Bioinformatic screening and detection of allergen cross‐reactive IgE‐binding epitopes

Protein allergens can be related by cross‐reactivity. Allergens that share relevant sequence can cross‐react, those lacking sufficient similarity in their IgE antibody‐binding epitopes do not cross‐react. Cross‐reactivity is based on shared epitopes that is based on shared sequence and higher level structure (charge and shape). Epitopes are important in predicting cross‐reactivity potential and may provide the potential to establish criteria that identify homology among allergens. Selected allergen's IgE‐binding epitope sequences were used to determine how the FASTA algorithm could be used to identify a threshold of significance. A statistical measure (expectation value, E‐value) was used to identify a threshold specific to identifying cross‐reactivity potential. Peanut Ara h 1 and Ara h 2, shrimp tropomyosin Pen a 1, and birch tree pollen allergen, Bet v 1 were sources of known epitopes. Each epitope or set of epitopes was inserted into random amino acid sequence to create hypothetical proteins used as queries to an allergen database. Alignments with allergens were noted for the ability to match the epitope's source allergen as well as any cross‐reactive or other homologous allergens. A FASTA expectation value range (1 × 10^?5–1 × 10^?6) was identified that could act as a threshold to help identify cross‐reactivity potential.     

Physicochemical properties and thermal stability of Lep w 1, the major allergen of whiff

Whiff (Lepidorhombus whiffiagonis) is a fish frequently consumed in Spain. Lep w 1, its major allergen, is a calcium‐binding β‐parvalbumin. The resistance of Lep w 1 to heat denaturation and to digestion were studied by circular dichroism spectroscopy and by in vitro gastric digestion systems. Purified Lep w 1 was thermally stable up to 65°C at neutral pH. Calcium depletion resulted in a change of its structure as determined by circular dichroism spectroscopy. A partial loss of structure was also observed at acidic pH; however, the allergen retained its full IgE‐binding ability. The partially denatured Lep w 1 was easily digested by pepsin within 2 min. Further, the IgE reactivity of proteins extracted from cooked fish and their stability to proteolysis were analyzed. The extract revealed a higher number of IgE reactive bands than an extract from uncooked fish. IgE binding to these proteins could not be inhibited by an extract from uncooked fish. In contrast to a raw fish extract, the cooked extract showed higher resistance to pepsinolysis. The stability of Lep w 1 to thermal denaturation and digestion explains the high allergenicity of whiff.     

The influence of different acids and pepsin on the extractability of collagen from the skin of Baltic cod (Gadus morhua)

Solutions of (0.5 M) citric, lactic and acetic acids and 0.15 M HCl were used for the extraction of collagen from the whole skins of Baltic cod (Gadus morhua). The extractions were performed at a temperature of 4 °C for 24, 48 and 72 h using a solid/solution ratio of 1:6 (w/v). Of the acids used, HCl was the least effective solvent for collagen. The maximal yield of collagen extracted with citric acid was 60%. Collagen extraction with acetic or lactic acid give a maximal yield of about 90% with HCl yielding of only 18%. After enzymatic treatment of cod skin the yield of protein extracted with HCl and citric acids increased to 40% and 20%, respectively. Collagen was completely solubilized under the same conditions in acetic and lactic acids. Electrophoretic analysis of collagens extracted in HCl and citric acids with enzymatic treatment proved that the isolated protein was denaturated. The solutions of acetic and lactic acids are solvents for native collagen.     

Immunoglobulin E epitope mapping by microarray immunoassay reveals differences in immune response to genetic variants of caseins from different ruminant species

The allergenicity of the caseins (CN), one of the major allergens in cow milk, is well characterized and their immunoglobulin E (IgE)-binding epitopes have been identified. However, investigations about the allergenic potential of the genetic variants occurring in the caseins are lacking. Therefore, this study determined the influence of the genetic polymorphism on IgE binding to epitopes of bovine casein variants. Furthermore, differences in IgE binding between epitopes of goats and water buffaloes were analyzed. A set of 187 peptides, covering the previously identified sequential IgE-binding epitopes of α_S1-, α_S2-, β-, and κ-CN variants from cows and the corresponding homologous peptides of water buffaloes and goats, were synthesized and tested by means of peptide microarray for IgE binding, using sera from 16 cow milk-sensitized individuals. Seven of the 16 sera samples showed positive signals on microarrays and were included in this study. In 5 α_S1-CN variants (A, B, C, E, and I), the AA substitution or deletion affected the immunoreactivity of epitopes AA 4 to 23, AA 17 to 36, AA 83 to 102, AA 173 to 192, and AA 175 to 194, as well as of the variant-specific peptides AA 184 to 196, AA 187 to 199, AA 174 to 193, and AA 179 to 198, which were found to resist gastrointestinal digestion. Variation in IgE binding was further detected for peptides AA 103 to 123 and AA 108 to 129 of 3 β-CN variants (A¹, A², and B). The majority of sera showed IgE binding to α_S1-CN peptides of cows and the homologous counterpart of goats and water buffaloes. However, α_S1- and β-CN epitopes from goats and water buffaloes had lower immunoreactivity than those of cows, but, in some cases, higher or exclusive IgE binding was observed. The results of this study indicate that genetic variants of the caseins differ in their allergenicity. This might be useful in the search for a suitable protein source for cow milk-allergic patients. In addition, milk from water buffaloes and goats harbor an allergenic potential due to cross-reactivity of IgE antibodies with cow milk caseins and are, therefore, not an acceptable alternative in the nutrition of cow milk-allergic patients.     

Development of an epitope-specific analytical tool for the major peanut allergen Ara h 2 using a high-density multiple-antigenic peptide strategy

Using the major peanut allergen Ara h 2 as an example, an analytical tool enabling the determination of immunoglobulin E (IgE)-epitopes in processed food allergens was developed. We synthesized a multiple-antigenic peptide (MAP) of the IgE-reactive linear epitope 3 (amino acid positions 27-36) of Ara h 2 and raised a monospecific antiserum against this epitope to obtain a positive control for future epitope resolved diagnostics. First, a MAP of epitope 3, having a molecular mass of 7770 Da, was synthesized, purified, and its structure confirmed by liquid chromatography-mass spectrometry (electrospray ionization) (LC-MS(ESI)), matrix assisted laser desorption/ionization-time of flight (MALDI-TOF), and Edman sequencing. The MAP was then used to raise high titer antibodies in rabbits using the adjuvant Titermax and to characterize the specificity of IgE from allergenic patients sensitized to Ara h 2. The antiserum exclusively detects Ara h 2 in crude peanut extract with a titer of 10(7) by Western blot and reacts specifically with epitope 3 shown by epitope mapping for a library of solid-phase-bound synthetic 15-mer peptides covering the entire sequence of Ara h 2. Such IgE-reactive epitopes are of high analytical relevance as they could constitute the basis for epitope-specific detection systems for use in quality control in the food industry or for forensic purposes in cases of fatal reactions to otherwise undetected peanut proteins.     

Comparison of different extraction solutions for the analysis of allergens in hen’s egg

An important requirement for the correct procedure of allergen analysis in hen’s egg is to obtain complete and unaltered protein extracts. Besides the aim of a quantitative extraction of the allergens from the matrix, it is equally important not to alter their allergenic potential during the extraction process. This paper describes and compares six extraction solutions for the analysis of whole-egg proteins and allergens. These requirements were examined via protein determination according to Bradford [Bradford, M. M. (1976). Rapid and sensitive method for quantitation of microgram quantities of protein utilizing principle of protein–dye binding. Analytical Biochemistry, 72, 248–254] and Kjeldahl [Meyer, A. H. (2006). Lebensmittelrecht, Verlag C.H. Beck München, Stand: 1. February 2006, § 64, Lebensmittel- und Futtermittelgesetzbuch, Amtliche Sammlung von Untersuchungsmethoden, Nr. L 06.00-7] as well as the EAST-inhibition method. It could be demonstrated that the extraction with a urea solution (8 M) led to significant interferences during the protein determination, and substantially reduced the allergenic potential of egg proteins. With all other extraction solutions adequate protein contents could be extracted. The highest protein content was achieved by the extraction with phosphate buffered saline followed by a Tween 20 solution, physiological saline, water, and acetate buffer. The results show that none of these extracts – except for the urea solution (8 M) – was altered in its’ allergenic potential.     

Facial skin mapping: from single point bio‐instrumental evaluation to continuous visualization of skin hydration,barrier function,skin surface pH,and sebum in different ethnic skin types

Dry skin is one of the most important concerns of consumers worldwide. Despite huge efforts over several decades, the personal care industry still does not offer a perfect solution to satisfy the unmet needs of consumers for moisturising treatments in different ethnic groups. The paucity of data for the underlying cellular and biochemical problems in, and the effects of moisturisers on photodamaged facial skin may partly explain this. Mainly, single point measurements are used to understand the effects of products on skin physiology even on surrogate skin sites such as the non‐photodamaged volar forearm. Some groups have developed discontinuous facial maps of skin biophysical properties, however, in 2014 a continuous facial analysis of bio‐instrumental evaluations was developed using a heat map approach. These maps enabled a continuous visualization of features that not only revealed an unexpected complexity of facial skin but also indicated that use of surrogate skin sites for facial skin is inappropriate. We have demonstrated that remarkable gradients of skin hydration, TEWL, skin surface pH and sebum exist within short distances across the face and the gradients are distinctive among different ethnic groups. In addition, these studies have demonstrated that darkly‐pigmented individuals do not necessarily have a better skin barrier function than their less‐pigmented counterparts and that Caucasians have a lower facial skin surface pH compared with more pigmented subjects. Overall, there are no correlations between capacitance, TEWL and skin surface pH including individual topology angle values. Novel 3D camera approaches have also been used to facilitate a more precise assignment of measurement sites and visualisation. The 3D facial colour mappings illustrated precisely the local moisturising effects of a moisturising cream. There were subtle ethnic differences in efficacy that may be related to underlying skin biochemistry and/or ethnic differences in product application. A placebo‐controlled study using conductance measurements in Chinese subjects is also reported. Finally, a new whole face statistical approach has been taken to prove differences in skin parameters but also of moisturiser treatment that adds further to our understanding of the ethnic differences in skin physiology and product application. This paper reviews the background of the development and application of this methodology.     

Antioxidant capacity of medicinal plants from the Province of Córdoba (Argentina) and their in vitro testing in a model food system

The total phenols content (Folin–Ciocalteau assay) and antioxidant capacity (ferric reducing/antioxidant power – FRAP) of 41 plants from Córdoba (Argentina) were analyzed. Phenol content ranged from 8.2 to 100.2 mg GAE/g. FRAP ranged from 85.2 to 1862.0 μmol of Fe(II)/g. Capparis atamisguea had the lowest values of total phenols content and antioxidant capacity (8.2 mg GAE/g and 85.2 μmol of Fe(II)/g, respectively), while Ligaria cuneifolia exhibited the highest values (100.2 mg GAE/g and 1862.0 μmol of Fe(II)/g, respectively). A significant linear correlation (p < 0.05) was found (0.9125) between phenols content and antioxidant capacity. Results support the idea that these plants may be a good source of natural antioxidants for food applications. Plants from the Asteraceae family (the most representative of the Córdoba flora) were further tested for their DPPH radical scavenging activity. Some plant extracts were tested in a simple food system to investigate to their potential use in foods.     

Residue analyses and exposure assessment of the Irish population to nitrofuran metabolites from different food commodities in 2009–2010

An exposure assessment to nitrofuran residues was performed for three human populations (adults, teenagers and children), based on residue analyses of foods of animal origin (liver, honey, eggs and aquaculture) covering the 2-year period 2009–2010. The occurrence of nitrofuran metabolites in food on the Irish market was determined for the selected period using the data from Ireland’s National Food Residue Database (NFRD) and from results obtained from the analysis of retail samples (aquaculture and honey). Laboratory analyses of residues were performed by methods validated in accordance with Commission Decision 2002/657/EC regarding performance of the analytical method and interpretation of results. Semicarbazide (SEM) was the contaminant most frequently identified and its content ranged from 0.09 to 1.27 μg kg^?1. SEM is currently used as a marker of nitrofuran abuse, but it may also occur from other sources. The presence of nitrofuran metabolite 3-amino-2-oxazolidinone (AOZ) was detected in two aquaculture samples (prawns) at 1.63 and 1.14 μg kg^?1, but such a low number of positive cases did not present sufficient data for a full AOZ exposure assessment. Therefore, the evaluation of exposure was focused on SEM-containing food groups only. Exposure assessments were completed using a probabilistic approach that generated 10 iterations. The results of both the upper- and lower-bound exposure assessments demonstrate that SEM exposure for Irish adults, teenagers and children from selected food commodities are well below EFSA-estimated safe levels.     

The effects of sex and seasonality on the metal levels of different muscle tissues of mature Atlantic blue crabs (Callinectes sapidus) in Mersin Bay,north‐eastern mediterranean

The objective of this study was to determine the seasonal and sexual effects on metal levels of lump crabmeat (LCM) and chela crabmeat (CCM) of mature Atlantic blue crabs, Callinectes sapidus, caught in the Mersin Bay, the north‐eastern Mediterranean. The findings indicated that the annual ranges of metal levels in the LCM of Atlantic blue crab were as follows: 0.44–0.61 μg Cd g^?1, 0.30–0.60 μg Cr g^?1, 0.24–0.52 μg Pb g^?1, 9.72–43.70 μg Cu g^?1, 39.52–97.26 μg Zn g^?1, 11.97–32.48 μg Fe g^?1. The annual range of metal levels in the CCM of Atlantic blue crab were as follows: 0.52–1.07 μg Cd g^?1, 0.24–0.61 μg Cr g^?1, 0.28–0.56 μg Pb g^?1, 22.17–68.09 μg Cu g^?1, 93.92–175.21 μg Zn g^?1, 8.81–19.47 μg Fe g^?1. Cd, Cu, Zn levels in CCM of Atlantic blue crabs were higher than in LCM, whereas Fe levels were found lower (P < 0.05). Fe metal specifically accumulated in LCM, and Cd, Cu and Zn metals accumulated in CCM. Metals such as Cu, Zn and Fe showed seasonal variations. It was found out that Cu, Zn and Fe levels of muscle tissues of the Atlantic blue crab in spring and summer seasons were higher than in autumn and winter seasons.     

Condensed tannins from Lotus corniculatus and Lotus pedunculatus exert different effects on the in vitro rumen degradation of ribulose‐1,5‐bisphosphate carboxylase/oxygenase (Rubisco) protein

Condensed tannins (CT) or proanthocyanidins (PA), which occur in a restricted range of forages, have the ability to interact with proteins and enzymes and can influence the digestion of plant protein in the rumen. We compared the effects of CT extracts from Lotus corniculatus and pedunculatus on degradation of the principal leaf protein, ribulose‐1,5‐bisphosphate carboxylase/oxygenase (Rubisco), by rumen microorganisms. Total soluble leaf protein extracted from white clover (Trifolium repens ) was incubated with fresh rumen fluid from sheep and a range of concentrations of each CT extract. The rate of degradation of the large (LSU) and small subunit (SSU) of Rubisco was quantified by fractionating the proteins in samples taken from in vitro rumen incubations using sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS‐PAGE) and imaging densitometry. To deduce the effects of the CT extracts, experiments were performed in the presence (CT inactive) and absence (CT active) of polyethylene glycol (PEG; MW 3350). The two CT extracts differed markedly in their effects on the degradation of the LSU and SSU of Rubisco. At concentrations of 0.89 and 1.79 mg CT mg ⁻¹ total soluble leaf protein, the CT extract from L pedunculatus was more effective at preventing the degradation of the LSU and SSU by rumen microorganisms than the CT extract from L corniculatus. At a concentration of 1.79 mg CT mg ⁻¹ total soluble leaf protein, the CT extracts from L corniculatus and pedunculatus prevented about 0.75 and 0.83 of the LSU and about 0.69 and 0.86 of the SSU, respectively, from being degraded. Addition of PEG removed the inhibition and almost complete degradation of these proteins occurred, as was the case in incubations without CT extracts. The results of this study suggest that the concentration of CT in the diet and the chemical structure which affects the activity of the CT needs to be considered when assessing the effects of CT on protein metabolism in ruminants. © 1999 Society of Chemical Industry