FIGURE 4.

Patient sera reveal reduced cross-reactive carbohydrate determinants (CCD) binding to MANII-RNAi fruit extracts. (A) Immunoblots were prepared with extracts of Micro-Tom wild-type (wt) and RNAi-silenced fruits (GNTI-RNAi #20; vINV #9; MANII-RNAi lines #11 and #14). CCD-positive sera of potato/tomato-allergic patients PT-02 and PT-06 (Kaulfürst-Soboll et al., 2011a) were used for the detection of CCD-specific IgE (sIgE) antibodies. A serum of a non-allergic individual, also containing sIgE for polygalacturonase 2A (46 kDa, a major glycoprotein of tomato fruits), but only minor CCD-sIgE, was included as control. The Ponceau S-stained blot (protein) is shown as the loading reference. The black arrowhead points to vacuolar invertase (vINV, 52 kDa), which was not detected in vINV-RNAi fruits. Note that the CCD-positive patient sera (left) show strong binding to wt and vINV-RNAi, but reduced binding to GNTI-RNAi and MANII-RNAi extracts. Apparent molecular masses are indicated in kDa (PageRuler Prestained Protein Ladder, Fermentas). (B) Whole blood of patient PT-02 was used for a basophil activation test (BAT) with the indicated fruit extracts, demonstrating about 10 times reduced stimulation by GNTI-RNAi and also MANII-RNAi. Horseradish peroxidase (HRP; vacuolar glycoprotein with nine complex N-glycans) in PBS served as a positive control for CCD-dependent basophil stimulation and PBS as negative control.