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

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Abstract
Background More than 70% of the patients allergic to grass pollen exhibit IgE‐reactivity against the high molecular mass fraction between 50 and 60 kDa 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 allergen, Phl p 13, has recently been purified and characterized at the cDNA level. Objective The relative importance of the two high molecular mass allergens has been characterized with respect to their IgE‐binding frequency and capacity. Methods Both high molecular mass allergens were biochemically purified and subjected to nitrocellulose strips. About 306 sera obtained from subjects allergic to grass pollens were used to determine specific IgE‐binding frequency to Phl p 4 and Phl p 13. IgE‐binding of allergens was quantified by ELISA measurements. Pre‐adsorption of sera with purified allergens and subsequent incubation of nitrocellulose‐blotted timothy grass pollen extract was performed to determine whether or not Phl p 4 and Phl p 13 represent the whole high molecular mass allergen fraction. Proteolytic stability of both allergens was investigated by addition of protease Glu‐C. Results More than 50% of 300 patients displayed IgE‐binding with both allergens. 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 strong reacting sera with Phl p 13 eliminated not only reactivity of the 55/60 kDa double band, but in addition a ‘background smear’. Whilst undenatured Phl p 4 was resistent to proteolytic digestion with Glu‐C, native Phl p 13 was 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 candidates for potential recombinant therapeutics that may provide a basis for improved immunotherapy.