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For the identification and characterization of allergen grass group VI we performed two-dimensional immunoblotting of timothy grass pollen (Phleum pratense). Two intense 13-kD protein spots of pi 5.2 and 5.5 were found to be IgE reactive. By N-terminal microsequencing and amino acid analysis we identified them as Phl p 6 isoallergens. An antiserum was raised against Phl p 6 by immunizing BALB/c mice with allergen bearing nitrocellulose particles of one isoform. The antiserum revealed an intense reactivity to Phi p 6 isoforms, but also showed a weak cross-reactivity with Phl p 5 allergens. After immunoabsorption of patients’ serum to Phl p 6 spots on the blotting membrane, we were able to demonstrate that the eluted human IgE antibodies cross-react with the grass group V allergens as well. Therefore, Phl p 5 and Phl p 6 possess one or more common IgE binding epitopes.

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Timothy pollen extract was separated by 2D PAGE blot for further characterization of the major allergen Phl p V. Using pooled patient serum, we demonstrated that Phl p V consists of 4 components at 32 kD and 4 components at 38 kD, each differing in their pIs. The primary structure and the amino acid composition of the 8 proteins were determined from the blotted samples. Proteins of the same molecular weight did not differ in the 20 N-terminal amino acid residues, whereas the 32- and 38-kD proteins showed only 60% sequence identity. Furthermore, the results of the amino acid analyses suggest differences in their protein structure. Therefore, it might be possible that both proteins express different IgE-reactive epitopes.

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