High-throughput NMR authentication of food allergens

For in vitro allergy diagnosis, purified food allergens have to meet high standard quality criteria. 1D 1H-NMR analysis can play a significant role to assess the conformation and to contribute to the authentication of allergens. Furthermore, this method allows to i) perform a structural comparison of allergens within a protein family, ii) identify the thermal stability of food allergens and iii) detect structural differences between recombinant and natural allergens. Methods: The most important food allergens from cow’s milk (Bos d 4, 5, 8) , hen’s egg (Gal d 1-5), fish (Gad m 1), shrimp (Pen a 1), peanut (Ara h 1-3), hazelnut (Cor a 1-4, Cor a 8,9,11), celery (Api g 1,4,5) and apple (Mal d 1-4) and peach (Pru p 1,3) were purified according to established purification protocols and their allergenic activity tested. Primary and secondary structures were verified by N-terminal sequencing, MALDI-TOF-MS analysis and far-UV CD spectroscopy. IgE binding capacity was tested in ELISA and immunoblotting assays using allergic patients’ sera. The presence and extent of the tertiary structure was assessed by two 1D-1H-NMR experiments, using a 700MHz field at 298K and under variable buffer conditions. If available the consistency of the allergen’s spectra was compared with structural information already available. Thermal stability of purified nsLTPs from different sources was investigated and NMR spectra compared. NMR spectra of recombinant Cor a 8 was compared to the natural counterpart as well as for the fish parvalbumin. For Bet v 1 related food allergens their NMR spectra were compared regarding relevant structural differences. Results and conclusions: The analysis of the 1D-1HNMR spectra allowed the classification of the allergens into molecules whose spectra showed the unquestionable features of a rigid and extended tertiary structure, molecules without a rigid tertiary structure and allergens which displayed both features, tertiary structure with flexible and mobile regions. Furthermore, differences regarding thermal stability within a protein family were detected based on NMR spectra. In summary, 1D-1H-NMR proved a highly useful method requiring low protein concentrations, without 15N and 13C labeling for the structural authentication of allergens even when there is a limited quantity of protein available. This work was carried out within the EuroPrevall project, funded by the EU (contract number FOODCT- 2005-514000)