Novel Polyclonal−Monoclonal-Based ELISA Utilized To Examine Lupine (<i>Lupinus</i> Species) Content in Food Products

Holden, Lise; Moen, Lena Haugland; Sletten, Gaynour B. G.; Dooper, Maaike M.B.W.

doi:10.1021/jf063320w

Cited by 35 publications

(30 citation statements)

References 25 publications

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“…In a previous study, a monoclonal antibody produced against lupin unexpectedly cross-reacted with almond [18], which indicated recognition of a well-conserved epitope or structure between lupin and almond. This prompted us to investigate IgE binding to almond in the sera from the 6 lupin-allergic patients.…”

Section: Discussionmentioning

confidence: 99%

“…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.…”

Section: Introductionmentioning

confidence: 99%

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

Holden¹,

Sletten²,

Lindvik

et al. 2008

Int Arch Allergy Immunol

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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.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

Holden¹,

Sletten²,

Lindvik

et al. 2008

Int Arch Allergy Immunol

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“…Both procedures are routinely used in our laboratory. Procedure 1 is applied in ELISA for the determination of specific proteins (for example lupin, peanut, hazelnut and shrimp), based on a study in which several extraction buffers (based on citrate, high salt buffer, PBS, sodium carbonate, urea, sodium borate or trisÁglycine) were compared for the extraction of lupin proteins (Holden et al, 2007). Procedure 2 has been used for the isolation of lupin globulins (Dooper et al, 2006).…”

Section: Discussionmentioning

confidence: 99%

Antibody binding to hazelnut (Corylus avellana) proteins: the effects of extraction procedure and hazelnut source

Dooper

Plassen

Holden

et al. 2008

Food and Agricultural Immunology

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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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“…Thus, all products containing even trace amounts of lupin must be labelled correctly [3] and the International Union of Immunological Societies (IUIS) allergen nomenclature subcommittee recently designated β-conglutin as the Lup an 1 allergen [4]. Currently available commerial enzyme linked immunosorbent assays exploit polyclonal antibodies that are not specific to ß-conglutin [5] and reports in the literature only detail monoclonal IgG antibodies against α-conglutin and IgM antibodies against ß-conglutin [6], [7], and there are no reports or commercial ELISAs for the specific detection of ß-conglutin. There is thus a need for an analytical tool/method that can specifically detect the Lup an 1 allergen, ß-conglutin.…”

Section: Introductionmentioning

confidence: 99%

DNA Aptamers against the Lup an 1 Food Allergen

et al. 2012

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Using in vitro selection, high affinity DNA aptamers to the food allergen Lup an 1, ß-conglutin, were selected from a pool of DNA, 93 bases in length, containing a randomised sequence of 49 bases. ß-conglutin was purified from lupin flour and chemically crosslinked to carboxylated magnetic beads. Peptide mass fingerprinting was used to confirm the presence of the ß-conglutin. Single stranded DNA was generated from the randomised pool using T7 Gene 6 Exonuclease and was subsequently incubated with the magnetic beads and the captured DNA was released and amplified prior to a further round of Systematic Evolution of Ligands by Exponential Enrichment (SELEX). Evolution was monitored using enzyme linked oligonucleotide assay and surface plasmon resonance. Once a plateau in evolution was reached, the isolated DNA sequences were cloned and sequenced. The consensus motif was identified via alignment of the sequences and the affinities of these sequences for immobilised ß-conglutin were determined using surface plasmon resonance. The selected aptamer was demonstrated to be highly specific, showing no cross-reactivity with other flour ingredients or with other conglutin fractions of lupin. The secondary structures of the selected aptamers were predicted using m-fold. Finally, the functionality of the selected aptamers was demonstrated using a competitive assay for the quantitative detection of ß-conglutin. . Future work will focus on structure elucidation and truncation of the selected sequences to generate a smaller aptamer for application to the analysis of the Lup an 1 allergen in foodstuffs.

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Novel Polyclonal−Monoclonal-Based ELISA Utilized To Examine Lupine (Lupinus Species) Content in Food Products

Cited by 35 publications

References 25 publications

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

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

Antibody binding to hazelnut (Corylus avellana) proteins: the effects of extraction procedure and hazelnut source

DNA Aptamers against the Lup an 1 Food Allergen

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