We prepared a protein extract of 0.6 mg/ml in phosphate buffered saline using frozen lingonberries in the presence of protease inhibitors. After informed consent patient's serum was obtained. Two independent lingonberry allergen extract-coated IgE ELISAs using goat anti-human IgE-labelled-peroxidase (Sigma-Aldrich, ST Louis, MO, USA) showed positive results. (OD 495): 0.343 (patient) vs 0.017 (control) (Mean values from 2 different assays).
SDS-PAGE[8] was performed with a 12% polyacrylamide gel and a stacking gel of 4%. It was applied 19.2 μg of lingonberry extract to every lane and electrophoresis was performed (Mini Protean II System, Bio-Rad laboratories, Richmond, USA). Then, proteins were electrophoretically transferred from the separating gel to Immobilon-P™ (PVDF, Millipore Corporation, Billerica, MA, USA) membranes in a transfer buffer. After blocking with a solution of gelatine 3% for 1 hour, the membranes were washed and incubated overnight with patient's and normal control sera. Next day, membranes were washed and incubated with goat anti-human IgE-labelled-peroxidase as mentioned above. Detection was performed with a chemiluminescence substrate (Pierce Chemical Company, Rockford, Illinois). The western-blot revealed IgE in the patient's serum that bound to some medium/high-molecular-weight protein bands (Figure 1b). Control sera were negative.