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Analysis of IgE Antibodies from a Patient with Atopic Dermatitis: Biased V Gene Usage and Evidence for Polyreactive IgE Heavy Chain Complementarity-Determining Region 3¹

Michael R. Edwards^*, Wandy Brouwer^*, Caroline H. Y. Choi^*, John Ruhno, Robyn L. Ward^, and Andrew M. Collins^2,*

Schools of ^* Microbiology and Immunology and Medicine, University of New South Wales, Kensington, Australia; Royal North Shore Hospital, St. Leonards, Australia; and Department of Medical Oncology, St. Vincent’s Hospital, Darlinghurst, Australia

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_H6 gene family. IGHV6-1 is rarely used in the expressed adult repertoire; however, it is associated with fetal derived Abs. Features of the V_H6 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_H1, V_H3, and V_H4 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.