Immunoglobulin E mediated food allergy.Modelling and application of diagnostic and predictive tests for existing and novel foods

It is known that some foods cause an allergenic response in certain individuals. Clinical and immunological tests are available for the diagnosis of food allergy and identification of food allergens. However, there are no valid tests for the prediction of the allergenic potential of foods not normally recognized as allergenic. Such foods include: food products developed from foods which may not be recognizable as allergenic in their modified forms; foods produced using novel processes (novel foods), for example genetically modified foods; and foods not normally consumed but that are being used increasingly as alternatives to more traditional foods. Both the risks associated with food allergy and the fact that foods such as the ones described above will become available to the consumer, highlight the need for methods to screen for potential food allergens. This review provides a general overview of food allergy including mechanism, development and prevalence, but focuses on and discusses: 1) the possible risks (with specific reference to food allergy) associated with new and novel foods; and 2) the development/use of food allergy models (in vivo and in vitro) to assess the allergenic potential of new and novel foods.     

Adverse reactions to foods

Allergic reactions to foods represent a prominent, actual and increasing problem in clinical medicine. Symptoms of food allergy comprise skin reactions (urticaria, angioedema, eczema) respiratory (bronchoconstriction, rhinitis), gastrointestinal (cramping, diarrhea) and cardiovascular symptoms with the maximal manifestation of anaphylactic shock. They can be elicited by minute amounts of allergens. The diagnosis of food allergy is done by history, skin test, in vitro allergy diagnosis and — if necessary — oral provocation tests, if possible placebo-controlled. Avoidance of respective allergens for the allergic patient, however, is often complicated or impossible due to deficits in declaration regulations in many countries. Increasing numbers of cases including fatalities, due to inadvertent intake of food allergens are reported. It is therefore necessary to improve declaration laws and develop methods for allergen detection in foods. Allergens can be detected by serological methods (enzyme immunoassays, in vitro basophil histamine release or in vivo skin test procedures in sensitized individuals). The problem of diagnosis of food allergy is further complicated by cross-reactivity between allergens in foods and aeroallergens (pollen, animal epithelia, latex etc.). Elicitors of pseudo-allergic reactions with similar clinical symptomatology comprise low-molecular-mass chemicals (preservatives, colorings, flavor substances etc.). For some of them (e.g. sulfites) detection assays are available. In some patients classic allergic contact eczema can be elicited systemically after oral intake of low-molecular-mass contact allergens such as nickel sulfate or flavorings such as vanillin in foods. The role of xenobiotic components in foods (e.g. pesticides) is not known at the moment. In order to improve the situation of the food allergic patient, research programs to elucidate the pathophysiology and improve allergen detection strategies have to be implemented together with reinforced declaration regulations on a quantitative basis.     

Immunological identification and characterization of individual food allergens

Food allergies of type-I-allergy are immunoglobulin E (IgE) mediated and caused by certain proteins or glycoproteins, which are called food allergens. An analytical marker of allergens is the IgE-reactivity to these substances. Normally food allergens are minor components in allergenic source material, which consist of a huge number of chemical different substances. Thus allergen extraction, separation and immunological detection methods are described which identify and characterize individual food allergens by a minimum of manipulation. Favoured separation methods of allergenic extracts are electrophoretic ones allowing the combination of highly resolved protein separations with immunological detection methods subsumed by the term immunoblotting. These techniques are a useful basis to characterize allergens by chemical methods. Once the primary protein structure of a food allergen is established, the way is cleared for the identification of epitopes. Epitopes are immunological detectable parts of a protein or glycoprotein generating the interface between chemical structure and immune-system. The nature of epitopes may differ, for instance, can be conformational, continuous, or built up by glycoconjugates, which determine the stability of food allergens, especially in the case of food processing. Progress in identification and characterization of food allergens will improve diagnostics and therapy of food allergy.     

Comparison of different purification procedure for extraction of staphylococcal enterotoxin A from foods.

Different procedures commonly used for extraction, purification, and concentration of staphylococcal enterotoxins from foods were investigated with 131I- and 125I-labeled staphylococcal enterotoxin A. Loss of labeled enterotoxin A was compared with loss of total nitrogen. The results showed that in most of the common procedures, such as gel filtration, ion exchange, and heat treatment, the percentage of loss of labeled enterotoxin A was greater than the loss of total nitrogen. Chloroform extraction and acid precipitation with hydrochloric acid had nearly the same effect on the purification of both labeled enterotoxin A and total nitrogen. Ammonium sulfate precipitation proved to be practical and was successfully used for purification of enterotoxin A from sausage extract. Simultaneous use of trypsin and Pseudomonas peptidase for treatment of food extracts considerably reduced food proteins capable of interfering with serological detection of enterotoxins but did not essentailly influence the loss of enterotoxin A.     

Food allergen detection by mass spectrometry: From common to novel protein ingredients

Food allergens are molecules, mainly proteins, that trigger immune responses in susceptible individuals upon consumption even when they would otherwise be harmless. Symptoms of a food allergy can range from mild to acute; this last effect is a severe and potentially life-threatening reaction. The European Union (EU) has identified 14 common food allergens, but new allergens are likely to emerge with constantly changing food habits. Mass spectrometry (MS) is a promising alternative to traditional antibody-based assays for quantifying multiple allergenic proteins in complex matrices with high sensitivity and selectivity. Here, the main allergenic proteins and the advantages and drawbacks of some MS acquisition protocols, such as multiple reaction monitoring (MRM) and data-dependent analysis (DDA) for identifying and quantifying common allergenic proteins in processed foodstuffs are summarized. Sections dedicated to novel foods like microalgae and insects as new sources of allergenic proteins are included, emphasizing the significance of establishing stable marker peptides and validated methods using database searches. The discussion involves the in-silico digestion of allergenic proteins, providing insights into their potential impact on immunogenicity. Finally, case studies focussing on microalgae highlight the value of MS as an effective analytical tool for ensuring regulatory compliance throughout the food control chain.     

MALDI based identification of soybean protein markers--possible analytical targets for allergen detection in processed foods

Soybean (Glycine max) is extensively used all over the world due to its nutritional qualities. However, soybean is included in the "big eight" list of food allergens. According to the EU directive 2007/68/EC, food products containing soybeans have to be labeled in order to protect the allergic consumers. Nevertheless, soybeans can still inadvertently be present in food products. The development of analytical methods for the detection of traces of allergens is important for the protection of allergic consumers. Mass spectrometry of marker proteolytical fragments of protein allergens is growingly recognized as a detection method in food control. However, quantification of soybean at the peptide level is hindered due to limited information regarding specific stable markers derived after proteolytic digestion. The aim of this study was to use MALDI-TOF/MS and MS/MS as a fast screening tool for the identification of stable soybean derived tryptic markers which were still identifiable even if the proteins were subjected to various changes at the molecular level through a number of reactions typically occurring during food processing (denaturation, the Maillard reaction and oxidation). The peptides (401)Val-Arg(410) from the G1 glycinin (Gly m 6) and the (518)Gln-Arg(528) from the α' chain of the β-conglycinin (Gly m 5) proved to be the most stable. These peptides hold potential to be used as targets for the development of new analytical methods for the detection of soybean protein traces in processed foods.     

Lab scale and medium scale production of recombinant allergens in Escherichia coli

Recombinant products have become invaluable tools for diagnostic as well as therapeutic purposes in modern medicine. Especially in cases where raw naturally derived products are difficult to standardize, well-defined recombinant single components represent the matter of choice. In the recent past, much effort has been undertaken to define individual proteins derived from various sources like pollen, spores of moulds, pet dander, and food causing Type 1 allergic reactions in humans. Therefore, methods for cloning, sequencing, and expressing cDNAs coding for allergens in Escherichia coli became of great interest to allergologists. For the recombinant production of allergens, suitable expression systems, growing conditions, and purification steps have to be established for each individual product. Finally, the purified recombinant allergen has to be carefully investigated for the biochemical, biophysical, and immunological properties. In the following paper, several prokaryotic expression systems, purification strategies, and analytical methods will be presented and pitfalls discussed.     

Reproducibility of atopy patch tests with food and inhalant allergens

Although atopy patch tests (APT) seem a valuable additional tool in the diagnostic work-up for food allergy in children with atopic eczema/dermatitis syndrome, the immunopathology and some technical aspects of testing remain controversial. Few published data are available on the reproducibility of APT with inhalants and only two studies include fresh food allergens. In this study we therefore investigated the reproducibility of duplicate APT (left versus right side of the back) with native and commercially available food (cow s milk, hen s egg, tomato, wheat flour) and with inhalant allergens (Dermatophagoides pteronyssinus and mixed grasses) in a large unselected population of children. We tested a population of 277 Italian school children with three APT allergens: fresh food (cow s milk, hen s egg, tomato and wheat flour), standardised food allergens in petrolatum (the same four foods) and standardised inhalant allergens routinely used for skin prick testing. For the four food allergens (applied in the natural form or as the standardised commercial preparation) from one- to three quarters of the APT gave positive results on one side and negative reactions on the opposite side (Cohen s K coefficient between 0.38, fresh tomato and 0.81, fresh cow s milk). Conversely, APT with inhalant allergens were invariably reproducible (Cohen s K = 1.00). The possible technical and immunologic reasons explaining why reproducibility of APT differed for the two types of allergens await an answer from extensive controlled studies.     

Mapping of fruit allergens by 2D electrophoresis and immunodetection

Proteomic analyses of fruits are confronted with a series of specific obstacles: a general low protein content in plant tissues, allergen extraction from highly complex matrices and protein determination in the presence of interfering compounds. Different methods are currently being introduced to achieve higher protein yields and a simultaneous removal of interfering substances, such as polyphenols and polysaccharides. However, no universal protocol suitable for protein purification from any given plant species is available. Protein profiling by 2DE-western blotting offers a powerful tool for the detection and characterization of known and novel plant allergens. Moreover, the detection of IgE-reactive proteins from fruits is improved by combining western blot and alternative visualization techniques. The recent developments in bioinformatics and databases facilitate the interpretation of profiling studies with regard to novel potential fruit allergens.     

Mapping of fruit allergens by 2D electrophoresis and immunodetection

Proteomic analyses of fruits are confronted with a series of specific obstacles: a general low protein content in plant tissues, allergen extraction from highly complex matrices and protein determination in the presence of interfering compounds. Different methods are currently being introduced to achieve higher protein yields and a simultaneous removal of interfering substances, such as polyphenols and polysaccharides. However, no universal protocol suitable for protein purification from any given plant species is available. Protein profiling by 2DE-western blotting offers a powerful tool for the detection and characterization of known and novel plant allergens. Moreover, the detection of IgE-reactive proteins from fruits is improved by combining western blot and alternative visualization techniques. The recent developments in bioinformatics and databases facilitate the interpretation of profiling studies with regard to novel potential fruit allergens.     

Stability of food allergens and allergenicity of processed foods

The allergenicity of food could be altered by several processing procedures. For various foods of animal and plant origin the available literature on this alteration is described. Investigations on hidden allergens in food products are also dealt with.     

The direct application of the polymerase chain reaction to DNA extracted from foods

Two methods for the successful extraction of DNA from foods are described. The rapid lysis method uses a proteinase K buffer system to lyse cells and solubilize food samples. DNA is then precipitated using isopropanol. The second method achieves cell lysis using toluene and mutanolysin, and solubilization using guanidium thiocyanate. Following protein removal with organic solvents DNA is precipitated with isopropanol. Both methods enabled the polymerase chain reaction to be applied directly to DNA extracted from samples of cheese, coleslaw and raw chicken and allowed the direct rapid, sensitive and specific detection of Yersinia enterocolitica, Aerococcus viridans and Listeria monocytogenes in these foods.     

Comparative assessment of DNA extraction methods for identification of glanders and melioidosis etiological agents by PCR

Pathogenic Burkholderia are considered as a cause of dangerous infections and potential agents of bioterrorism. Comparative assessment of different methods of extraction and purification of DNA for PCR analysis of pure cultures and samples contaminated by etiological agents of glanders and melioidosis was performed. Samples of soil and food artificially contaminated by pathogenic Burkholderia as well as organs of infected animals were tested. DNA was extracted by methods of boiling, nucleosorption with presence of guanidine thiocyanate, guanidine thiocyanatephenol extraction, guanidine thiocyanate-phenol extraction with additional purification of DNA by nucleosorption. Amplification was performed by "Flash" technique and detector of fluorescence was used for analysis of PCR products. Utilization of the recommended methods of preparation depending on the nature of sample let to detect by the "Flash" technique the etiological agents of glanders and melioidosis in concentration =10(3) microbial cells per ml. Choice of DNA extraction and purification methods is determined by type of a sample and presence in it of admixtures inhibiting PCR.     

Inhibitory effects of collagen on the PCR for detection of Clostridium perfringens

It is essential to identify specific food components that inhibit PCR in order to increase the sensitivity of the PCR method for rapid detection of pathogens contaminating a food. We found that collagen, a major component of several foods, inhibited PCR. The inhibitory action of collagen on PCR could be partially reversed by adjusting the concentration of magnesium ion in the reaction mixture and by the use of various DNA extraction methods to remove the collagen from the DNA. Also, the source of thermostable DNA polymerase was affected by the presence of collagen. These results suggest the need to optimize the extraction and assay conditions for rapid detection of enterotoxigenic Clostridium perfringens by PCR with respect to the kind of food being analyzed.     

Detection of Staphylococcal Enterotoxin in Food

Methods are described for the extraction and serological detection of trace amounts of enterotoxins A and B in foods incriminated in outbreaks of staphylococcal food poisoning. Evidence is presented for the probable applicability of the methods for the detection of unidentified enterotoxins.     

Isolation and characterization of antimicrobial food components

Nowadays there is an evident growing interest in natural antimicrobial compounds isolated from food matrices. According to the type of matrix, different isolation and purification steps are needed and as these active compounds belong to different chemical classes, also different chromatographic and electrophoretic methods coupled with various detectors (the most used diode array detector and mass spectrometer) have to be performed. This review covers recent steps made in the fundamental understanding of sample preparation methods as well as of analytical tools useful for the complete characterization of bioactive food compounds. The most commonly used methods for extraction of natural antimicrobial compounds are the conventional liquid-liquid or solid-liquid extraction and the modern techniques such as pressurized liquid extraction, microwave-assisted extraction, ultrasound-assisted extraction, solid-phase micro-extraction, supercritical fluid extraction, and matrix solid phase dispersion. The complete characterization of the compounds is achieved using both monodimensional chromatographic processes (LC, nano-LC, GC, and CE coupled with different type of detectors) and, recently, using comprehensive two-dimensional systems (LC×LC and GC×GC).     

Inhibitory Effects of Collagen on the PCR for Detection of Clostridium perfringens

It is essential to identify specific food components that inhibit PCR in order to increase the sensitivity of the PCR method for rapid detection of pathogens contaminating a food. We found that collagen, a major component of several foods, inhibited PCR. The inhibitory action of collagen on PCR could be partially reversed by adjusting the concentration of magnesium ion in the reaction mixture and by the use of various DNA extraction methods to remove the collagen from the DNA. Also, the source of thermostable DNA polymerase was affected by the presence of collagen. These results suggest the need to optimize the extraction and assay conditions for rapid detection of enterotoxigenic Clostridium perfringens by PCR with respect to the kind of food being analyzed.     

Recombinant barley-produced antibody for detection and immunoprecipitation of the major bovine milk allergen, β-lactoglobulin

Recombinant allergens and antibodies are needed for diagnostic, therapeutic, food processing and quality verification purposes. The aim of this work was to develop a barley-based production system for β-lactoglobulin (BLG) specific immunoglobulin E antibody (D1 scFv). The expression level in the best barley cell clone was 0.8–1.2 mg/kg fresh weight, and was constant over an expression period of 21 days. In the case of barley grains, the highest stable productivity (followed up to T2 grains) was obtained when the D1 scFv cDNA was expressed under a seed-specific Glutelin promoter rather than under the constitutive Ubiquitin promoter. Translational fusion of ER retention signal significantly improved the accumulation of recombinant antibody. Furthermore, lines without ER retention signal lost D1 scFv accumulation in T2 grains. Pilot scale purification was performed for a T2 grain pool (51 g) containing 55.0 mg D1 scFv/kg grains. The crude extract was purified by a two-step purification protocol including IMAC and size exclusion chromatography. The purification resulted in a yield of 0.47 mg of D1 scFv (31 kD) with high purity. Enzyme-linked immunosorbent assay revealed that 29 % of the purified protein was fully functional. In immunoprecipitation assay the purified D1 scFv recognized the native 18 kD BLG in the milk sample. No binding was observed with the heat-treated milk sample, as expected. The developed barley-based expression system clearly demonstrated its potential for application in the processing of dairy milk products as well as in detecting allergens from foods possibly contaminated by bovine milk.     

Prediction of food protein allergenicity: a bioinformatic learning systems approach

Food hypersensitivity is constantly increasing in Western societies with a prevalence of about 1-2% in Europe and in the USA. Among children, the incidence is even higher. Because of the introduction of foods derived from genetically modified crops on the marketplace, the scientific community, regulatory bodies and international associations have intensified discussions on risk assessment procedures to identify potential food allergenicity of the newly introduced proteins. In this work, we present a novel biocomputational methodology for the classification of amino acid sequences with regard to food allergenicity and non-allergenicity. This method relies on a computerised learning system trained using selected excerpts of amino acid sequences. One example of such a successful learning system is presented which consists of feature extraction from sequence alignments performed with the FASTA3 algorithm (employing the BLOSUM50 substitution matrix) combined with the k-Nearest-Neighbour (kNN) classification algorithm. Briefly, the two features extracted are the alignment score and the alignment length and the kNN algorithm assigns the pair of extracted features from an unknown sequence to the prevalent class among its k nearest neighbours in the training (prototype) set available. 91 food allergens from several specialised public repositories of food allergy and the SWALL database were identified, pre-processed, and stored, yielding one of the most extensively characterised repositories of allergenic sequences known today. All allergenic sequences were classified using a standard one-leave-out cross validation procedure yielding about 81% correctly classified allergens and the classification of 367 non-allergens in an independent test set resulted in about 98% correct classifications. The biocomputational approach presented should be regarded as a significant extension and refinement of earlier attempts suggested for in silico food safety assessment. Our results show that the framework described here is powerful enough to become useful as part of a multiple-procedure test scheme that also depicts other evaluation approaches such as solid phase immunoassay and tests for stability to digestions.