Cinzia Ballabio

Cross-reactivity between mammalian proteins

BACKGROUND Cross-reactivity between food allergens occurs when they share part of their amino acid sequence, or when their three-dimensional molecular structure causes them to have a similar capacity to bind specific antibodies. OBJECTIVES To review data from our laboratory on cross-reactivity between mammalian proteins (milk and meat allergens). METHODS Studies used immunoelectrophoresis (sodium dodecyl sulfate-polyacrylamide gel electrophoresis/polyacrylamide gel electrophoresis and immunoblotting), and animal monoclonal antibodies. RESULTS The findings suggest that animal monoclonal antibodies specific for cows milk proteins are able to recognize the major part of milk proteins from mammals bred in Mediterranean countries (sheep, goat, and buffalo); weak cross-reactivity was observed with milk proteins from mares and donkeys. None of the antibodies used in our studies reacted with proteins from an exotic mammalian species: the camel. Similar cross-reactions were found with human circulating immunoglobulin E from children allergic to milk. With regard to beef allergy, monoclonal antibodies specific for bovine serum albumin cross-reacted only with ovine serum albumin, whereas the number of sera from allergic children able to recognize other mammalian serum albumins depended directly on the closeness of phylogenetic relationship between animal species and inversely on the percent identity with human serum albumin in the main epitopic sequence. CONCLUSION An area of heterogeneity between animal and human species in a critical amino acid sequence (epitope) of an allergen can determine the degree of immunogenic activity.

Molecular aspects of milk allergens and their role in clinical events.

AbstractMilk allergy is the most frequent food allergy in childhood. Even though cases of newly developed milk allergy in adulthood are known, this allergy is less frequent in adults since it is normally outgrown by children during the first years of life. One of the reasons why allergy to cow’s milk shows its highest prevalence in children is its early introduction into the diets of babies when breast feeding is not possible. The major allergens are caseins and β-lactoglobulin, but allergies to other minor proteins (immunoglobulins, bovine serum albumin) have also been reported. Milk allergenicity can be reduced by various treatments (mainly hydrolysis), meaning that formulas based on cow’s milk can often be safely fed to children allergic to milk proteins. Cross-reactivity has been described between different mammalian milks and between milk and meat or animal dander. Cross-contamination can result from inadequate cleaning of industrial equipment and constitutes a hidden danger for allergic subjects who unknowingly ingest milk proteins. FigureInvolvement (expressed as percentage of total subjects) of the most abundant milk proteins in the sensitization of 80 children allergic to cow’s milk. The upper panel includes all positive responses, even minor ones; data in the lower panel are restricted to the most severe positive responses (see text for details). SPT, skin prick test; CAP, CAP test; IMM, immunoblotting; alpha-LA, α-lactalbumin; beta-LG, β-lactoglobulin; cas, caseins; BSA, bovine serum albumin

Characterization of bovine serum albumin epitopes and their role in allergic reactions.

Objective:  This review provides updated information on conformational and sequential epitopes identified in bovine serum albumin (BSA) and summarizes available data about the role of structural modifications on BSA antigenicity/allergenicity.

Ochratoxin A in conventional and organic cereal derivatives: a survey of the Italian market, 2001–02

Ochratoxin A is a mycotoxin produced mainly by Penicillium verrucosum and Aspergillus ochraceus. Although typically considered a cereal contaminant, it has also been detected in dried fruit, nuts, meat and derivatives. To estimate the quantity of ochratoxin A that might be ingested by Italian consumers from these foods, 211 cereal derivatives (flours and bakery products) were analysed by high-performance liquid chromatography. Products were from conventional and organic agriculture and from integrated pest management agriculture. All commercial flours and derivatives examined contained ochratoxin A at concentrations very much below the legal limit (3 μg kg−1): the highest value, 0.816 μg kg−1, was detected in a sample of spelt whole flour from organic agriculture. In many samples, the ochratoxin content was below the limit of detection; only rarely did values exceed 0.5 μg kg−1. In baby foods, four samples were above the particularly restrictive Italian legal limit of 0.5 μg kg−1. Although some significant differences were found between samples from conventional and organic agriculture when some product categories were examined (namely, baby foods as semolina and rice creams), no important difference was found between the two types of agricultural practice when all types of cereal derivatives were considered together.

Identification of the basic subunit of Ara h 3 as the major allergen in a group of children allergic to peanuts

BACKGROUND Several proteins have been identified as peanut allergens; among them, Ara h 1 (7S globulin) and Ara h 2 (2S globulin) are usually considered the major allergens. OBJECTIVE To identify the major allergens in a group of children selected for their specific pattern of immunoreactivity. METHODS We identified the dominant allergen by using (1) amino acid sequencing of the bands that show the strongest IgE immunoreactivity in 1-dimensional electrophoresis and immunoblotting and (2) specific animal IgGs raised against the dominant immunoreactive band to pinpoint the allergen(s) in peanut proteins separated by 2-dimensional electrophoresis and immunoblotting. To confirm these data, we further examined the peanut proteome using serum samples from the children with the unusual immunoreactivity. RESULTS We found a group of children with marked peanut allergy who are specifically sensitized to the basic subunit of Ara h 3 (11S globulin family). CONCLUSION That the dominant immunoreactivity in these patients is in a basic subunit of Ara h 3 was unexpected, because previous studies had indicated that Ara h 3 was only a minor peanut allergen and that the identified allergenic epitopes occurred mainly in the acidic Ara h 3 subunit.

Tolerance of heat-treated kiwi by children with kiwifruit allergy

Kiwifruit allergy is increasing among children but whether heating affects clinical tolerance to kiwifruit is unknown. To assess tolerance to heated kiwifruit in children allergic to fresh kiwifruit. In this prospective trial, 20 children (median age 9.4 yr) with a history of immediate allergic reactions to fresh kiwifruit underwent double‐blind placebo‐controlled food challenges with steam‐cooked (100 °C for 5′) and industrially homogenised kiwifruit. Skin prick tests with a commercial kiwifruit allergen, raw kiwifruit and double‐blind placebo‐controlled food challenge with 25 g of fresh kiwifruit were used to confirm the history. Specific kiwifruit IgE to native and homogenized fruit were identified by immunoblotting. Fresh kiwifruit induced positive skin prick wheals in all children (confirmed during challenge in 19 patients). Commercial skin prick test elicited a positive response in five children, steam‐cooked kiwifruit in five, and the homogenised kiwifruit preparation in none. UniCAP© determinations were positive for kiwifruit in three patients. All childrens sera showed specific IgE at immunoblotting with raw kiwifruit and one with the homogenised preparation (major allergens identified: Act c 1 and Act c 2). There was no clinical reactivity following challenge with homogenised kiwifruit but one child reacted to cooked kiwifruit challenge. Industrial heat treatment and homogenisation can make kiwifruit safe for children who are allergic to this increasingly popular fruit. This has dietary implications for children who are allergic to several fruit and vegetable proteins.

Antigenic Determinants of Bovine Serum Albumin

Background: Bovine serum albumin (BSA) is one of the most widely studied proteins; its structure is well known and its antigenic characteristics have been described in several papers. The aim of this research was the identification of the BSA antigenic determinants. Methods: This study was performed using limited proteolysis and an immunoblotting technique, in which a commercial murine antibody and sera from children sensitized to BSA were used. Results: Findings suggest amino acids (aa) 524–598 as an epitopic area for human species. The most critical sequence seems to be aa 524–542, even if it must be included in a longer fragment to be recognized by antibodies. Murine IgG antibodies also recognize fragments contained in the first half (NH2-terminal portion) of BSA. Conclusions: The results presented in this study indicate that the epitopic sites of an antigenic protein can be different when different species are considered, so that data obtained with antibodies from animal species cannot be directly extrapolated to the behavior of human IgEs.

Assessment of the tolerance to lupine-enriched pasta in peanut-allergic children

Background Reports of allergy to lupine derivatives (as de novo sensitization or cross‐reactivity in subjects allergic to peanut) are increasing as their use in food products increases.

Biochemical and immunochemical characterization of different varieties of amaranth (Amaranthus L. ssp.) as a safe ingredient for gluten-free products.

Celiac disease is a food intolerance triggered by the ingestion of gluten-containing cereals; the only therapy is a strict gluten-free diet for life. In recent years, amaranth flour has received considerable attention as an interesting source for the formulation of gluten-free products due to its high nutritional value and low content of prolamins, the toxic proteins for celiacs. The aim of this study was to characterize 40 amaranth varieties using both SDS-PAGE/immunoblotting and ELISA to assess their possible tolerance by celiac subjects. All of the amaranth samples studied showed similar binding affinities for both specific anti-gliadin antibodies and human IgAs. In most amaranth grains, the content of gluten-like proteins measured by ELISA was <20 ppm. The molecular characterization of amaranth proteins suggests that amaranth is safe for celiacs to consume. It is recommended that the most suitable amaranth varieties are those having the lowest content of proteins cross-reacting with anti-gliadin antibodies.

Evaluation of the Residual Antigenicity of Dairy Whey Hydrolysates Obtained by Combination of Enzymatic Hydrolysis and High-Pressure Treatment

Dairy whey was hydrolyzed for 15 min with five food-grade enzymes (Alcalase, Neutrase, Corolase 7089, Corolase PN-L, and Papain) at atmospheric pressure (0.1 MPa) and in combination with high pressure (HP) at 100, 200, and 300 MPa, applied prior to or during enzymatic digestion. The peptide profile of the hydrolysates obtained was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and their residual antigenicity was assessed by immuno-blotting with anti-beta-lactoglobulin monoclonal antibodies and the sera from pediatric patients allergic to cows milk proteins. Moreover, to evaluate the presence of residual trace amounts of casein in bovine whey hydrolysates, immunoblotting with anti-cows milk protein polyclonal antibodies was performed. SDS-PAGE analysis showed that HP treatment increased hydrolysis by the proteases assayed, especially when it was applied during the enzymatic digestion. Positive reactions at the band corresponding to beta-lactoglobulin were detected for Corolase PN-L and Corolase 7089 hydrolysates, except for those obtained under 300 MPa by the last protease. However, the immunochemical reaction was lower in the hydrolysis products obtained under HP than in those obtained at atmospheric pressure and after the HP treatment. On the contrary, no residual immunochemical reactivity associated with beta-lactoglobulin was observed in the hydrolysates obtained by Alcalase and Neutrase under HP, and none was observed in any of the hydrolysis products obtained by Papain. The presence of traces of casein was not significant. These results suggest that HP combined with selected food-grade proteases is a treatment that can remove the antigenicity of whey protein hydrolysates for their use as ingredients of hypoallergenic infant formulae.