CONSTRUCTION OF A COMBINATORIAL IGE LIBRARY FROM AN ALLERGIC PATIENT - ISOLATION AND CHARACTERIZATION OF HUMAN IGE FABS WITH SPECIFICITY FOR THE MAJOR TIMOTHY GRASS-POLLEN ALLERGEN, PHL-P-5

To characterize human IgE antibodies with specificity for a major alle rgen at the molecular level, we have constructed an IgE combinatorial library from a grass pollen allergic patient. cDNAs coding for IgE hea vy chain fragments and for light chains were reverse-transcribed and p olymerase chain reaction-amplified from RNA of peripheral blood lympho cytes and randomly combined in plasmid pComb3H to yield a combinatoria l library of 5 x 10(7) primary clones. IgE Fabs with specificity for P hl p 5, a major timothy grass pollen allergen, were isolated by pannin g. Sequence analysis showed that the 4 of the Fabs used the same heavy chain fragments which had combined with different kappa light chains. Soluble recombinant IgE Fabs were purified by affinity chromatography to Phl p 5 and, like natural IgE antibodies, cross-reacted with group 5 allergens from different grass species. The described approach shou ld facilitate studies on the molecular interaction between IgE antibod ies and allergens and encourages the consideration of specific IgE Fab s that are capable of interfering with allergen-IgE binding as potenti al therapeutic tools.