D3.351 - Characterization of peanut roasting-enhanced Ara h 2 - Ara h 1 complexes
Background
Cross-reactivity between structurally unrelated peanut storage proteins Ara h 1, 2 and 3 has been reported. We have proposed an alternative explanation for this unexpected finding: roasting-enhanced complexation of via disulphide-bridge swapping misinterpreted as cross-reactivity.
Method
Extracts of unroasted and roasted peanuts were studied by SDS-PAGE, immunoblotting and sandwich ELISA assays, using human monoclonal IgE and IgG antibodies (HuMabs) against Ara h 1 and Ara h 2. Complexes were purified by a combination of ultrafilration/size-exclusion chromatography (UF/SEC) and affinity purification.
Results
A hybrid sandwich ELISA was developed using anti-Ara h 1 HuMab as capturing and anti-Ara h 2 HuMab as detecting antibody. SDS-PAGE, immunoblotting with anti-Ara h 1 and anti-Ara h 2 HuMabs, and hybrid ELISA confirmed enhancement of Ara h 1 – Ara h 2 complexation upon roasting. Roasted peanut extract was separated into high and low molecular weight fractions (cut-off ~50 kDa) by both UF and SEC. A combination of the hybrid ELISA and normal Ara h 2 ELISA revealed that a large fraction of Ara h 2 is complexed in roasted peanut. From the >50 kDa fraction, Ara h 1 – Ara h 2 complexes were enriched by affinity chromatography using Sepharose-immobilized HuMab IgG 38B7 directed against Ara h 2. These complexes will be used to study their role in the process of sensitization by co-cultures of specific B- and T-cell clones from peanut allergic patients.
Conclusion
The presence of Ara h 1 and Ara h 2 allergen complexes was confirmed by immunoblotting and sandwich-ELISA's. The roasting of peanuts increased the presence of these complexes.
