Allergy Asthma Immunol Res.  2015 Jan;7(1):60-68. 10.4168/aair.2015.7.1.60.

Cross-Reactivity Between the Soybean Protein P34 and Bovine Caseins

• Candreva A
• Smaldini P
• Curciarello R
• Cauerhff A
• Fossati CA
• Docena GH
• Petruccelli S

• KMID: 2260153
• DOI: http://doi.org/10.4168/aair.2015.7.1.60

Abstract

PURPOSE
Soy-based formulas are widely used as dairy substitutes to treat milk allergy patients. However, reactions to soy have been reported in a small proportion of patients with IgE-mediated milk allergies. The aim of this work was to explore whether P34, a mayor soybean allergen, is involved in this cross-reactivity.
METHODS
In vitro recognition of P34 was evaluated by immunoblotting, competitive ELISA and basophil activation tests (BAT) using sera from allergic patients. In vivo cross-reactivity was examined using an IgE-mediated milk allergy mouse model.
RESULTS
P34 was recognized by IgE antibodies from the sera of milk allergic patients, casein-specific monoclonal antibodies, and sera from milk-allergic mice. Spleen cells from sensitized mice incubated with milk, soy or P34 secreted IL-5 and IL-13, while IFN-gamma remained unchanged. In addition, the cutaneous test was positive with cow's milk proteins (CMP) and P34 in the milk allergy mouse model. Moreover, milk-sensitized mice developed immediate symptoms following sublingual exposure to P34.
CONCLUSIONS
Our results demonstrate that P34 shares epitopes with bovine casein, which is responsible for inducing hypersensitivity symptoms in milk allergic mice. This is the first report of the in vivo cross-allergenicity of P34.

Keyword

Food allergy; soybeans; milk; P34 protein; cross-reactivity

MeSH Terms

Animals
Antibodies
Antibodies, Monoclonal
Basophils
Caseins*
Enzyme-Linked Immunosorbent Assay
Epitopes
Food Hypersensitivity
Humans
Hypersensitivity
Immunoblotting
Immunoglobulin E
Interleukin-13
Interleukin-5
Mice
Milk
Milk Hypersensitivity
Milk Proteins
Soy Milk
Soybeans*
Spleen
Antibodies
Antibodies, Monoclonal
Caseins
Epitopes
Immunoglobulin E
Interleukin-13
Interleukin-5
Milk Proteins

Figure

• 

  Fig. 1 Immunochemical analysis of soy protein. (A) IgE immunoblotting of P34 and MBP revealed with human sera of milk-allergic patients (1-10), a representative serum of a non-milk-allergic patient (11), and of a non-allergic subject (12). (B) Immunoblotting of P34 and MBP revealed with casein-specific monoclonal antibodies. Ponceau S staining of the blotted membrane shows the transferred protein. (C) Inhibition ELISA; wells were coated with α-casein (0.25 µg/well) and 1D5 antibody was pre-incubated with various concentrations of α-casein, P34 or OVA as soluble inhibitors. MBP, maltose-binding protein; 1D5, α-casein-specific monoclonal antibody; 4C3, β-casein-specific monoclonal antibody; 3B5, κ-casein specific monoclonal antibody; MW of MBP: 44 kDa; MW of P34-MBP: 83.6 kDa.

• 

  Fig. 2 Basophil activation test by flow cytometry. Basophils from healthy donor subjects were stripped and passively sensitized with milk-specific IgE-containing sera from milk-allergic patients (n=5). (A) Cells were selected according to physical parameters (AI), live cells (AII) and IgE-membrane-bound cells (AIII). (B) Double-positive cells expressing CD63 and CD203c were analyzed in the later gate when different allergens at various concentrations were used to induce basophil activation. Results corresponding to a representative experiment are shown. (C) Percentages of CD63+ CD203c+ cells with all positive sera analyzed. Data represent means±SEM % of double-positive basophils. Statistically significant difference by ANOVA: *P<0.05 , **P<0.01, ***P<0.005, vs PBS group. CMP, cow's milk proteins; SP, soy proteins; OVA, ovalbumin.

• 

  Fig. 3 Experimental design, clinical symptoms and histamine levels. (A) Schematic drawing of the experimental protocol: BALB/c mice (n=8 per group) were subjected to weekly intragastric sensitization with cholera toxin and CMP from day 0 through 35. Challenge was performed at day 45 by intragastric (CMP or SP) (n=7 per group) or sublingual (β-Lg, P34 or OVA) (n=5 per group) protein administration. Control mice received only CMP and were then orally challenged. (B) Symptoms were observed 30 minutes following challenge with CMP or SP and scored according to Table 1. (C) Quantification by ELISA of histamine following oral challenge (mean values±SEM). (D) Cutaneous test: sensitized and control mice were subcutaneously injected with CMP, SP and saline (n=4 per group). The presence of blue color in the skin within minutes after injection was considered a positive cutaneous test. Statistically significant difference by ANOVA: ***P<0.005. CMP, cow's milk proteins; SP, soy proteins; CT, cholera toxin.

• 

  Fig. 4 Immune responses to milk and soy proteins. (A) Specific IgE (by EAST), IgG1 and IgG2a (by ELISA) in serum of milk-sensitized mice (mean values±SEM). (B) Levels of IL-5, IL-13 and IFN-γ (by ELISA) in supernatants of stimulated spleen cells (mean values±SEM). Results correspond to a single experiment with at least three mice per condition representative of three separate experiments with similar results. (C) Cutaneous test in sensitized mice subcutaneously injected with P34 in the right flank and MBP in the left flank. (D) Clinical scores assigned to symptoms observed within 30 minutes following the sublingual challenge with β-Lg, P34 or OVA. (A) and (B) were analyzed by Student's t-test and (D) with ANOVA test. Statistically significant differences are shown as: *P<0.05, **P<0.01, ***P<0.005, CMP, cow's milk proteins; SP, soy proteins; MBP, maltose-binding protein, β-Lg, beta lactoglobulin; OVA, ovalbumin.

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