The high molecular mass allergen fraction of timothy grass pollen (Phleum pratense) between 50-60 kDa is comprised of two major allergens: Phl p 4 and Phl p 13

Background More than 70% of the patients allergic to grass pollen exhibit I gE-reactivity against the high molecular mass fraction between 50 and 60 kD a of timothy grass pollen extracts. One allergen from this fraction is Phl p 4 that has been described as a basic glycoprotein. A new 55/60 kDa allerg en, Phl p 13, has recently been purified and characterized at the cDNA leve l. Objective The relative importance of the two high molecular mass allergens has been characterized with respect to their IgE-binding frequency and capa city. Methods Both high molecular mass allergens were biochemically purified and subjected to nitrocellulose strips. About 306 sera obtained from subjects a llergic to grass pollens were used to determine specific IgE-binding freque ncy to Phl p 4 and Phl p 13. IgE-binding of allergens was quantified by ELI SA measurements. Pre-adsorption of sera with purified allergens and subsequ ent incubation of nitrocellulose-blotted timothy grass pollen extract was p erformed to determine whether or not Phl p 4 and Phl p 13 represent the who le high molecular mass allergen fraction. Proteolytic stability of both all ergens was investigated by addition of protease Glu-C. Results More than 50% of 300 patients displayed IgE-binding with both aller gens. Clear differences concerning the immunological properties of Phl p 4 and Phl p 13 were confirmed by individual IgE reactivities. Quantification of specific IgE for both allergens revealed comparable values. For complete inhibiton of IgE-binding in the high molecular mass range preincubation of sera with both allergens was necessary. Interestingly, inhibition of stron g reacting sera with Phl p 13 eliminated not only reactivity of the 55/60 k Da double band, but in addition a 'background smear'. Whilst undenatured Ph l p 4 was resistent to proteolytic digestion with Glu-C, native Phl p 13 wa s degraded rapidly. Conclusion Phl p 4 and Phl p 13 are immunologically different and must both be considered as major allergens. They are judged to be important candidat es for potential recombinant therapeutics that may provide a basis for impr oved immunotherapy.