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Keywords:

  • cross-reactivity;
  • food allergy;
  • lupine allergy;
  • lupine immunoblotting;
  • oral allergic syndrome

Dried and boiled lupine seed (Lupinus albus) is a traditional snack in some Mediterranean countries, namely in Portugal. The mixture of lupine with wheat flour was authorized in Europe in the late 1990s in bread, pasta and bakery (1, 2).

Several allergic reactions to lupine ingestion have been reported since 1994, but they were generally considered as a result of cross-reaction to peanut and/or other legumes (1–5).

We have previously presented two atopic patients complaining of oral allergic syndrome following lupine ingestion, denying symptoms associated with peanut or other legumes (6). Patient 1 was a 50-year-old man, presenting allergic rhinitis and asthma, sensitized to house mites and patient 2, a 52-year-old woman, with allergic seasonal rhinitis and asthma, sensitized to house dust mite, grass, parietaria and mugwort pollens. They had negative skin prick tests to peanut, soy, beans and lentil and positive prick-to-prick tests to raw lupine seed. Specific IgE (ImmunoCAP, Pharmacia, Uppsala, Sweden) to lupine were 46.0 and 26.8 kUA/l and to peanut 0.39 and 0.84 kUA/l respectively. Sera from the two patients were submitted to L. albus IgE inhibition by lupine and peanut extracts (Pharmacia UniCAP protocol). Inhibition values with lupine extract were 90%, while with peanut extract were 75% and 65%.

In this work, we tried to find lupine antigens responsible for a primary sensitization. Lupine and peanut protein extracts were obtained from smashed dry seeds. Protein separation was performed by isoelectrofocusing (IEF) and by SDS-PAGE, being blotted onto 0.2-μm-pore nitrocellulose membranes (Bio-Rad, Hercules, CA, USA) (7, 8), using a Nova Blot semi-dry system (Pharmacia Biotech, Uppsala, Sweden) for the latter (9). Each patient’s serum was tested towards IEF and SDS-PAGE lupine separations by immunoblotting. Two controls were applied, one without serum and the other with neither serum nor secondary antibodies. Afterwards, immunoblotting inhibition with lupine and peanut extracts was performed, followed by antigen detection in blots from lupine IEF and SDS-PAGE separations (7).

  • image(1)

[  IgE immunoblots with Lupinus albus extract submitted to pH 3–10 IEF in polyacrylamide gel and blotted onto nitrocellulose membranes. B1, control without serum; B2, control without serum and secondary antibodies; 1 and 2, serum from patients 1 and 2, respectively. Serum inhibited by lupin extract (1L and 2L) and by peanut extract (1P and 2P). ]

IgE-binding proteins migrated to a pH ranging from 5 to 7. Those ones not inhibited by peanut extract migrated to pH 5 and to pH 6.05–6.3. Sera IgE from both patients recognized proteins of approximately 34 kDa and in the 40–65 kDa range. Patient 2 serum also recognized 7–15, 38, 77, 162 and 195 kDa proteins. Inhibition with lupine extract was complete for both sera. Serum preincubation with peanut extract did not inhibit patient 1 bands. On the other hand, it completely inhibited the 7–15 and 77 kDa bands and partially the 38, 40–65, 162 and 195 kDa components recognized by patient 2. Band of 34 kDa was never inhibited.

In this study, the clinical picture, skin prick tests and serum IgE to lupine and to peanut were suggestive of a sensitization to lupine not related to peanut allergy. The complete inhibition of lupine-specific IgE by lupine extract (90%) vs the only partial inhibition by peanut extract (65% and 75%) strongly supports this hypothesis. Immunoblotting studies performed by IEF as well as SDS-PAGE showed that some lupine IgE-binding proteins were inhibited by peanut extract, but they also evidenced bands only partially inhibited and others that suffered no inhibition at all, namely the 34 kDa protein and some of the proteins in the range of 40–65 kDa. These results clearly support that lupine is capable of inducing allergic sensitization out of the context of cross-reactivity with peanut. Taking them into account, we reinforce the need of including the lupine content in mandatory food labeling.

References

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  2. References
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