IMMUNOLOGICAL CHARACTERIZATION OF PURIFIED RECOMBINANT TIMOTHY GRASS-POLLEN (PHLEUM-PRATENSE) ALLERGENS (PHL-P-1, PHL-P-2, PHL-P-5)

Background: Grass pollen allergens belong to the potent elicitors of t ype I allergy. Approximately 40% of allergic individuals display IgE r eactivity with grass pollen allergens. In previous studies we have rep orted the complementary DNA cloning and expression in Escherichia coli of three of the most relevant timothy grass pollen allergens: Phl p 1 , Phl 2, and Phl p 5. Objective: To achieve high level expression of i mmunologically active timothy grass pollen allergens in E. coli, the c DNAs were inserted into expression plasmids. Methods: The three recomb inant grass pollen allergens were expressed at high levels in E. coli as recombinant nonfusion proteins, purified by conventional protein by conventional protein chemical methods and tested for their IgE-bindin g capacity by immunoblot and ELISA, as well as in histamine release as says. Results: Milligram amounts of pure recombinant allergens were ob tained from cultured E. coli. IgE binding to purified recombinant Phl p 1, Phl p 2, and Phl p 5 could be demonstrated by immunoblot and ELIS A. With ELISAs the percentage of grass pollen-specific IgE directed ag ainst the individual recombinant allergens induced dose-dependent and specific histamine release from patients' blood basophils. Conclusion: Purified recombinant timothy grass pollen allergens useful tools for diagnosis and therapy of grass pollen allergy.