Identification and characterisation of the proteins bound by specific phage-displayed recombinant antibodies (scFv) obtained against Brazil nut and almond extracts.

BACKGROUND: Almonds and Brazil nuts are widely consumed allergenic nuts whose presence must be declared according to food labelling regulations. Their detection in food products has been recently achieved by ELISA methods with recombinant antibodies (scFv) isolated against complete Brazil nut and almond protein extracts. The screening of phage-scFv libraries against complete protein extracts confers a series of advantages over the use of purified proteins, as recombinant proteins might alter their native folding. However, using this strategy, the nature of the target detected by phage-displayed antibodies remains unknown, and requires further research to identify whether they are nut allergens or other molecules present in the extract, but not related to their allergenic potential. RESULTS: Electrophoretic, chromatographic, immunological and spectrometric techniques revealed that the Brazil nut (BE95) and almond (PD1F6 and PD2C9) specific phage-scFvs detected conformational epitopes of the Brazil nut and almond 11S globulins, recognised by WHO/IUIS as Ber e 2 and Pru du 6 major allergens. Circular dichroism data indicated that severe heat treatment would entail loss of epitope structure, disabling scFv for target detection. CONCLUSIONS: The presence of important Brazil nut and almond allergens (Ber e 2 and Pru du 6) in foodstuffs can be determined by using phage-display antibodies BE95, PD1F6 and PD2C9 as affinity probes in ELISA. © 2017 Society of Chemical Industry.

Selection of recombinant antibodies by phage display technology and application for detection of allergenic Brazil nut (Bertholletia excelsa) in processed foods.

Current immunological methods for detection of Brazil nut allergens in foods are based on polyclonal antibodies raised in animals. Phage display technology allows the procurement of high-affinity antibodies avoiding animal immunization steps and therefore attaining the principle of replacement supported by animal welfare guidelines. In this study, we screened Tomlinson I and J libraries for specific binders against Brazil nut by employing a Brazil nut protein extract and a purified Brazil nut 2S globulin, and we successfully isolated a phage single chain variable fragment (named BE95) that specifically recognizes Brazil nut proteins. The selected phage scFv was further used as affinity probe to develop an indirect phage-ELISA for detection of Brazil nut in experimental binary mixtures and in commercial food products, with a limit of detection of 5 mg g(-1). This study describes for the first time the isolation of recombinant antibody fragments specific for an allergenic tree nut protein from a naïve library and paves the way to develop new immunoassays for food analysis based on probes that can be produced in vitro when required and do not rely on animal immunization.

Solution structure, copper binding and backbone dynamics of recombinant Ber e 1-the major allergen from Brazil nut.

BACKGROUND: The 2S albumin Ber e 1 is the major allergen in Brazil nuts. Previous findings indicated that the protein alone does not cause an allergenic response in mice, but the addition of components from a Brazil nut lipid fraction were required. Structural details of Ber e 1 may contribute to the understanding of the allergenic properties of the protein and its potential interaction partners. METHODOLOGY/PRINCIPAL FINDINGS: The solution structure of recombinant Ber e 1 was solved using NMR spectroscopy and measurements of the protein back bone dynamics at a residue-specific level were extracted using (15)N-spin relaxation. A hydrophobic cavity was identified in the structure of Ber e 1. Using the paramagnetic relaxation enhancement property of Cu(2+) in conjunction with NMR, it was shown that Ber e 1 is able to specifically interact with the divalent copper ion and the binding site was modeled into the structure. The IgE binding region as well as the copper binding site show increased dynamics on both fast ps-ns timescale as well as slower µs-ms timescale. CONCLUSIONS/SIGNIFICANCE: The overall fold of Ber e 1 is similar to other 2S albumins, but the hydrophobic cavity resembles that of a homologous non-specific lipid transfer protein. Ber e 1 is the first 2S albumin shown to interact with Cu(2+) ions. This Cu(2+) binding has minimal effect on the electrostatic potential on the surface of the protein, but the charge distribution within the hydrophobic cavity is significantly altered. As the hydrophobic cavity is likely to be involved in a putative lipid interaction the Cu(2+) can in turn affect the interaction that is essential to provoke an allergenic response.

Ber e 1 protein: the versatile major allergen from Brazil nut seeds.

Due mainly to its extremely high content of sulphur amino acids, Ber e 1 protein, the major allergen from Brazil nut, has attracted much scientific and press attention. Ber e 1 was the main target protein in early biotechnology transgenic work, in early processing studies of plant storage proteins, in plant vacuolar targeting studies and as the main protein in early nutritional supplementation experiments. Ber e 1 was also one of the first food allergens to be unintentionally transferred from one plant to another and was involved in the first reported case of systemic allergic reaction caused by a food allergen transferred in semen. In this review, many of the Ber e 1 unique biotechnological and structural functions are discussed with a particular emphasis on its use as model protein for studies of intrinsic allergenicity of food proteins.

Single nut or total nut avoidance in nut allergic children: outcome of nut challenges to guide exclusion diets.

BACKGROUND: Children with diagnosed nut allergy are typically advised by health professionals to exclude all nuts from their diets, irrespective of the outcome of allergy testing, to avoid inadvertent contact through contamination or the possible development of new allergies. METHODS: In our service, as we feel greater diagnostic accuracy prevents dietary risk taking, we provide the facility for children with nut allergy the opportunity of controlled exposure to 'other' nuts irrespective of whether their allergy prick test (PT) results are positive or negative. We performed open food challenges on our paediatric day ward. The challenge food was administered by way of a homemade biscuit containing 8 g of each nut challenged and given in increasing visually measured doses. RESULTS: Over the 5-year period from 2006, we challenged 145 children diagnosed as peanut allergic or tree nut allergic. In those with peanut allergy challenged to tree nuts, none of the 72 with negative PTs to tree nuts reacted on challenge whilst 7 of 22 (31.2%) with positive PTs did. In patients with tree nut allergy challenged to peanuts and/or other tree nuts, 3 of 38 (7.9%) with negative PT results and 5 of 13 (38.4%) with positive PT results reacted. CONCLUSION: Children allergic to peanuts with negative allergy tests to tree nuts had no co-existing allergy, but were at risk of tree nut allergy where PTs were positive. Children with tree nut allergy were at risk of co-existing peanut or other tree nut allergy whether PTs were positive or negative. Oral challenges to clarify allergy status in all nuts show co-existing allergies even in young children and in so doing may reduce anxiety, minimize unnecessary dietary restrictions and prevent later episodes of anaphylaxis through uninformed exposure.

A novel real-time polymerase chain reaction method for the detection of Brazil nuts in food.

A qualitative real-time PCR-based method for the detection of the Brazil nut (Bertholletia excelsa) component in food is described. The method consists of DNA isolation by chaotropic SPE and the subsequent PCR with Brazil nut-specific primers and a TaqMan fluorescent probe. The primers and the probe are targeted to the gene encoding for the 11s globulin of the Brazil nut. The method was positive for eight Brazil nut samples from the market and negative for all other tested plant and meat materials used in the food industry (36 samples). The intrinsic LOD of the method was 10 pg Brazil nut DNA. Using a series of model nut paste mixtures with defined Brazil nut contents, a practical detection limit of 0.1% (w/w) Brazil nut was estimated. Practical applicability of the entire method was tested by qualitative analysis of nine food samples; no discrepancies between the declared and detected Brazil nut contents were found. The presented PCR-based method is useful for sensitive and selective detection of Brazil nut in food samples.

Influence of plant lipids on immune responses in mice to the major Brazil nut allergen Ber e 1.

BACKGROUND: Lipids, particularly bacterial lipopolysaccharide, can impact on immune responses to proteins, with low doses enhancing type 2 responses. OBJECTIVE: We have examined the influence of natural plant lipid extracts on antibody responses provoked in mice by recombinant Ber e 1, the major allergen in Brazil nuts. METHODS: BALB/c strain mice were immunized (by intraperitoneal injection) with natural or recombinant Ber e l produced in Pichia pastoris and admixed with various lipid fractions isolated from Brazil nuts. Serum samples were analysed for specific IgE antibody by homologous passive cutaneous anaphylaxis assay and for IgG by enzyme-linked immunosorbant assay. RESULTS: Exposure to recombinant (lipid-free) Ber e 1 alone failed to induce detectable IgG or IgE antibody. Co-administration of the total lipid fraction (with reduced triglyceride levels), sterol-rich, or polar lipid fractions, resulted in marked adjuvant effects on IgG and IgE. However, the beta-sitosterol and glycolipid-rich fractions were associated with only low-level IgG antibody, and had little impact on IgE antibody production. Natural Ber e 1 containing endogenous lipids also provoked IgG and IgE antibody responses. Identical IgE and IgG antibody responses were detected regardless of whether natural or recombinant Ber e 1 was used as substrates for analyses. CONCLUSION: Endogenous Brazil nut lipids are required for the induction of optimal antibody responses to Ber e 1 in the BALB/c strain mouse. Appropriate antibody binding sites are present on both natural and recombinant forms of Ber e 1, suggesting that the impact of lipid is at the induction phase, rather than antibody recognition, and is possibly required for efficient antigen presentation.

IgE cross-reactivity between the major peanut allergen Ara h 2 and tree nut allergens.

Allergy to peanut and tree nuts is characterised by a high frequency of life-threatening anaphylactic reactions and typically lifelong persistence. Although peanut is the most common cause of nut allergy, peanut allergic patients are frequently also sensitive to tree nuts. It is not known if this is due to cross-reactivity between peanut and tree nut allergens. In this study, the major peanut allergen Ara h 2 was cloned from peanut cDNA, expressed in E. coli cells as a His-tag fusion protein and purified using a Ni-NTA column. Immunoblotting, ELISA and basophil activation indicated by CD63 expression all confirmed the IgE reactivity and biological activity of rAra h 2. To determine whether or not this allergen plays a role in IgE cross-reactivity between peanut and tree nuts, inhibition ELISA was performed. Pre-incubation of serum from peanut allergic patients with increasing concentrations of almond or Brazil nut extract inhibited IgE binding to rAra h 2. Purified rAra h 2-specific serum IgE antibodies also bound to proteins present in almond and Brazil nut extracts by immunoblotting. This indicates that the major peanut allergen, Ara h 2, shares common IgE-binding epitopes with almond and Brazil nut allergens, which may contribute to the high incidence of tree nut sensitisation in peanut allergic individuals.

Anaphylaxis due to Brazil nut skin testing in a walnut-allergic subject.

The diagnosis and management of nut allergy can be difficult because of the possible severity of the clinical manifestations and the cross reactivity between different species. We report a case of anaphylaxis due to skin testing in a young adult with clinically ascertained walnut allergy. After an episode of anaphylaxis due to walnut ingestion, a routine diagnostic workup was carried out, involving skin prick test with commercial extracts, prick by prick with fresh food and CAP-RAST assay for different nuts. Immediately after pricking with fresh Brazil nut, a severe episode of anaphylaxis occurred, that required epinephrine and intravenous steroids. The subject had never eaten Brazil nut before. Therefore we hypothesize a cross reactivity effect, since this phenomenon is well known for tree nuts. Our case suggests that in vivo diagnosis, especially if fresh nuts are used, should be performed only if adequate equipment to treat anaphylaxis is available.

Functional analysis of cross-reactive immunoglobulin E antibodies: peanut-specific immunoglobulin E sensitizes basophils to tree nut allergens.

BACKGROUND: Peanut and tree nuts are a major cause of food-induced anaphylaxis with an appreciable mortality. Co-sensitization to peanuts and tree nuts is a common clinical observation and may be because of peanut-specific serum IgE antibodies that cross-react with tree nut allergens. It is, however, unclear whether these cross-reactive IgE antibodies are involved in effector-cell activation. OBJECTIVE: To determine if cross-reactivity of peanut-specific IgE antibodies with tree nuts can cause effector cell activation using an in vitro basophil activation assay. METHODS: Two peanut allergic subjects with positive specific IgE for peanut and tree nuts (as measured by CAP-FEIA) were studied. Basophil activation to peanut and tree nuts, as indicated by CD63 expression, was assessed by flow cytometry to confirm co-sensitization to peanut and tree nuts. Inhibition ELISA using sera from the subjects was performed to detect peanut-specific IgE antibodies that cross-reacted with tree nut proteins. To determine whether cross-reactive tree nut allergens can induce effector-cell activation, peanut-specific antibodies were affinity purified from the subject sera and used to resensitize non-peanut/tree nut allergic donor basophils stripped of surface IgE. Basophil activation was then measured following stimulation with peanut and tree nut extracts. RESULTS: The two peanut allergic subjects in this study showed positive basophil activation to the peanut and tree nut extracts. Inhibition ELISA demonstrated that pre-incubation of the peanut allergic subject sera with almond, Brazil nut and hazelnut extracts inhibited IgE binding to peanut extract. IgE-stripped basophils from non-peanut/tree nut allergic subjects resensitized with affinity-purified peanut-specific antibodies from the peanut allergic subject sera became activated following stimulation with peanut, almond and Brazil nut extracts, demonstrating biological activity of cross-reactive IgE antibodies. CONCLUSION: Peanut-specific IgE antibodies that cross-react with tree nut allergens can cause effector-cell activation and may contribute to the manifestation of tree nut allergy in peanut allergic subjects.

Efficacy of a management plan based on severity assessment in longitudinal and case-controlled studies of 747 children with nut allergy: proposal for good practice.

BACKGROUND: There are few data on the long-term management of children with peanut/nut allergy. Advice is variable and often inadequate; further reactions are common. There is no consensus on the criteria for prescription of rescue medication, particularly adrenaline. METHOD: A longitudinal prospective and case-control study in a tertiary allergy clinic. Patients/parents/school staff of 747 children with confirmed peanut or tree nut allergy received detailed verbal and written advice on nut avoidance, training in recognition and (self-) treatment of reactions and a written treatment plan. The severity of nut allergy was graded (mild-severe) and emergency medication was allocated according to our criteria: all received oral antihistamines, injected adrenaline (EpiPen) was given to those with reactions with airway narrowing, milder reactions to low-dose exposure or concomitant asthma. At annual follow-up over 25 906 patient-months (median: 39 months) retraining was given and details of further reactions (frequency, severity and treatment) were obtained. Criteria for allocation of EpiPen were evaluated. RESULTS: The worst reaction pre-enrolment was mild in 64% and moderate/severe in 36% (airway narrowing). Of 615 subjects followed up, 21% had a further reaction (eightfold reduction in frequency), mostly mild. There was a 60-fold reduction in the frequency of severe reactions. Of those with a moderate-severe initial reaction, 99.5% had no or a less severe follow-up reaction. No child with a mild or severe index reaction had a severe follow-up reaction. Only 1/615 (0.2%) had a severe follow-up reaction and only 2/615 (0.3%) used adrenaline, both successfully and had it available according to our criteria. Of mild-moderate reactions, 77% required oral antihistamines alone and 15% no treatment. Children who had follow-up reactions had more frequent and severe reactions pre-enrolment. CONCLUSION: The management plan greatly reduced the frequency and severity of further reactions and was successful for all children. Our criteria for selective prescription of EpiPen in the context of this management plan were appropriate. This is the first study to provide evidence on which to inform practice.

The major human structural IgE epitope of the Brazil nut allergen Ber e 1: a chimaeric and protein microarray approach.

A protein microarray system containing different dilutions of 77 related and non-related proteins was used to show that IgE from subjects allergic to Brazil nut specifically recognise the seed 2S albumin protein (Ber e 1). Further, correctly folded chimaeric 2S albumin proteins containing structural epitope replacement were constructed and directed to the secretion pathway of the methylotropic yeast Pichia pastoris. Through the use of a chimaeric protein microarray system together with sera from a panel of 18 well-characterised Brazil nut allergic subjects, a structural IgE epitope of Ber e 1 was mapped to a helix-loop-helix region. The same structural region has been previously reported as the immunodominant region in related food allergens by different techniques. In conclusion, the combination of chimaeric proteins and protein microarrays will greatly facilitate the screening of a large number of individuals for a particular structural epitope and help to further our understanding of how proteins are recognised by the adaptive immune system.

Immunological analysis of allergenic cross-reactivity between peanut and tree nuts.

BACKGROUND: Peanut and tree nut allergy is characterized by a high frequency of life-threatening anaphylactic reactions and typically lifelong persistence. Peanut allergy is more common than tree nut allergy, but many subjects develop hypersensitivity to both peanuts and tree nuts. Whether this is due to the presence of cross-reactive allergens remains unknown. OBJECTIVE: The aim of this study was to investigate the presence of allergenic cross-reactivity between peanut and tree nuts. METHODS: Western blotting and ELISA were performed using sera from subjects with or without peanut and tree nut allergy to assess immunoglobulin E (IgE) reactivity to peanut and tree nut extracts. Inhibition ELISA studies were conducted to assess the presence of allergenic cross-reactivity between peanut and tree nuts. RESULTS: Western blot and ELISA results showed IgE reactivity to peanut, almond, Brazil nut, hazelnut and cashew nut for peanut- and tree nut-allergic subject sera. Raw and roasted peanut and tree nut extracts showed similar IgE reactivities. Inhibition ELISA showed that pre-incubation of sera with almond, Brazil nut or hazelnut extracts resulted in a decrease in IgE binding to peanut extract, indicating allergenic cross-reactivity. Pre-incubation of sera with cashew nut extract did not cause any inhibition. CONCLUSION: These results show that multiple peanut and tree nut sensitivities observed in allergic subjects may be due to cross-reactive B cell epitopes present in different peanut and tree nut allergens. The plant taxonomic classification of peanut and tree nuts does not appear to predict allergenic cross-reactivity.