title: Analysis of IgE antibodies from a patient with atopic dermatitis: biased V gene usage and evidence for polyreactive IgE heavy chain complementarity-determining region 3.
abstract: To better understand V gene usage, specificity, and clonal origins of IgE Abs in allergic reactions, we have constructed a combinatorial Ab library from the mRNA of an adult patient with atopic dermatitis. Sequence analysis of random clones revealed that 33% of clones used the IGHV6-1 H chain V gene segment, the only member of the V(H)6 gene family. IGHV6-1 is rarely used in the expressed adult repertoire; however, it is associated with fetal derived Abs. Features of the V(H)6 rearrangements included short complementarity-determining region 3, frequent use of IGHD7-27 D gene, and little nucleotide addition at the D-J junction. There was also a low level of mutation compared with V(H)1, V(H)3, and V(H)4 rearrangements. The library was expressed as phage-Fab fusions, and specific phage selected by panning on the egg allergen ovomucoid. Upon expression as soluble IgE Fabs, 12 clones demonstrated binding to ovomucoid, skim milk, and BSA by ELISA. Nucleotide sequencing demonstrated that the IGHV6-1 V gene segment encoded each of the 12 multiply reactive IgE Fabs. A cyclic peptide was designed from the complementarity-determining region 3 of several of these clones. The cyclic peptide bound both self and nonself Ags, including ovomucoid, human IgG, tetanus toxoid, and human and bovine von Willebrand factor. These results suggest that some IgE Abs may bind more than one Ag, which would have important implications for understanding the multiple sensitivities seen in conditions such as atopic dermatitis.
authors.affiliation: School of Microbiology and Immunology, University of New South Wales, Kensington, Australia.
authors.forename: Michael R
authors.initials: MR
authors.lastname: Edwards
authors.forename: Wandy
authors.initials: W
authors.lastname: Brouwer
authors.forename: Caroline H Y
authors.initials: CH
authors.lastname: Choi
authors.forename: John
authors.initials: J
authors.lastname: Ruhno
authors.forename: Robyn L
authors.initials: RL
authors.lastname: Ward
authors.forename: Andrew M
authors.initials: AM
authors.lastname: Collins
mesh_terms: D000328:Adult; D000595:Amino Acid Sequence; D000916:Antibody Diversity / Q000235:genetics; D000918:Antibody Specificity / Q000235:genetics; D001666:Binding Sites, Antibody / Q000235:genetics; D001667:Binding, Competitive / Q000235:genetics / Q000276:immunology; D003001:Cloning, Molecular; D022801:Complementarity Determining Regions / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D004252:DNA Mutational Analysis; D003876:Dermatitis, Atopic / Q000235:genetics* / Q000276:immunology*; D005260:Female; D015723:Gene Library; D015326:Gene Rearrangement, B-Lymphocyte, Heavy Chain; D006801:Humans; D007073:Immunoglobulin E / Q000032:analysis* / Q000235:genetics* / Q000378:metabolism; D007140:Immunoglobulin Fab Fragments / Q000096:biosynthesis; D007128:Immunoglobulin Fragments / Q000096:biosynthesis / Q000235:genetics; D007143:Immunoglobulin Heavy Chains / Q000235:genetics* / Q000378:metabolism; D007144:Immunoglobulin J-Chains / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D009711:Nucleotides / Q000378:metabolism; D010059:Ovomucin / Q000378:metabolism; D010456:Peptides, Cyclic / Q000138:chemical synthesis / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics / Q000276:immunology; D017422:Sequence Analysis, DNA; D020539:Sequence Analysis, Protein
chemical_list: D022801:Complementarity Determining Regions; D007140:Immunoglobulin Fab Fragments; D007128:Immunoglobulin Fragments; D007143:Immunoglobulin Heavy Chains; D007144:Immunoglobulin J-Chains; D007135:Immunoglobulin Variable Region; D009711:Nucleotides; D010456:Peptides, Cyclic; C100277:immunoglobulins, Fd; D010059:Ovomucin; D007073:Immunoglobulin E