in the group of mice that underwent the 28-day protocol than in those that underwent the 14- or 35-day protocols (p < 0.01 and p < 0.05, respectively). Anti-CMP IgE antibodies were detected in both the 14- and 28-day protocols, but the response was higher in the group that underwent the 28-day protocol (p < 0.001). The anti-CMP IgE antibody response detection was improved after serum IgG depletion (p < 0.001). Anti-PAP IgE antibodies were not detected. Mice with undetectable serum levels of protein-specific IgE triggered anti-OVA, -CMP, and -PAP IgG responses. An adjuvant-free 28-day protocol with five ip immunizations seems appropriate for evaluation of the inherent sensitizing or allergenic capacity of the studied proteins. Reproducible results were obtained utilizing the BALB/c mouse strain. Inter-laboratory studies including a larger number of proteins should be carried out to validate this model.

Assessing the Sensitizing and Allergenic Potential of the Albumin and Globulin Fractions from Amaranth (Amaranthus hypochondriacus) Grains before and after an Extrusion Process

“…We and others have shown that the dose of allergen, as well as the number and frequency of immunizations, are key factors to sensitize BALB/c mice to food proteins [14,25,26]. The adjuvant-free 28-day protocol with five immunizations used in the present study was shown to be effective to detect even allergens that hardly trigger IgE immune responses after intraperitoneal administration [14]. The globulin fraction from amaranth was tested at three doses, but no anti-globulin IgE responses were detected either under extruded or non-extruded conditions.…”

“…A5503) and potato acid phosphatase (PAP, 0.5–3.0 unit/mg solid, Sigma-Aldrich, P3752) were used as allergenic and hypoallergenic proteins, respectively [21,22]. The proteins were dissolved in PBS (pH 7.4) in order to obtain a 0.2 mg/mL OVA solution and 2.0 mg/mL PAP solution, as previously described [14]. All protein concentrations were measured using the bicinchoninic acid method following the manufacturer’s instructions (BCA assay, Thermo Scientific Pierce TM , Rockford, IL, USA).…”

“…Taking into account the doses that triggered the highest anti-non-extruded albumin and globulin IgE response (0.05 and 0.25 mg per mouse, respectively), the albumin and globulin fractions from extruded amaranth were administered. OVA and PAP were administered as previously described (0.05 and 0.5 mg per mouse, respectively) [14].…”

“…Evidence suggests that other unidentified proteins with molecular mass greater than profilin are the triggers of allergy reactions in such cases [9]. Notably, recent studies have shown the ability of a murine model to discriminate between common and rarely allergenic proteins [14], and this allows the evaluation of the sensitizing and allergenic potential of proteins before and after food processing.…”

Assessment of allergenicity of irradiated dairy products in a Balb/c mice model

“…Animal models in vivo are frequently used in food allergies research (Dearman and Kimber 2009;Van Gramberg et al 2013;Arámburo-Galvez et al 2018;Kanagaratham et al 2018). They are a mandatory element in the preclinical studies, thus overcoming not only potentially risky and difficult manipulations, but also ethical limitations in clinical studies (Oyoshi et al 2014).…”