Two-dimensional gel electrophoretic analysis with immobilized pH gradients of Dactylis glomerata pollen allergens

We studied Orchard grass pollen allergens (Dactylis glomerata) by a new two-dimensional electrophoretic technique which is the combination of isoelectric focusing (IEF) in immobilized pH gradient (IPG) and horizontal SDS-PAGE. The IPG-Dalt (two-dimensional gel electrophoresis with immobilized pH gradients in the first dimension) electrophoresis was made with four different but complementary pH gradients: 3-10.5, 4-9, 4-7, 6.3-10.5. We also compared the behavior of allergens in native and denaturing conditions during IEF. This technique followed by electroblotting on nitrocellulose membrane and immunodetection with an allergic patient serum was shown to increase the number of IgE-binding components up to 100 compared to one-dimensional techniques. Major allergen groups could be identified. New allergens not yet described could be characterized by their isoelectric points and their molecular masses. This highly resolving and reproducible technique, should allow an easy allergen standardization.