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 allergen at the molecular level, we have constructed an IgE combinatorial library from a grass pollen allergic patient. cDNAs coding for IgE heavy chain fragments and for light chains were reverse-transcribed and polymerase chain reaction-amplified from RNA of peripheral blood lymphocytes and randomly combined in plasmid pComb3H to yield a combinatorial library of 5 x 10(7) primary clones. IgE Fabs with specificity for Phl p 5, a major timothy grass pollen allergen, were isolated by panning. 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 should facilitate studies on the molecular interaction between IgE antibodies and allergens and encourages the consideration of specific IgE Fabs that are capable of interfering with allergen-IgE binding as potential therapeutic tools.
Steinberger
P
P
Institute of General and Experimental Pathology, AKH, University of Vienna, Austria.
Kraft
D
D
Valenta
R
R
D000485:Allergens / Q000276:immunology*; D000595:Amino Acid Sequence; D001483:Base Sequence; D001666:Binding Sites, Antibody; D001667:Binding, Competitive; D018076:DNA, Complementary; D006801:Humans; D006967:Hypersensitivity / Q000276:immunology*; D007073:Immunoglobulin E / Q000235:genetics*; D007140:Immunoglobulin Fab Fragments / Q000235:genetics; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000276:immunology*; D006109:Poaceae / Q000276:immunology; D011058:Pollen / Q000276:immunology*; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology; D013045:Species Specificity
D000485:Allergens; D018076:DNA, Complementary; D007140:Immunoglobulin Fab Fragments; C076049:Phl p V protein, Phleum pratense; D010940:Plant Proteins; D011994:Recombinant Proteins; D007073:Immunoglobulin E