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Repertoire cloning of a human IgG inhibitor ofαIIbβ3function. The OG idiotype

Molecular Immunology
Publication Date
DOI: 10.1016/0161-5890(95)00034-c
  • Platelet
  • Isoantibody
  • Inhibitor
  • Idiotype
  • Human
  • Thrombasthenia
  • Integrin
  • αIibβ3


Abstract A patient (OG) with Glanzmann thrombasthenia became refractory to platelet transfusion following the production of IgG antibodies (Ab1) specific for the integrin subunit β 3. We generated recombinant V H and V L cDNA libraries using IgG-specific mRNA isolated from OG peripheral blood B-lymphocytes that had been selected for binding to antigen ( α IIb β 3 adsorbed to plastic dishes). These antigen-specific B-lymphocytes contain rearranged V H DNA segments that belong exclusively to the V H4 gene family. Recombinant Fab were expressed on the surface of filamentous phage coinfected with V H and V L segments cloned into the phagemid pHEN1 or the phage fd-tet-DOG1. To facilitate selection of the desired recombinant Ab1 Fab, we developed a rabbit polyclonal antibody specific for affinity-purified OG anti- β 3 Fab (Ab2). Ab2 reacts specifically with Ab1, and this interaction is inhibited by purified α IIb β 3. Following three rounds of phage selection on Ab2 adsorbed to plastic dishes and random reassociation of heavy and light chains, we isolated Ab1 Fab and tested their binding to α IIb β 3. Five Id-positive Fab were selected for further characterization. These Fab use one of two V H genes (H21 or H23) complexed with one of three Vλ genes. Subsequent sequence data demonstrated that all three lambda genes are the same clone L22 which uses a germline Vλ gene segment. Fab using H23 bind to α IIb β 3, while those using H21 do not. Based on sequence homology, both H21 and H23 use V H gene segments belonging to the V H4 gene family. Thus, the idiotype OG is restricted to the V H4 gene family and is the first sequenced prototype of human antibodies that bind close to or at a functional epitope(s) of α IIb β 3.

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