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.

The use of lupine in foods has increased considerably during the past decade, reflected by a corresponding increase in reported lupine-induced allergic incidents. Lupine allergy may arise either by primary sensitization or by clinical cross-reactivity in peanut-allergic persons. Detection of lupine proteins in food has previously been based on the use of patient serum. A novel sandwich enzyme-linked immunosorbent assay (ELISA) for the detection and quantification of lupine in processed foods was developed, using a polyclonal rabbit antilupine capture antibody and a biotinylated conjugate of the same antibody for detection. The antibody was highly specific for lupine, apart from minor cross-reactivities to other legumes. The assay had a detection limit of 1 mug/g and was successfully used to quantify lupine protein in various food matrixes. Recoveries ranged from 60 to 116%, while the intra-and interassay coefficients of variation were <6% and <21%, respectively.

Sweet lupines are increasingly used in food production. Cause for concern has been expressed due to the increase in reported lupine-induced allergic incidents and the association between lupine and peanut allergies. In the current study, a polyclonal-monoclonal antibody-based sandwich ELISA for the detection of lupine proteins in foods was developed. The assay was sensitive to both native and processed proteins from Lupinus angustifolius and Lupinus albus and had a detection limit of 1 mug/g. Intra- and interassay coefficients of variation were <5 and <17%, respectively. A selection of 112 food samples, both with and without lupine declaration, was evaluated for their content of lupine. The data showed that the majority were in agreement with the respective labeling. However, some inconsistency was seen, typically in bread/rolls and soy flours.

Background: The ingestion of dietary products containing sweet lupin (such as Lupinus albus or Lupinus angustifolius) has been reported to cause IgE-mediated allergic reactions. Recent studies have indicated lupin globulins as important IgE binding proteins. The aim of the present study was to generate and characterize monoclonal antibodies (mAbs) against lupin seed proteins. Methods: Mice were immunized with a protein isolate from L. albus and mAbs were obtained by hybridoma techniques. Albumins and globulins were extracted, and the globulin fraction was separated further into conglutins by anion exchange chromatography. Specificities, binding patterns and applications of the mAbs were investigated by immunochemical methods. Results: Five mAbs were produced: Lu11 (an IgG2b antibody), Lu8, Lu18, Lu34 and Lu35 (all IgM antibodies). The mAbs reacted strongly with protein isolates from both L. albus and L. angustifolius. All mAbs are directed towards the lupin globulin fraction; Lu11 and Lu18 recognize α-conglutin, while Lu8, Lu34 and Lu35 recognize β-conglutin. In addition, Lu11 inhibited the binding of IgE from patients with positive skin prick tests to lupin proteins in a competitive ELISA by approximately 30%. Furthermore, preliminary results show that Lu11 can be used to develop a sensitive method for the detection of α-conglutin in foods. Conclusions: Lupin globulins are immunogenic and α-conglutin is a potential allergen. This is the first study describing mAbs against the candidate lupin allergens, emphasizing the importance of additional studies on conglutins in lupin allergy.

Children with sensitization to lupin are not likely to have a clinical lupin allergy. Avoidance of lupin on the basis of lupin sensitization or peanut allergy would lead to unnecessarily strict diets. Food challenge is currently necessary to diagnose lupin allergy.

The quality of an applied protein extract is important in both serological and in vivo diagnosis of allergy, and for allergen detection methods. In the present study the effects of the extraction procedure and hazelnut source on antibody binding to hazelnut (Corylus avellana) proteins were investigated. An overnight extraction procedure in trisÁ glycine buffer at 458C was compared to a quick, vigorous extraction in a tris buffer at room temperature (RT). Antibody binding was studied by the use of polyclonal antiserum, a monoclonal antibody against the pollen-unrelated allergen Cor a 9, and human IgE from hazelnut allergic individuals. The extraction procedure, as well as the storage time and degree of processing of hazelnuts affected the protein constitution of the extract, as visualised by SDSÁPAGE. Fresh and roasted (10 min at 1708C) hazelnuts generally contained a higher amount of intact hazelnut proteins than elder ( 6 months) raw hazelnuts stored at RT. The time of storage of raw hazelnuts was inversely related to the amounts of Cor a 9 and IgE-binding proteins. In general, a rapid extraction procedure increased the efficacy of protein extraction and antibody-binding capacity to the proteins compared to an overnight extraction protocol. The present study indicates that for the production of hazelnut protein extracts, the use of fresh hazelnuts is important. A quick, vigorous extraction in a tris buffer might contribute positively, at least for extraction of Cor a 9.

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