Monoclonal Antibodies against the Candidate Lupin Allergens α-Conglutin and β-Conglutin

Abstract

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.

Characterization of IgE Binding to Lupin, Peanut and Almond with Sera from Lupin-Allergic Patients

“…Protein extracts were stored at –20°C. Procedures for the isolation of globulins and the generation of protein fractions containing α- or β-conglutins (indicated as α- or β-conglutins in the text) have been described previously [16]. For the isolation of γ-conglutin, L. angustifolius seeds were soaked in deionized H 2 O for 16 h and heated at 60°C for 4 h. Precipitated γ-conglutin was obtained by centrifugation and dissolved in 50 m M Tris buffer, pH 7.5. δ-Conglutin was principally isolated as described elsewhere [19].…”

“…In earlier studies performed in this laboratory, anti-lupin polyclonal and monoclonal antibodies have been generated, characterized and used in the development of enzyme-linked immunosorbent assay (ELISA) methods for the detection of lupin in foods [16,17,18]. In the comprehensive specificity testing of the antibodies, the monoclonal antibody was found to cross-react with almond, which was a somewhat unexpected finding because these plants are phylogenetically distant.…”

Cross-reactivity between peanut and lupin proteins

“…a-, b-and c-Conglutins were purified according to a published method (Dooper, Holden, Faeste, Thompson, & Egaas, 2007), using a preparative HPLC 1200 (Agilent Technologies, Santa Clara, CA). The purity of the column fractions was confirmed by MS/MS analysis.…”

Lupin Allergen Detection

“…Proteins from seeds of L. albus and L. angustifolius have been analyzed and identified as potential allergens (Parisot et al, 2001;Magni et al, 2005a;Dooper et al, 2007;Lindvik et al, 2008). Detailed information can be found in the Allergome database, www.allergome.org.…”

“…Lupin seed proteins have been fractionated by chromatographic methods (Sironi et al, 2005;Dooper et al, 2007). The separated conglutins have been studied for IgE-binding affinities (Magni et al, 2005a;Holden et al, 2008), and cross-reactivity with major peanut allergens has been elucidated (Dooper et al, 2009).…”