Christiane Kruse Fæste

Liquid chromatography and mass spectrometry in food allergen detection.

Food allergy is an important issue in the field of food safety because of the hazards for affected persons and the hygiene requirements and legal regulations imposed on the food industry. Consumer protection and law enforcement require suitable analytical techniques for the detection of allergens in foods. Immunological methods are currently preferred; however, confirmatory alternatives are needed. The determination of allergenic proteins by liquid chromatography and mass spectrometry has greatly advanced in recent years, and gel-free allergenomics is becoming a routinely used approach for the identification and quantitation of food allergens. The present review provides a brief overview of the principles of proteomic procedures, various chromatographic set ups, and mass spectrometry instrumentation used in allergenomics. A compendium of published liquid chromatography methods, proteomic analyses, typical marker peptides, and quantitative assays for 14 main allergy-causing foods is also included.

A case of peanut cross-allergy to lupine flour in a hot dog bread

Background: In a case monitored by the Norwegian National Register for Severe Allergic Reactions to Food, a patient with peanut allergy experienced an allergic reaction after eating a particular brand of hot dog bread. The aim of this study was to identify the eliciting allergen. Methods: Extracts from the hot dog bread and reference material from peanut, lupine and lupine-fortified food products were analysed by immunochemical methods with patient serum and a new polyclonal anti-lupine antibody. Results: Evidence could be provided that the hot dog bread contained proteins from lupine but not from peanut. Conclusion: Crossed peanut-lupine allergy can have clinical significance. A peanut-allergic patient reacted against hidden lupine protein in a hot dog bread. Presented with our results, the producer confirmed the use of lupine flour and changed the ingredient list.

Characterization of IgE binding to lupin, peanut and almond with sera from lupin-allergic patients.

Background: The increasing number of applications of sweet lupins in food is paralleled by an increase in immunoglobulin E (IgE)-mediated allergic reactions to lupin proteins. In particular, lupin allergy seems to appear in patients with an existing peanut allergy. In the present study, IgE-binding studies towards fractionated lupin seed proteins, and peanut and almond proteins were performed using sera from patients with confirmed lupin allergy. Methods:Immunoblotting and indirect ELISA were performed to investigate IgE binding to protein extracts. ELISA inhibition experiments were performed to investigate the presence of cross-reactive allergens in the protein extracts. Results: Immunoblotting and ELISA experiments demonstrated IgE binding to all lupin conglutins (α, β, γ and δ) as well as to peanut and almond proteins, with a unique IgE-binding profile for each patient. High IgE binding to α-conglutin was observed and IgE from the majority of patients similarly recognized two proteins within the α-conglutin-containing fraction, 40 and 43 kDa in size. Inhibition ELISA experiments showed that preincubation of sera with lupin conglutins, peanut and almond resulted in decreased IgE binding to lupin flour. Conclusions: Overall, these results indicate that α-, β-, γ- and δ-conglutins are candidate allergens in lupin and suggest a particularly strong allergenicity of α-conglutins. Furthermore, the results indicate the presence of cross-reactive allergens in lupin, peanut and almond.

Allergenicity and antigenicity of fenugreek (Trigonella foenum-graecum) proteins in foods

BACKGROUND Fenugreek is an ingredient in Indian-style spiced foods. Reports of adverse reactions reflect a trend toward a more international cuisine. Fenugreek allergy has not been systematically investigated so far. OBJECTIVE Study the allergenicity and antigenicity of fenugreek proteins using patient sera and a newly developed polyclonal antifenugreek antibody. METHODS Allergenic fenugreek proteins were identified by immunoblotting, using sera from 29 patients with specific IgE to peanut and other legumes. In addition, 2 patients were evaluated by skin prick test and open food challenge with native fenugreek powder. Spiced and flavored food products were analyzed for fenugreek by semiquantitative IgE and IgG immunoblotting. RESULTS High levels of specific IgE to both peanut and fenugreek were seen in most sera. Fenugreek sensitization is believed to be a consequence of cross-reactivity in patients with peanut allergy. Primary fenugreek allergy was suspected in only 1 case. The fenugreek dose eliciting objective symptoms was about 2 mg in the open food challenge. Major fenugreek allergens were identified at 50, 52, and 74 kd and peanut proteins at 22, 36, and 40 kd. A specific polyclonal antifenugreek antibody was found suitable for food analysis. In a food survey, about 1/3 of the fenugreek-containing products were labeled correctly. CONCLUSION Fenugreek seed powder, an ingredient in spiced foods, contains several potential allergens. There is evidence for a high rate of cross-reactivity to peanut.

Characterization of potential allergens in fenugreek (Trigonella foenum-graecum) using patient sera and MS-based proteomic analysis

BACKGROUND Fenugreek is a legume plant used as an ingredient of curry spice. Incidents of IgE-mediated food allergy to fenugreek have been reported. Coincidence with allergy to peanut, a major food allergen, seems to be common suggesting a rather high rate of cross-reactivity. OBJECTIVE Characterization of fenugreek allergens using patient sera and mass spectrometry-based proteomic analysis. METHODS Allergenic fenugreek proteins were detected by immunoblotting, using sera from 13 patients with specific IgE to peanut and fenugreek. IgE-binding proteins were analyzed by peptide mass fingerprinting and peptide sequencing. RESULTS A fenugreek protein quintet in the range from 50 kDa to 66 kDa showed high IgE-affinity, the protein at 50 kDa reaching the strongest signals in all patients. Proteomic analyses allowed the classification of several fenugreek proteins to a number of allergen families. Fenugreek 7S-vicilin and 11S-legumin were partly sequenced and revealed considerable homologies to peanut Ara h 1 and Ara h 3, respectively. The presence of a fenugreek 2S albumin and pathogenesis-related (PR-10) plant pollen protein was assumed by database searching results. CONCLUSION In this study, individual fenugreek proteins were characterised for the first time. Observed homologies to major peanut allergens provide a molecular explanation for clinical cross-reactivity.

Characterisation of potential novel allergens in the fish parasite Anisakis simplex

The parasitic nematode Anisakis simplex occurs in fish stocks in temperate seas. A. simplex contamination of fish products is unsavoury and a health concern considering human infection with live larvae (anisakiasis) and allergic reactions to anisakid proteins in seafood. Protein extracts of A. simplex produce complex band patterns in gel electrophoresis and IgE-immunostaining. In the present study potential allergens have been characterised using sera from A. simplex-sensitised patients and proteome data obtained by mass spectrometry. A. simplex proteins were homologous to allergens in other nematodes, insects, and shellfish indicating cross-reactivity. Characteristic marker peptides for relevant A. simplex proteins were described.

In vitro and in vivo hepatic metabolism of the fungal neurotoxin penitrem A.

Penitrem A is a potent neurotoxin produced by several species in the genus Penicillium, which primarily affects the central nervous system. The toxin has several effects on neurotransmitter release, both at the central and peripheral level, as well as on ion channels. We have evaluated the hepatic metabolism of penitrem A by rat hepatocytes and rat-liver microsomes in vitro. In addition, we have conducted an in vivo study in mice and determined metabolites in several organs. According to our results, penitrem A is extensively metabolized in the liver to at least five metabolites more hydrophilic than the parent compound.

Mapping of the Immunodominant Regions of Shrimp Tropomyosin Pan b 1 by Human IgE-Binding and IgE Receptor Crosslinking Studies

Background: Epitope mapping of an allergen is generally done by IgE-binding assays with short synthetic peptides, but this provides little information about which domains are responsible for IgE receptor crosslinking on effector cells. Our aim was to map the immunodominant regions of shrimp tropomyosin by both IgE-binding and IgE-receptor crosslinking studies. Methods: Five overlapping fragments covering Pandalus borealis tropomyosin were cloned, expressed in Escherichia coli and characterized by circular dichroism spectroscopy, native PAGE and bis(sulfosuccinimidyl) suberate-crosslinking. IgE binding was detected by Western blot, indirect ELISA and inhibition ELISA, and IgE receptor crosslinking was investigated by basophil activation test and skin prick test with Norwegian shrimp allergic adults. Results: The N- and C-terminal fragments of tropomyosin showed the highest amount of secondary structure. Western blot studies showed preferential binding to the terminal fragments, while indirect and inhibition ELISA studies showed binding to all fragments, but with individual variations. Basophil CD63 expression was upregulated by all fragments at high concentrations (1 µg/ml) and showed individual variations comparable to ELISA results. A mixture of the fragments with equal molar ratios induced comparably strong CD63 activation as for tropomyosin. Skin prick test studies showed positive responses to the terminal and middle fragments and increased responses to the fragment mixture compared to whole tropomyosin. Conclusions: The terminal and middle fragments of tropomyosin had the highest IgE reactivity, but overall no clear immunodominant region was observed in this study. These results correlated well with previous studies with short peptides. Dividing shrimp tropomyosin into five fragments did not reduce the allergenicity of the protein.

Differential influence of the degree of processing on immunogenicity following proteolysis of casein and β-lactoglobulin

Abstract Simulated gastric fluid digestion was used to investigate how heat processing and fat content influence effects of proteolytic digestion on milk protein immunogenicity in milk with varying fat content and degree of processing. Changes in antigenicity and allergenicity of casein and β-lactoglobulin (β-LG) were demonstrated, using polyclonal antibodies and milk-allergic patient sera in immunoblot and enzyme-linked immunosorbent assay (ELISA) inhibition. Heat-induced changes in ultra-high temperature processed (UHT) milk increased stability of IgE and IgG casein epitopes to digestive proteolysis, but had little effect on specific IgE or IgG binding. Native κ-casein may be more resistant to digestion, but this was abrogated by heat treatment. Although β-LGs digestive stability decreased in UHT milk, immunogenicity was retained after hydrolysis in both processed (UHT) and non-processed (semi-skimmed) milks. Differences in fat levels in three milk matrices which had been processed similarly did not appear to influence digestibility. The possible clinical significance of these findings remains is discussed.

Development of liquid chromatography-tandem mass spectrometry methods for the quantitation of Anisakis simplex proteins in fish.

The parasite Anisakis simplex is present in many marine fish species that are directly used as food or in processed products. The anisakid larvae infect mostly the gut and inner organs of fish but have also been shown to penetrate into the fillet. Thus, human health can be at risk, either by contracting anisakiasis through the consumption of raw or under-cooked fish, or by sensitisation to anisakid proteins in processed food. A number of different methods for the detection of A. simplex in fish and products thereof have been developed, including visual techniques and PCR for larvae tracing, and immunological assays for the determination of proteins. The recent identification of a number of anisakid proteins by mass spectrometry-based proteomics has laid the groundwork for the development of two quantitative liquid chromatography-tandem mass spectrometry methods for the detection of A. simplex in fish that are described in the present study. Both, the label-free semi-quantitative nLC-nESI-Orbitrap-MS/MS (MS1) and the heavy peptide-applying absolute-quantitative (AQUA) LC-TripleQ-MS/MS (MS2) use unique reporter peptides derived from anisakid hemoglobin and SXP/RAL-2 protein as analytes. Standard curves in buffer and in salmon matrix showed limits of detection at 1μg/mL and 10μg/mL for MS1 and 0.1μg/mL and 2μg/mL for MS2. Preliminary method validation included the assessment of sensitivity, repeatability, reproducibility, and applicability to incurred and naturally-contaminated samples for both assays. By further optimization and full validation in accordance with current recommendations the LC-MS/MS methods could be standardized and used generally as confirmative techniques for the detection of A. simplex protein in fish.