Repertoire cloning of a human IgG inhibitor of alpha IIB beta 3 function. The OG idiotype

Molecular Immunology
F IshidaT J Kunicki

Abstract

A patient (OG) with Glanzmann thrombasthenia became refractory to platelet transfusion following the production of IgG antibodies (Ab1) specific for the integrin subunit beta 3. We generated recombinant VH and VL cDNA libraries using IgG-specific mRNA isolated from OG peripheral blood B-lymphocytes that had been selected for binding to antigen (alpha IIb beta 3 adsorbed to plastic dishes). These antigen-specific B-lymphocytes contain rearranged VH DNA segments that belong exclusively to the VH4 gene family. Recombinant Fab were expressed on the surface of filamentous phage coinfected with VH and VL 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-beta 3 Fab (Ab2). Ab2 reacts specifically with Ab1, and this interaction is inhibited by purified alpha IIb beta 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 alpha IIb beta 3. Five Id-positive Fab were selected for further characterization. These Fab use one of two VH genes (H21 or H23) complexed wi...Continue Reading

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Citations

Sep 10, 2003·The Journal of Laboratory and Clinical Medicine·Y S MehtaDipika Mohanty
Jun 6, 2000·Advanced Drug Delivery Reviews·H R Hoogenboom de Haard H
Sep 23, 2003·Clinical Immunology : the Official Journal of the Clinical Immunology Society·M J JacobinG Clofent-Sanchez
Feb 23, 1999·British Journal of Haematology·Y Y YangP P Chen
Mar 31, 1999·Baillière's Clinical Haematology·H DeckmynK Peerlinck
Feb 2, 2002·The Journal of Immunology : Official Journal of the American Association of Immunologists·Marie-Josée JacobinGisèle Clofent-Sanchez

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