BIOCHEMICAL AND IMMUNOLOGICAL CHARACTERIZATION OF RECOMBINANT ALLERGEN LOL-P-1

Pollen from perennial rye grass (Lolium perenne), a major cause of typ e-I allergy worldwide, contains a complex mixture of allergenic protei ns among which Lol p 1 is one of the most important. We describe the e xpression, purification and characterization of a recombinant Lol p1 o verproduced in Escherichia coli. The recombinant allergen, expressed i n high yields and purified in milligram amounts, bound to specific IgE antibodies from human sera, induced histamine release from sensitized human basophils, and elicited rabbit antisera that recognize specific ally recombinant Lol p 1 and natural Lol p 1 of pollen extract. Recomb inant Lol p 1 was used to develop ImmunoCAP assays for analysis of 150 sera that were Radioallergosorbent test positive to L. perenne pollen . In 130 of them (87%) the assay detected a significant level of IgE a ntibodies to Lol p 1, reaching on average 37% of the level obtained wi th a test for IgE to the whole grass pollen extract. To map epitopes o n Lol p 1, we produced three deletion mutants [des(116-240)-Lol p 1, d es-(1-88)-Lol p 1 and des-(133-189)-Lol p 1], which were efficiently e xpressed in bacteria. These all showed a strong reactivity with the sp ecific rabbit IgG antibodies, but lacked most or all the allergenic pr operties of recombinant Lol p 1. A study of the antigenic structure of Lol p 1 was performed using the three deletion mutants and a set of 1 7-18-residue overlapping synthetic peptides covering the whole allerge n sequence. The results indicate that human IgE and rabbit IgG antibod ies bind to distinct regions of Lol p 1, and that at least some import ant IEE epitopes are mainly conformational. The findings suggest that recombinant allergens constitute useful reagents for further developme nt of serological diagnosis of allergy, and that it should be possible to produce immunogenic fragments of allergenic proteins without aller genic properties.