**4. Conclusions**

50 Current Insights in Pollen Allergens

similar quantities.

(Bio-Rad).

**3.3. IgE-binding analysis by 2-D Western blotting** 

Ole e 1 isoforms showed the highest quantitative differences (Fig. 3A). Thus, the 12 Ole e 1 isoforms detected on 2D blots could be grouped on the basis of their intensities into 3 quantitative classes: high (>50% intensity; isoforms Ole e 1a, c, e, g and i), medium (20-50%; Ole e 1b, d, j, k and l) and low abundant (<20%; Ole e 1f and h). On the other hand, the six Ole e 9 isoforms were discriminated as high (Ole e 9a, b, c and d) or low (Ole e 9e and f) abundant (Fig. 3D). Ole e 2a and b isoforms showed similar intensities, while the amount of Ole e 2c was lower but still significant. Finally, the two Ole e 7 isoforms identified showed

The IgE reactivity of 'Picual' pollen proteins was assayed on 2-D blots using a pool of three olive allergic patients' sera and an anti-human IgE peroxidase-conjugated secondary antibody. Chemiluminiscent detection of IgE-binding was carried out with the Immun-StarTM WesternCTM Chemiluminescence Kit (Bio-Rad) according to the manufacturer´s instructions. Chemiluminiscent spots were imaged with a ChemiDoc XRS Molecular Imager

**Figure 4.** IgE-binding analysis by 2-D Western blotting of olive (cv. 'Picual') pollen total proteins. The blot was probed with a pool of sera from three patients allergic to olive pollen. Reactive spots were visible on the 2-D blot after detection by chemiluminiscence. Protein markers are displayed on the left.

The IgE-binding pattern is shown in Fig. 4. Incubation of the 2-D blot with patient's sera revealed up to 31 IgE-reactive spots with molecular weights ranging from 18,5 to 32 kDa. Allergic patients' IgEs recognized the 12 Ole e 1 isoforms detected by 10H1 antibody (Fig. 4, arrows). In addition, 9 new spots that might correspond to other Ole e 1 isoforms were also Two-dimensional Western blotting is a suitable method for olive pollen allergen isoform profiling, and might help in the standardization of protein extracts used for allergy diagnosis and immunotherapy. The 2-D electrophoresis-based immunodetection of allergens has a few advantages over classical 1-D Western-blotting, since they provide: 1) detailed information about individual isoforms of polymorphic allergens (i.e. number of isoforms, pIs and their positions on the 2-D map), and 2) relative quantitative data of each isoform. In addition, using a pool or individual sera from an allergic population, it is possible to identify which are the most reactive isoforms of a given allergen. In the present work, four allergens, namely Ole e 1, Ole e 2, Ole e 7 and Ole e 9, have been studied in a single variety (i.e. 'Picual'). These analyses should extend to other olive allergenic proteins and a higher number of cultivars. Moreover, this method can be also applied to other allergenic pollens and other type of allergies (e.g. food allergens).
